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Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses

Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses Nadia Terrazzini, Paola Mantegani, Florian Kern, Claudio Fortis, Anna Mondino, Stefano Caserta Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 The Journal of Infectious Diseases MAJOR ARTICLE Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses 1 2 3 2 4,a 3,4,5,a Nadia Terrazzini, Paola Mantegani, Florian Kern, Claudio Fortis, Anna Mondino, and Stefano Caserta 1 2 School of Pharmacy and Biomolecular Sciences, University of Brighton, United Kingdom; Laboratory of Clinical Immunology, Clinic of Infectious Diseases, San Raffaele 3 4 Scientific Institute, Milan, Italy; Brighton and Sussex Medical School, The University of Sussex, Falmer, East Sussex, United Kingdom; Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy; School of Life Sciences, The University of Hull, United Kingdom Background. Interleukin (IL)-7 promotes the generation, expansion, and survival of memory T cells. Previous mouse and human studies showed that IL-7 can support immune cell reconstitution in lymphopenic conditions, expand tumor-reactive T cells for adoptive immunotherapy, and enhance effector cytokine expression by autoreactive T cells. Whether pathogen-reactive T cells also benefit from IL-7 exposure remains unknown. June Methods. In this study, we investigated this issue in cultures of peripheral blood mononuclear cells (PBMCs) derived from patients infected with various endemic pathogens. Aer s ft hort-term exposure to IL-7, we measured PBMC responses to antigens derived from pathogens, such as Mycobacterium tuberculosis, Candida albicans, and cytomegalovirus, and to the superantigen Staphylococcus aureus enterotoxin B. Results. We found that IL-7 favored the expansion and, in some instances, the uncovering of pathogen-reactive CD4 T cells, by promoting pathogen-specific interferon- γ, IL-2, and tumor necrosis factor recall responses. Conclusions. Our findings indicate that IL-7 unveils and supports reactivation of pathogen-specific T cells with possible diagnos - tic, prognostic, and therapeutic significance of clinical value, especially in conditions of pathogen persistence and chronic infection. Keywords. candida infection; CMV infection; pathogen-specific responses; SEB responses; TB infection. Interleukin (IL)-7 [1] is a pleiotropic cytokine [2] regulating (eg, Bcl-2) [14, 20], IL-7 promotes the long-term survival of lymphopoiesis and T-cell homeostasis [3, 4]. It binds to a het- naive and memory-phenotype cells. Recombinant IL-7 elicits a erodimeric receptor formed by an α-chain (CD127 [3]), which marked increase of central memory (T ) and effector memory CM is private to the IL-7 receptor (IL-7R) and the common-γ-chain T cells (T ) when administered to aged nonhuman primates EM cytokine-receptor (CD132 [5, 6]). In humans, mutations neg- [21] and lymphopenic patients and macaques infected with atively ae ff cting the levels of CD127 have been correlated with human immunodeficiency virus (HIV) or simian immunode - severe immunodeficiency [ 7]. Conversely, increased serum lev- ficiency virus, respectively [ 22–24]. Similar effects are reported els of IL-7 and/or dysregulated activation of CD127 are reported in patients undergoing stem cell transplantation [25, 26], OA-CC-BY in patients with autoimmune conditions, including multiple where IL-7 levels correlate with the generation of stem-cell sclerosis [8, 9], rheumatoid arthritis [10], type-I diabetes [11], memory T cells [26]. In cancer patients, IL-7 preferentially inflammatory bowel disease [ 12], and psoriasis [13]. increases naive but not regulatory T-cell numbers [27], main- Upon antigen (Ag) encounter, IL-7 sustains the generation taining T-cell receptor (TCR) repertoire diversity [28]. Hence, of memory T lymphocytes in vitro [14] and in vivo [14–16] the immunotherapeutic use of IL-7 is increasingly proposed and favors the transition of effector to central memory cells to favor immune-cell reconstitution and function aer l ft ym - [17, 18] while driving their proliferation [19]. Via the JAK3/ pho-depleting chemotherapy or in the elderly. In the setting of STAT5 pathway and the upregulation of antiapoptotic factors adoptive T-cell therapy (ACT), IL-7 has been used in primary cultures to engineer and expand tumor-reactive T cells [29, 30]. We previously found that IL-7 selectively expands tumor-reac- Received 3 November 2017; editorial decision 14 February 2018; accepted 19 February 2018; tive CD4 T cells capable of promoting tumor protection in ACT published online February 28, 2018. Presented in part: 4th European Congress of Immunology, 6–9 September 2015, Vienna, [29]. Whether similar results could be extendible to patho- Austria. gen-specific T cells remains unknown. Therefore, we sought to A. M. and S. C. contributed equally to this work. Correspondence: S. Caserta, PhD, School of Life Sciences, Hardy Building, The University of investigate the expansion of pathogen-reactive CD4 T cells in Hull, Hull, United Kingdom, HU6 7RX UK ([email protected]). individuals ae ff cted by recurrent or persistent/chronic bacterial The Journal of Infectious Diseases 2018;217:1997–2007 (Mycobacterium tuberculosis [MTB] and Staphylococcus aur- © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons eus), fungal (Candida albicans [Ca]), or viral (cytomegalovirus Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted [CMV]) infections. We report that, in all cases, IL-7 enriched reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/infdis/jiy096 pathogen-specific CD4 T cells, enabling their detection and IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 1997 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 sensitizing them to Ag-specific recall responses. Furthermore, numbers of viable (0.1% Trypan blue-negative) cells were resus- IL-7 rescued chronically activated pathogen-specific effectors pended in complete media (Roswell Park Memorial Institute enhancing their Ag-recall responses. We believe that these data medium containing penicillin, streptomycin, glutamine, and open new avenues for diagnostic, prognostic, and therapeutic 10% FCS [all from Thermo Fisher Scientific] or autologous applications. serum) with or without human recombinant IL-7 (50  ng/mL, unless specified otherwise in individual figures; R&D Systems/ MATERIALS AND METHODS Biotechne, Minneapolis, MN) for 7 days. Where indicated, cells were stained with the fluorescent dye 5-(and-6)-carboxyu fl - Classification of Tuberculosis Patients orescein diacetate succinimidyl ester ([CFSE] Thermo Fisher Human immunodeficiency virus-seronegative patients with Scientific; 1 µM), in accordance with the manufacturer’s instruc - active tuberculosis ([TB] clinic and culture confirmed) were tions, using autologous serum or FCS to quench the labeling. recruited at the Clinic of Infectious Diseases, San Raffaele Where indicated, cells were first cultured in complete medium Hospital (Milan, Italy). They underwent tuberculin skin test - with or without bacterial SEB (1  μg/mL, Sigma/Merck). Aer ft ing (TST) administered by the Mantoux method with 0.1  mL 5 days, cells were harvested, washed and counted. Equal num- (5 tuberculin units) of Biocinetest-PPD tuberculin (Chiron ber of viable cells were finally seeded in culture with or with - Italia, Milan, Italy). The size of induration was evaluated aer ft out IL-7, for additional 7 days (day 12). Cyclosporine A ([CSA] 48–72 hours (an induration ≥10  mm was classified as posi - 0.5  µg/mL; Calbiochem/Merck) or anti-lymphocyte func- tive). Peripheral blood was drawn before starting any therapy tion-associated antigen (LFA)-1 blocking antibody (5  µg/mL; and following written informed consent. Healthy controls were a gift from Professor Ruggero Pardi, University Vita-Salute San selected among HIV-seronegative individuals with no history Raffaele, Milan) were added to the cultures. of TB exposure, no infection, and with negative reaction to the TST. Healthy controls were tested for the presence of Ca-Ag Mycobaterium tuberculosis Peptides and Candida albicans responses. Antigen-Specific Enzyme-Linked Immunospot Assay e enzy Th me-linked immunospot (ELISPOT) assay for interferon Ethics Statement (IFN)γ detection was performed as described previously [31]. Written informed consent or consultee approval to enroll was In brief, equal numbers of viable cells (5 × 10 cells/well) were secured for all study participants (patients and healthy donors). seeded in duplicate in 96-well plates (MAIPS4510; Millipore/ This study was approved by the Ethical Committee of the San Merck), precoated with anti-IFNγ capture monoclonal antibody Raffaele Scientific Institute, the UK National Research Ethics ([mAb] B-B1; Diaclone, Besançon, France), together with autol- Service (reference: 13/LO/1270), and the Brighton  and Sussex ogous irradiated PBMCs (5 × 10 cells/well), and Mycobacterium Medical School  (BSMS) Research Governance and Ethics tuberculosis peptides ([MTPs] a pool of 6 synthetic peptides, Committee (reference: 13/182/LLE) and carried out in accord- 2  µg/mL per peptide [Primm, Milan, Italy])  or Ca-Ag (25  µg/ ance with the approved guidelines. All data were anonymized. mL [Bio-Rad, Hercules, CA]) for 18 hours at 37°C, in 5% CO Cytomegalovirus Serology atmosphere. Biotinylated anti-IFNγ detection mAb (B-G1; Healthy donors were screened for the presence of CMV-specific Diaclone) was added (4 hours), followed by the streptavidin-al- antibody in serum. Cytomegalovirus immunoglobulin (Ig)G kaline phosphatase conjugate (1 hour) (Amersham Pharmacia serology (Architect CMV IgG; Abbot, Maidenhead, UK) was Biotech Europe GmbH, Freiburg, Germany). Aer ft a wash - performed at the Brighton and Sussex University Hospital Trust ing step, the nitroblue tetrazolium-5-bromo-4-chloro-3-in- virology laboratory. Cytomegalovirus-seropositive and -seroneg- dolylphosphate (Sigma) chromogenic substrate was added. + − ative individuals are referred to as CMV and CMV , respectively. Individual spot-forming cells were counted using an auto- mated image analysis system ELISPOT reader (AID-GmbH, Human Samples and T-Cell Cultures Strassberg, Germany). Mycobacterium tuberculosis peptides (20 Patients and healthy donors used for MTB- and Candida- amino acids) were derived from the ESAT-6 and CFP-10 secre- specific responses were part of a previously published tory proteins of MTB, purified (>70%), and previously validated cohort [31]. Donors (male/female, 10:9) used for CMV and [31]. Peripheral blood mononuclear cells in medium alone or Staphylococcus aureus enterotoxin B (SEB)-specific responses stimulated with phytohemagglutinin ([PHA-P] Sigma; 5 µg/mL) were 68  ±  17  years old. Peripheral blood mononuclear cells were used to assess assay background and functionality. (PBMCs) were isolated by blood centrifugation over Ficoll- Hypaque (Sigma/Merck, Darmstadt, Germany) density gradi- Flow Cytometry Analysis and Intracellular Cytokine Staining ent. Cultures were derived either from freshly isolated (CMV/ Cells were harvested, washed with staining buffer (phos - SEB responses) or cryopreserved (90% fetal calf serum [FCS] and phate-buffered saline supplemented with 0.5% FCS and 10% dimethyl sulfoxide; MTB/Ca-Ag responses) PBMCs. Equal 0.02% NaN ), and incubated with mAbs directed against 1998 • JID 2018:217 (15 June) • Terrazzini et al Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 surface Ags for 15 minutes at room temperature. e Th follow - Patients with detectable (patient [Pt]#1 and Pt#2; Figure  1) or ing fluorescence-conjugated mAbs were used: anti-CD3-v500, undetectable (Pt#3; Figure 1) MTB-specific IFN γ-producing T anti-CD8-allophyocyanine-H7, anti-CD27-phycoerythrine cells were analyzed. Cryopreserved cells were tested in MTP- (PE), IL-2-fluorescein isothiocyanate, TNF α Alexa700 (all recall assays immediately aer t ft hawing (day [d]0; Figure  1A from BD Biosciences, Franklin Lakes, NJ); anti-CD4-peridi- and B) or at the end of a culture (d7) in IL-7 compared with nin chlorophyll, anti-IFNγ PE-Cy7 (Cyanine-7), anti-CD154 complete media (med). Not all patients with detectable MTP- Pacific-Blue (BioLegend, Cambridge, UK); anti-CD45RA ECD specific T-cell responses on fresh PBMCs [ 31] had a detect- (Beckman Coulter, UK); and Yellow live-dead stain (Thermo able MTP response aer t ft hawing, as seen in the case of Pt#2, Fisher Scientific). er Th eaer ft , cells were washed in staining compared with Pt#1. Nonetheless, we found that IL-7 selec- buffer before acquisition. tively enriched cultures for MTP-specific IFN γ T cells by 4- to Intracellular cytokine staining was used to measure MTP- 10-fold in all cases (Figure 1B). Although MTP-specific T cells specific cytokine release at single-cell level. Equal numbers of could also expand in control cultures (as seen for Pt#1, med), 6 + + CFSE-labeled cytokine-cultured cells (0.6  ×  10 ) were stimu- this was best explained by the increase of total CD3 CD4 T lated (6 hours) with unpulsed (nil) or MTP-pulsed (4  µg/mL) cells in some patients (data not shown). It is important to note autologous irradiated (5000 rad) PBMCs (3 × 10 cells), in the that the frequency of MTP-specific cells increased in IL-7 cul - presence of human anti-CD28-stimulating mAb (2  µg/mL; tures more than the levels found in control cultures, even if the + + BD Biosciences). In the last 5 hours of stimulation, Brefeldin percentage of total CD3 CD4 T cells remained similar. Of note, A  (10  µg/mL; Sigma) was added. To measure CMV-/SEB- IL-7 also allowed us to detect MTP-specific T cells in samples specific cytokine release, equal numbers of cells were stimu - derived from immunosuppressed patients (ie, anergic; Pt#3). lated with CMV lysate (2  µg/mL; Advanced Biotechnologies, Sensitization with IL-7 significantly increased absolute numbers Eldersburg, MD) or SEB (1  µg/mL) for 2 hours followed by of MTP-specific T cells in all the TB patients analyzed (n = 5; 14 hours in the presence of Brefeldin A.  er Th eaer ft , cells were Figure  1C) compared with non-Bacillus Calmette-Guérin- washed, surface-stained as described above, fixed, permeabi - vaccinated healthy donors (non-MTB-infected controls, n = 8). lized, and stained with anti-CD4, anti-IL-2, anti-IFNγ, and/or To independently confirm the presence of MTP-specific T anti-TNFα mAbs. Events were acquired on a BD FACSCalibur cells, we additionally performed ICS after MTP stimulation. or LSRII, and data were analyzed using the FlowJo-v9.x soft - Mycobacterium tuberculosis peptide-pulsed irradiated autol- ware (TreeStar Inc., Ashland, OR). ogous PBMCs were used as Ag-presenting cells (APCs). As with ELISPOT assays, in ICS we found that the frequency Statistical Analysis + + of MTP-specific, IL-2 IFNγ CD4 T cells increased in IL-7 GraphPad Prism 7.03 was used for statistical analyses. The cultures, compared with freshly thawed PMBCs (d0) and D’Agostino-Pearson and Shapiro-Wilk tests were used to deter- control cultures (med; Supplementary Figure  1). Thus, mine normality of data distribution. For normally distributed MTB-specific T-cell recall responses are enhanced after sen- data that passed both tests, means and standard deviation are sitization with IL-7. shown, and paired t tests are used to compare 2 groups. For non-normally distributed data, non-parametric paired tests Interleukin-7 Supports Fungal and Viral-Specific CD4 T-Cell Accumulation (Wilcoxon test) were used to compare 2 groups. For multi- in Peripheral Blood Mononuclear Cells From Infected Individuals ple-group comparisons, 2-way ANOVA with Sidak’s or Tukey’s We then asked whether exposure to IL-7 would also drive the multiple comparison corrections were used, as indicated in accumulation of CD4 T cells specific for fungal Ags derived the figures. Levels of significance are as follows: *, P  ≤  .05; **, from endemic pathogens, such as Ca. Freshly thawed PBMCs P ≤ .005; and ***, P ≤ .0005, unless specified differently in indi - derived from individuals with recurrent Ca infections were vidual figures. immediately analyzed for IFNγ expression in Ca-Ag-recall ELISPOT, in the presence of Ag-pulsed irradiated autologous RESULTS PBMCs as APCs (d0; Figure  2), or seeded in culture with or without IL-7 (d7 and med, respectively; Figure 2), and analyzed Interleukin-7 Enhances Recall Responses of Mycobaterium tuberculo- aer 7  ft days. As in the case of TB-specific T cells, Ca-Ag-specific sis-Specific CD4 T Cells IFNγ spots were also remarkably increased in IL-7-driven cul- To investigate putative effects of IL-7 on pathogen-specific T tures, compared with controls (d0 and med; Figure  2A). In cells, we first analyzed chronically infected TB patients. These multiple individuals (n = 4), Ca-Ag-specific effectors were sig - were chosen based on clinical history and manifestation of acute nificantly increased by ~10-fold over the levels found at d0 aer ft MTB infection (clinic and culture confirmed), positive reaction IL-7 exposure compared with control medium (Figure 2B). to the TST, and ability of PBMCs to respond to stimulation with Because CMV-specific CD4 T cells have been recently shown major histocompatibility complex-II-restricted MTB-specific to significantly expand in elderly individuals chronically infected promiscuous peptides (MTPs) in IFNγ ELISPOT assays [31]. IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 1999 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 ex vivo (d0 equivalent) Cryopreserved PBMCs ELISPOT (18 + 4 hrs) unstimulated (nil) IL-7 vs control medium (med) MTP Ag-restimulation d0 d7 d7 ** d0 med IL-7 C nil Pt.#1 Pt.#2 Pt.#3 d0 IL-7 d0 IL-7 healthy MTB donors infected Figure  1. Interleukin (IL)-7 enhances Mycobacterium tuberculosis (MTB)-specific T-cell responses. (A) Cryopreserved peripheral blood mononuclear cells (PBMCs) from tuberculosis (TB) patients were analyzed for interferon (IFN)γ release by MTB-specific peptide (MTP) enzyme-linked immunospot (ELISPOT) assay at the time of thawing (day [d]0) and after a 7-day culture in the absence (control medium [med], dashed line) or presence of human recombinant IL-7 (solid line), in parallel to unstimulated controls (nil). For each patient, 2 duplicate vials were sequentially thawed, staggered in time (dash dotted lines): the first vial was used to set up the cultures at d0 (left dashdotted arrow); the second vial was thawed 7 days later (right dash dotted arrow) to provide for the following: (1) ex vivo control cells to be analyzed in parallel to cultured cells and (2) autologous feeder cells for the restimulation assays (refer to Materials and Methods for details). (B) Background IFNγ release was measured for any condition in antigen (Ag)-unpulsed control wells (nil), as representatively depicted for patient (Pt.)#1 (top row). The IFNγ-spots after restimulation with MTP promiscuous peptides were detected in 3 TB patients (Pt. #1, #2, and #3) after thawing (d0) and/or a 7-day culture (d7) in IL-7 or med. (C) The effect of the IL-7 treatment (d0 vs d7, IL-7) on MTP-specific IFN γ-spot accumulation in PBMCs derived from healthy donors (open circles, n = 8) and MTB-infected patients (open triangles, n = 5) was analyzed by the ELISPOT assay in multiple individuals. Statistical significance was determined using a 2-way analysis of variance with Sidak’s multiple comparison correction to evaluate (1) the effect of IL-7 treatment in the 2 different groups over time and (2) the diagnostic power of the discrimination of the 2 groups before and after IL-7 treatment, as indicated: *, P ≤ .05; **, P ≤ .005. A B d7 ** d0 med IL-7 10 IL-7 med nil Ca-Ag med IL-7 vs d0 vs d0 Figure 2. Interleukin (IL)-7 supports the expansion and effector function of Candida albicans (Ca)-specific T cells. (A) After thawing, peripheral blood mononuclear cells from a donor with recurrent Ca infection were analyzed for the release of the effector cytokine, interferon (IFN)γ, using a Ca-antigen (Ag)-specific enzyme-linked immunospot assay at the time of thawing (day [d]0) and after a 7-day culture in the absence (d7, control medium [med]) or presence of human recombinant IL-7 (d7, IL-7). Background IFNγ release was measured in Ag-unpulsed control wells (nil, top wells) in the presence of irradiated autologous feeder cells. (B) The range and average fold increase of Ca-Ag- specific IFN γ spots in control medium (med vs d0, white bar) and IL-7 (IL-7 vs d0, dotted bar) cultures over the levels found after thawing (d0) is shown. Statistically significant accumulation of Ca-Ag-specific IFN γ-spots was evaluated in multiple donors (n = 4) using a paired t test. *P ≤ .05; **, P ≤ .005; ***, P ≤ .0005. 2000 • JID 2018:217 (15 June) • Terrazzini et al MTP MTP-specific IFNγ spots/well Ca-specific IFNγ spot fold increase Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 with the endemic CMV [32, 33], we investigated CMV-specific their proliferation [29]. Thus, we asked whether IL-7-driven CD4 T-cell responsiveness to IL-7 in cultures derived from accumulation of human, pathogen-specific T cells also requires + − CMV-seropositive (CMV ) patients. CMV-seronegative (CMV ) cell division. To this aim, we set up CFSE-labeled PBMC cul- individuals were used as controls. Freshly derived PBMCs were tures (Figure  5A and B). We found that although a small frac- cultured in the presence of IL-7 or control medium (med) for tion of CD4 T cells derived from healthy donors (~1%–10%) 7 days, and then CMV-recall responses were tested in ICS, using underwent several rounds of “spontaneous” (ie, in the absence total CMV lysate as a source of viral Ags (Figure 3A, top). Again, of any introduced stimulation) cell division in complete cells cultured in IL-7 tended to show higher CMV-recall responses media (Figure  5A, top panel), dividing cells were dramatically compared with controls (Figure 3A). Of note, these CMV-specific increased upon IL-7 addition (Figure 5A, bottom panel). These + + T cells were mostly polyfunctional, TNFα IFNγ -producing cells cells expressed high levels of Bcl-2 (Supplementary Figure 3), in (Figure 3A) and remained undetectable in cultures derived from agreement with the prosurvival role of the cytokine [20]. More CMV individuals (Supplementary Figure 2A). Because we found importantly, IL-7 favored the expansion of 2 cell populations, that T cells express varying amounts of CD127 and upregulate it distinguishable for their proliferation potential (ie, either fast in control medium (Supplementary Figure 2B), as expected [34], or slow; F and S, respectively, in the figures) and with distinct we first rested freshly thawed PBMCs for 5 days, and then we sub - mechanistic requirements. Indeed, although both populations jected them to IL-7-driven cultures (Figure 3B, top). Under these were best detected in high-density cell cultures (Supplementary conditions, we found that IL-7 more potently and reproducibly Figure  4), they proved differentially sensitive to CSA. Fast- enabled the accumulation of CMV-specific T-cell responses from but not slow-dividing cells were sensitive to CSA inhibition + + CMV individuals (n = 10; Figure 3C). The IFN γ cells expressed (Figure  5B). In addition, fast-dividing cells were sensitive to low levels of CD127, indicative of IL-7-driven receptor down- anti-LFA-1 mAb blockade (Supplementary Figure 5), suggesting modulation [34], upon cytokine-driven activation (Figure  3D) that cell-to-cell contacts are also required for their proliferation. while maintaining high levels of LFA-1 (data not shown) and Of note, IL-7 elicited similar effects in autologous sera and FCS polyfunctional cytokine expression (Figure 3B). Thus, in addition (Supplementary Figure 4). We reckoned that, although slow-di- to bacterial responses, fungal and viral-specific responses are also viding cells represent cells undergoing homeostatic expan- enhanced by IL-7 signals. sion, known to occur via CSA-insensitive mechanisms [19] (Figure  5B, right panel in top row), fast-dividing cells may Interleukin-7 Promotes Responsiveness of Staphylococcus represent a distinct population of Ag-experienced memory aureus- Specific T Cells cells proliferating via a CSA-sensitive mechanism (Figure  5B, Bacterial superantigens, such as SEB, can lead to T-cell anergy middle-right panel in top row). Flow cytometry analysis indi- − + [35] or suppression [36]. We asked whether culturing the cells cated that although both T (CD45RA , CD62L ) and T CM EM − − in IL-7 might also help SEB-specific T-cell responses ( Figure 4). (CD45RA , CD62L ) cells accumulated in response to IL-7 The PBMCs from healthy donors were rested 5 days in control (Figure  5B, middle and bottom panel), only fast-dividing T CM − + medium and then left untreated or cultured in IL-7 for 7 days. (CD45RA , CD62L ) cells appeared sensitive to CSA (Figure 5B, The SEB-recall responses were then tested in ICS ( Figure  4A, bottom panel). It is interesting to note that the IL-7-driven top). We found that although SEB-specific T cells could be accumulation of CMV (Figure 5C, top), SEB (n = 15; Figure 5C, + + detected in control PBMC cultures (med), IFNγ or TNFα and bottom and right), and MTB-specific ( Supplementary Figure 6) + + TNFα IFNγ cells were all enriched for in IL-7-driven cultures polyfunctional memory T cells was also completely dependent from many individuals, significantly ( Figure 4A and B, n = 17). upon CSA-sensitive signaling. Taken together, our data indicate As seen for CMV-specific T cells ( Figure  3D), cytokine SEB- that IL-7-driven cultures might recapitulate the events account- specific cells expressed lower levels of CD127 ( Figure 4C) while ing for the maintenance of Ag-experienced memory T-cell sub- maintaining high levels of LFA-1 (data not shown). sets in vivo [15] and improve their identification and selection We also tested whether IL-7 is capable of supporting SEB- in vitro. responsiveness even after Ag-driven expansion, in vitro DISCUSSION (Figure  4D). We found that IL-7 significantly enhanced responses after SEB-restimulation (Figure 4D). Thus, cultur- In this study, we demonstrate that IL-7 supports the in vitro ing T cells in IL-7 either before or after Ag recall allows for a expansion of human pathogen-specific T cells, favoring and, higher frequency of Ag-specific cells to be identified. in some instances, enabling their enumeration and charac- terization. Interleukin-7-sensitive pathogen-specific effectors Interleukin-7 Drives Cyclosporine A-Sensitive Central Memory T-Cell included cells specific for endemically diffused pathogens (eg, Division S aureus, C albicans, CMV, and MTB, in parts of the world), In a previous mouse study, we found that IL-7 favored the in infecting humans recurrently during the lifetime, persisting vitro expansion of tumor-Ag-experienced T cells, by promoting (MTB) and/or reactivating in the body (CMV). u Th s, our data IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 2001 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 A B d0 d7 d0 d5 d12 PBMCs (CMV+ donor) CMV (ICS) PBMCs wash CMV (ICS) IL-7 unstimulated (nil) IL-7 unstimulated (nil) med control medium (med) med CMV Ag-recall CMV Ag-recall 5 5 0.0133 10 10 0.137 4 0.177 8.85e-3 nil 3 med IL-7 5 5 0.027 0.21 0.14 1.82 10 10 2 2 10 10 4 4 10 10 99.8 0.0111 99.8 0.0155 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 3 3 10 10 5 0.0326 0 0 0.21 4 0.0802 4 0.128 3 3 10 med 99.7 0.11 96.6 1.40 –10 –10 3 3 4 5 3 3 4 5 +CMV 3 –10 01 10 10 10 –10 0 10 10 0 TNFα 2 2 10 10 0 80.4 0.15 5 0 5 4.91 0.088 99.7 0.0576 99.8 7.52e-3 10 2 3 4 5 2 3 4 5 (2815) (2033) (285) (316) 0 10 10 10 10 0 10 10 10 10 4 4 10 10 0.259 3 3 0.191 4 10 10 4 0.138 0.489 IL-7 0 0 +CMV 3 3 19.4 0.064 93.2 1.76 –10 –10 2 3 3 4 5 3 3 4 5 10 –10 01 10 10 10 –10 0 10 10 0 99.3 0.268 99.3 0.0236 IFNγ 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 TNFα C D 1 * –1 –2 med IL-7 med IL-7 Figure 3. Interleukin (IL)-7 supports the accumulation of antiviral cytomegalovirus (CMV)-specific T cells. (A) Freshly derived peripheral blood mononuclear cells (PBMCs) from CMV donors were analyzed for inflammatory cytokine release after CMV-lysate intracellular cytokine staining (ICS) assay after a 7-day culture in the absence (d7, con - trol medium [med], dashed black line) or presence of human recombinant IL-7 (d7, IL-7, black line). Left and right dot plots show the levels of IL-2 and tumor necrosis factor (TNF)α or interferon (IFN)γ and TNFα in gated CD4 T cells, respectively. Background levels of cytokine secretion were typically measured in unstimulated controls (nil). (B) Freshly isolated PBMCs from CMV donors were rested for 5 days in plain medium (dashed black line) before a 7-day culture in the absence (d12, med, dashed black line) or presence of human recombinant IL-7 (d12, IL-7, black line). At day 12 (d12), CD4 T cells were analyzed for IFNγ and TNFα release (top row) alongside expression of CD127 + + (bottom row), after CMV-lysate ICS assay. Cytomegalovirus-specific IFN γ CD4 T cells show high CD127 expression in the resting cultures, whereas they downregulated CD127 expression upon exposure to IL-7. Background levels of cytokine secretion were typically measured in ICS unstimulated (nil) controls. A representative of >10 inde- + + + + pendent experiments is shown. (C) After subtraction of individual background levels of IFNγ and TNFα and IFNγ TNFα CD4 T cells detected in unstimulated controls (nil), + + + + the percentage of CMV-specific cytokine (IFNγ and TNFα ) CD4 T cells was evaluated in 10 independent CMV donors in IL-7 (IL-7, open triangles) compared with control medium ([med], open circles) cultures. The graph shows a statistically significant (paired Wilcoxon test, * P = .03) increase of the frequency (Log ) of CMV-specific CD4 T + + cells after IL-7 culture. (D) CD127 expression (Log mean fluorescence intensity [MFI]) is significantly downregulated in CMV-specific IFN γ CD4 T cells exposed to IL-7 (IL-7) compared with control medium (med) at d12 in 6 biologically independent replicates (paired Wilcoxon test, *P = .03). 2002 • JID 2018:217 (15 June) • Terrazzini et al CMV + + CMV-specific cytokine CD4 IL-2 (Log10 %) IFNγ + + CD127 on IFNγ CD4 (Log10 MFI) CD127 IFNγ Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 + + A B Single IFNγ Single TNFα 1 *** d0 d5 d12 10 PBMCs wash SEB (ICS) IL-7 Unstimulated (nil) med med SEB Ag-recall –1 –1 med IL-7 5 5 0.024 0.043 0.024 5.32E–3 10 10 –2 –2 4 4 10 10 10 10 med IL-7 med IL-7 nil 3 3 10 10 + + + IFNγ TNFα Total cytokine 0 0 2 2 3 3 10 10 –10 99.8 0.11 –10 99.9 0.11 *** *** 3 3 4 5 3 3 4 5 –10 0 10 10 10 –10 0 10 10 10 1 1 10 10 5 5 1.34 8.90 0.41 2.41 10 10 4 4 10 10 0 0 10 10 SEB 3 3 10 10 0 0 –1 –1 10 10 3 3 92.1 5.11 77.5 12.3 –10 –10 3 3 4 5 3 3 4 5 –10 0 10 10 10 –10 0 10 10 10 –2 –2 10 10 TNFα med IL-7 med IL-7 C D SEB d0 d5 d12 med IL-7 PBMCs wash SEB (ICS) 5 54.6 1.25 5 1.59 0.079 10 10 IL-7 SEB (2220) (1585) (338) (211) 4 4 10 10 med SEB Ag-recall 3 3 10 10 SEB d12 0 0 3 3 42.6 1.54 88.2 10.1 –10 –10 4.81 6.59 3 3 4 5 3 3 4 5 5 11.1 –10 0 10 10 10 –10 0 10 10 10 40 IFNγ 4 IL-7 *** 10 –10 3 3 4 5 –10 0 10 10 10 0.80 0.68 2.68 med –10 3 3 4 5 –10 0 10 10 10 med IL-7 med IL-7 TNFα Figure  4. Interleukin (IL)-7 supports superantigen-specific responses. (A) Freshly isolated peripheral blood mononuclear cells (PBMCs) were rested for 5  days in plain medium (dashed black line) then incubated for 1 week in IL-7 or control medium ([med] respectively IL-7, black line and med, dashed black line). At day (d)12, cells were stimulated with Staphylococcus aureus enterotoxin B (SEB) followed by an intracellular cytokine staining (ICS) assay to test antigen (Ag)-specific tumor necrosis factor (TNF) α and interferon (IFN)γ release, compared with unstimulated controls (nil). Dot plots show that the frequency of SEB-specific CD4 T cells producing TNFα and/or IFNγ increased + + + + upon exposure to IL-7 in 1 representative donor. (B) After subtraction of individual background levels of IFNγ and TNFα and IFNγ TNFα CD4 T cells detected in unstimulated + + + + + controls (nil), the percentage of SEB-specific cytokine (IFNγ , TNFα , IFNγ TNFα and total) CD4 T cells was evaluated in 17 independent donors in IL-7 (IL-7, open triangles) compared with control medium (med, open circles) cultures. The graphs show a statistically significant increase of the frequency (Log ) of SEB-specific cytokine CD4 T cells after IL-7 culture. All tests are paired Wilcoxon tests with the exception of the single TNFα analysis (paired t test): *, P ≤ .05; **, P ≤ .005; ***, P ≤ .0005. (C) At d12, cells cultured as in (A) were tested for CD127 expression in parallel to cytokine release in SEB-specific ICS assay. CD127 expression (Log mean fluorescence intensity [MFI]) is + + significantly decreased in SEB-specific IFN γ CD4 T cells exposed to IL-7 (IL-7, downward triangles) compared with control medium (med, upward triangles) in 12 independent biological replicates (paired Wilcoxon test; ***, P = .0005). (D) Freshly isolated PBMCs (d0) were stimulated with the SEB superantigen (dotted black line) for 5 days (d0–5). At d5, cells were washed and incubated for 1 week in IL-7 (IL-7, black line; d5–12) or control medium (med, dashed black line; d5–12). At d12, cells were stimulated with SEB (overnight) to test Ag-specific TNF α and IFNγ release, by ICS. Dot plots show that the frequency of SEB-specific CD4 T cells producing TNFα and/or IFNγ increased upon exposure to IL-7 (IL-7 d5–12, top row). The graph on the right shows data from the same cultures derived from 6 independent biological replicates. Statistically significant + + accumulation of SEB-specific, cytokine CD4 T cells was evaluated using a Wilcoxon matched-pairs signed-ranked test (P = .03). IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 2003 CD127 IFNγ + + CD127 on IFNγ CD4 (Log10 MFI) IFNγ + + + + % cytokine of CD4 % cytokine of CD4 (Log10) (Log10) + + %cytokine of CD4 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 A B med CSA IL-7 IL-7+CSA 4 4 4 4 4 10 10 10 10 10 0.01 0.25 0.32 0.46 44.6 0.003 0.36 56.9 2.67 10.2 31.1 0.48 9.18 34.9 3 3 3 3 3 10 10 10 10 10 FS 2 2 2 2 2 10 10 10 10 10 med FS 1 1 1 1 1 10 10 10 10 10 0.92 0.41 98 0 0 0 0 0 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CFSE 0.27 0.16 4 4 4 10 10 10 10 50 0.862 35.6 1.61 41.1 1.75 3 3 3 10 10 10 IL-7 2 2 2 10 10 10 S 1 1 1 18 26 55 10 10 10 0 1 2 3 4 47.7 1.44 60.5 2.31 55.5 1.66 0 0 0 10 10 10 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CFSE 4 4 4 10 10 10 55 1.25 39 1.93 8.33 0 3 3 3 10 10 10 2 2 2 10 10 10 1 1 1 10 10 10 0 43.3 0.417 0 56.2 2.89 0 89.9 1.72 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD45RA + + Single IFNγ Single TNFα 1 *** IL-7 IL-7+CSA 5 5 0.14 1.82 0.16 0.64 0 10 10 4 4 10 10 –1 10 –1 CMV 3 3 10 10 0 0 3 3 96.6 1.40 99.1 0.062 –10 –10 –2 –2 10 10 3 3 4 5 3 3 4 5 –10 0 10 10 10 –10 0 10 10 10 IL-7 IL-7+CSA IL-7 IL-7+CSA 5 1.72 24.2 5 1.99 0.42 10 10 + + + IFNγ TNFα Total cytokine 4 4 10 10 2 2 10 10 *** *** 3 3 SEB 10 10 1 1 10 10 0 0 3 3 –10 69.4 4.72 –10 96.8 0.98 3 3 4 5 3 3 4 5 0 0 –10 0 10 10 10 –10 0 10 10 10 10 10 TNFα –1 –1 10 10 –2 –2 10 10 IL-7 IL-7+CSA IL-7 IL-7+CSA Figure 5. Sensitization by interleukin (IL)-7 promotes pathogen-specific CD4 T cell proliferation in a cyclosporine A (CSA)-sensitive manner. 5-(and-6)-carboxyfluorescein diacetate succinimilyl ester (CFSE)-labeled peripheral blood mononuclear cells (PBMCs) (A–C) from healthy donors (A and B) were cultured for 7 days in the absence (med) or presence of human recombinant IL-7 (IL-7). (A) Proliferation of viable CD4 T cells in high-density cultures (5 × 10 cells/mL) was determined by flow cytometry after staining with anti-CD4 monoclonal antibody (mAb) and TO-PRO-3 (an intercalant agent of deoxyribonucleic acid entering necrotic and apoptotic cells). Contour plots depict the rela- − + 6 tive CFSE content within the same number of viable, TO-PRO-3 CD4 T cells. (B) Cells cultured (4 × 10 cells/mL) in the presence of IL-7 for 7 days in the absence or in the presence of CSA. At d7, cells were stained with anti-CD4, anti-CD45RA, and anti-CD62L mAb, and the relative CFSE content was analyzed by flow cytometry in total CD4 T + hi cells (top row). Fast- (F), slow- (S), and nondividing cells were determined based on CSA inhibition, as indicated. The percentage of naive (CD45RA CD62L ), central memory − hi − lo (CD45RA CD62L ), and effector memory (CD45RA CD62L ) cells were then investigated in slow- compared with fast-proliferating CD4 T cells. (C) Freshly isolated PBMCs were rested for 5 days then incubated for 1 week in IL-7 or IL-7 plus CSA (IL-7+CSA). At d12, cells were stimulated with Staphylococcus aureus enterotoxin B (SEB) during an intracellular cytokine staining (ICS) assay to test antigen-specific tumor necrosis factor (TNF) α and interferon (IFN)-γ release, compared with unstimulated controls (refer to Figures 3 and 4). Left dot plots show that the IL-7-driven accumulation of (top) cytomegalovirus (CMV)- and (bottom) SEB-specific CD4 T cells producing TNFα and/or IFNγ + + + + is reversed in the presence of CSA, by d12. After subtraction of individual background levels of IFNγ , TNFα , and IFNγ TNFα CD4 T cells detected in unstimulated controls + + + + + (nil), the percentage of SEB-specific cytokine (IFNγ , TNFα , IFNγ TNFα , and total) CD4 T cells was evaluated in 15 independent biological replicates in IL-7 (open circles) compared with IL-7+CSA cultures (open triangles). The right graphs show a statistically significant decrease in the frequency (Log ) of SEB-specific cytokine (except for IFNγ ) CD4 T cells in the presence of CSA at d12. All tests are paired Wilcoxon tests; *, P ≤ .05; **, P ≤ .005; ***, P ≤ .0005. 2004 • JID 2018:217 (15 June) • Terrazzini et al TO-PRO-3 IFNγ CD62L CD4 + + + + %cytokine of CD4 %cytokine of CD4 (Log10) (Log10) Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 will help the study and isolation of such pathogen-specific T developing systemic lupus erythematosus aer 3 dos ft es of IL-7 cells and others relevant to various clinical settings, includ- [45]. We speculate that the risk of developing autoimmunity ing harmful infections, especially in the case of immune-sup- aer IL ft -7 treatment may vary dependent on individual clin - pressed (HIV, transplantation, aged etc) or chronically infected ical history, genetic predisposition, and the administration individuals, in which pathogen-specific T cells might be low in regimen. Further studies are needed to define the long-term frequency and/or hyporesponsive. consequences of IL-7 administration. Previous studies showed that IL-7 can support immune- It is interesting to note that among memory cells, poly- + + cell reconstitution in lymphopenic conditions [22–25], restore functional T cells (double positive for IFNγ and IL-2 or sepsis-induced lymphocyte dysfunctions ([37, 38]), enhance TNFα ) were mostly enriched for by IL-7. Such cells were effector function of autoreactive T cells [ 8–13], and expand detected in subjects with chronic viral (including CMV [32]) tumor-reactive T cells [29, 30]. We then asked whether patho- infections and previously referred to as intermediate poly- gen-reactive T cells also benefited from IL-7 exposure. Our functional memory cells [46]. It is possible that IL-7 favors results show that IL-7 promotes the selective expansion of a differentiation of these cells in vitro (and possibly also in fraction of memory CD4 T cells containing pathogen-specific vivo), starting from IFNγ-producing cells. With respect to cells, best observed in high cell-density cultures. Although IL-7 maintaining polyfunctional T cells, IL-7 appears superior significantly enhanced overall cell recovery (by ~1.5-fold), cyto - to the cognate Ag by favoring (central) memory cell sur- kine-producing CD4 T cells were not enriched at a polyclonal vival [15–17], rather than terminal differentiation, acti- level (as detected by 12-O-tetradecanoyl-phorbol-13-acetate vation-induced cell death, and/or exhaustion [29]. Thus, and ionomycin stimulation; Supplementary Figure  7) unlike IL-7 may be useful for the expansion of human polyclonal that seen for pathogen-specific cells. These cells showed a fast and polyfunctional pathogen-specific CD4 (and to a lower + − + rate of proliferation, sensitive to CSA and LFA-1 inhibition, extent, CD3 CD4 or CD8 ; Supplementary Figure  8A–E) both in autologous sera and FCS. u Th s, neither homeostatic cell T cells that are hard to identify, even in the case of a rel- division [19, 39–42], known to be CSA insensitive [19, 42], nor atively well studied pathogen (such as CMV [47, 48]). food-related, bovine Ags seem to account for the accumulation Accordingly, exposing cells to IL-7 enabled better enumer- of fast-dividing cells. However, our results suggest the existence ation of in vivo-primed CMV-/SEB-specific cells, although of a cell-associated ligand capable of synergizing with IL-7 sig- these trends were less apparent in CD8 T cells, which did nals to promote the proliferation and responsiveness of patho- not undergo fast proliferation to the extent of CD4 T cells gen-specific T cells. We speculate that self-Ag/TCR-initiated (Supplementary Figure  8F). This was also the case when T signals may play a role in the IL-7 cultures. cells were Ag-restimulated in vitro before the IL-7 culture, Although further research is needed to identify such sig- opening the possibility that IL-7 (with or without Ag) might nals in IL-7 cultures, our data support a role for IL-7 in T cell- be superior to Ag alone in expanding T cells derived from in driven immunopathology in chronic and persistent infections, vivo-primed individuals. Our data also support the hypoth- coinfections, or autoimmunity. Accordingly, IL-7 is expressed esis that IL-7 sustains the preferential accumulation of poly- in inflamed tissues of patients with (rheumatic) autoimmune functional T-cell subsets within the repertoire of certain diseases, where it can be produced by several cell types [43] individuals, including perhaps the inflated responses of CD4 (including macrophages, dendritic cells, and fibroblasts) and T cells detected in elderly CMV individuals [32, 33]. Future favor pathogenic Th1- and Th17-associated cytokine secre - studies are needed to address this possibility. tion. Furthermore, dysregulated IL-7 expression or activation CONCLUSIONS of CD127 were found in patients with autoimmune conditions [8–13], suggesting that IL-7 supports the function of patho- We previously suggested that among other CD132-cytokines, genic effector cells in autoimmunity. In agreement  with this, IL-7 played nonredundant roles and outperformed IL-2 in blocking the IL-7R in experimental animal models amelio- driving Ag-experienced T-cell accumulation and mediating rated autoimmune disease manifestations [44]. Thus, together the expansion of less differentiated cells useful for gene therapy with available data, our results suggest that IL-7 might awaken [26, 30]. We now provide evidence supporting the use of IL-7 to auto-reactive T cells, or pathogen-specific effector T cells with reveal and expand in vivo-primed pathogen-specific lympho - cross-reactivity to self-Ag, hence contributing to autoimmu- cytes of clinical relevance, either as biomarkers of viral infection nity. However, this might not equally apply to patients with and disease activity [49] or as therapeutic tools. This may be rel - immune dysfunctions due to sepsis or chronic HIV/hepatitis evant for the treatment of chronic infectious diseases and can- C virus infection. Indeed, IL-7 administration mainly in HIV- cer, because adoptive immunotherapy with less differentiated T infected or immunosuppressed patients was generally well tol- cells is preferable over the transfer of terminally differentiated erated [22, 23, 25, 27, 28, 45], with a single report of a patient ee ff ctors [ 50]. IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 2005 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 Supplementary Data 6. Noguchi M, Nakamura Y, Russell SM, et  al. Interleukin-2 receptor gamma chain: a functional component of the Supplementary materials are available at The Journal of interleukin-7 receptor. Science 1993; 262:1877–80. Infectious Diseases online. Consisting of data provided by 7. Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective the authors to benefit the reader, the posted materials are not IL7R expression in T(-)B(+)NK(+) severe combined immu- copyedited and are the sole responsibility of the authors, so nodeficiency. Nat Genet 1998; 20:394–7. questions or comments should be addressed to the corre- 8. Nuro-Gyina PK, Rieser EL, Granitto MC, et al. Regulation of sponding author. effector function of CNS autoreactive CD4 T cells through inhibitory receptors and IL-7Rα. J Neuroinflammation Notes 2016; 13:302. Acknowledgments. We thank George Morrow and Dr. 9. Arbelaez CA, Glatigny S, Duhen R, Eberl G, Oukka M, Helen Stewart (University of Sussex) for technical support and Bettelli E. IL-7/IL-7 receptor signaling differentially ae ff cts reading the manuscript, respectively. We also thank Antonio effector CD4+ T cell subsets involved in experimental auto - Lanzavecchia and members of the San Raffaele Institute for use - immune encephalomyelitis. J Immunol 2015; 195:1974–83. ful discussions and suggestions. 10. Hartgring SA, Bijlsma JW, Lafeber FP, van Roon JA. Disclaimer. e Th funders had no role in study design, data Interleukin-7 induced immunopathology in arthritis. Ann collection and analysis, decision to publish or preparation of the Rheum Dis 2006; 65(Suppl 3):iii69–74. manuscript. 11. Penaranda C, Kuswanto W, Hofmann J, et al. IL-7 receptor Financial support. S.  C.  is supported by a Brighton and blockade reverses autoimmune diabetes by promoting inhi- Sussex Medical School Internal Fellowship (University of bition of effector/memory T cells. Proc Natl Acad Sci U S A Sussex). This work was funded by grants from the follow - 2012; 109:12668–73. ing: “Associazione Italiana Ricerca sul Cancro (AIRC)”; the 12. Watanabe M, Yamazaki M, Okamoto R, et  al. 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IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 2007 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses

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Oxford University Press
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Copyright © 2022 Infectious Diseases Society of America
ISSN
0022-1899
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1537-6613
DOI
10.1093/infdis/jiy096
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Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses Nadia Terrazzini, Paola Mantegani, Florian Kern, Claudio Fortis, Anna Mondino, Stefano Caserta Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 The Journal of Infectious Diseases MAJOR ARTICLE Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses 1 2 3 2 4,a 3,4,5,a Nadia Terrazzini, Paola Mantegani, Florian Kern, Claudio Fortis, Anna Mondino, and Stefano Caserta 1 2 School of Pharmacy and Biomolecular Sciences, University of Brighton, United Kingdom; Laboratory of Clinical Immunology, Clinic of Infectious Diseases, San Raffaele 3 4 Scientific Institute, Milan, Italy; Brighton and Sussex Medical School, The University of Sussex, Falmer, East Sussex, United Kingdom; Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy; School of Life Sciences, The University of Hull, United Kingdom Background. Interleukin (IL)-7 promotes the generation, expansion, and survival of memory T cells. Previous mouse and human studies showed that IL-7 can support immune cell reconstitution in lymphopenic conditions, expand tumor-reactive T cells for adoptive immunotherapy, and enhance effector cytokine expression by autoreactive T cells. Whether pathogen-reactive T cells also benefit from IL-7 exposure remains unknown. June Methods. In this study, we investigated this issue in cultures of peripheral blood mononuclear cells (PBMCs) derived from patients infected with various endemic pathogens. Aer s ft hort-term exposure to IL-7, we measured PBMC responses to antigens derived from pathogens, such as Mycobacterium tuberculosis, Candida albicans, and cytomegalovirus, and to the superantigen Staphylococcus aureus enterotoxin B. Results. We found that IL-7 favored the expansion and, in some instances, the uncovering of pathogen-reactive CD4 T cells, by promoting pathogen-specific interferon- γ, IL-2, and tumor necrosis factor recall responses. Conclusions. Our findings indicate that IL-7 unveils and supports reactivation of pathogen-specific T cells with possible diagnos - tic, prognostic, and therapeutic significance of clinical value, especially in conditions of pathogen persistence and chronic infection. Keywords. candida infection; CMV infection; pathogen-specific responses; SEB responses; TB infection. Interleukin (IL)-7 [1] is a pleiotropic cytokine [2] regulating (eg, Bcl-2) [14, 20], IL-7 promotes the long-term survival of lymphopoiesis and T-cell homeostasis [3, 4]. It binds to a het- naive and memory-phenotype cells. Recombinant IL-7 elicits a erodimeric receptor formed by an α-chain (CD127 [3]), which marked increase of central memory (T ) and effector memory CM is private to the IL-7 receptor (IL-7R) and the common-γ-chain T cells (T ) when administered to aged nonhuman primates EM cytokine-receptor (CD132 [5, 6]). In humans, mutations neg- [21] and lymphopenic patients and macaques infected with atively ae ff cting the levels of CD127 have been correlated with human immunodeficiency virus (HIV) or simian immunode - severe immunodeficiency [ 7]. Conversely, increased serum lev- ficiency virus, respectively [ 22–24]. Similar effects are reported els of IL-7 and/or dysregulated activation of CD127 are reported in patients undergoing stem cell transplantation [25, 26], OA-CC-BY in patients with autoimmune conditions, including multiple where IL-7 levels correlate with the generation of stem-cell sclerosis [8, 9], rheumatoid arthritis [10], type-I diabetes [11], memory T cells [26]. In cancer patients, IL-7 preferentially inflammatory bowel disease [ 12], and psoriasis [13]. increases naive but not regulatory T-cell numbers [27], main- Upon antigen (Ag) encounter, IL-7 sustains the generation taining T-cell receptor (TCR) repertoire diversity [28]. Hence, of memory T lymphocytes in vitro [14] and in vivo [14–16] the immunotherapeutic use of IL-7 is increasingly proposed and favors the transition of effector to central memory cells to favor immune-cell reconstitution and function aer l ft ym - [17, 18] while driving their proliferation [19]. Via the JAK3/ pho-depleting chemotherapy or in the elderly. In the setting of STAT5 pathway and the upregulation of antiapoptotic factors adoptive T-cell therapy (ACT), IL-7 has been used in primary cultures to engineer and expand tumor-reactive T cells [29, 30]. We previously found that IL-7 selectively expands tumor-reac- Received 3 November 2017; editorial decision 14 February 2018; accepted 19 February 2018; tive CD4 T cells capable of promoting tumor protection in ACT published online February 28, 2018. Presented in part: 4th European Congress of Immunology, 6–9 September 2015, Vienna, [29]. Whether similar results could be extendible to patho- Austria. gen-specific T cells remains unknown. Therefore, we sought to A. M. and S. C. contributed equally to this work. Correspondence: S. Caserta, PhD, School of Life Sciences, Hardy Building, The University of investigate the expansion of pathogen-reactive CD4 T cells in Hull, Hull, United Kingdom, HU6 7RX UK ([email protected]). individuals ae ff cted by recurrent or persistent/chronic bacterial The Journal of Infectious Diseases 2018;217:1997–2007 (Mycobacterium tuberculosis [MTB] and Staphylococcus aur- © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons eus), fungal (Candida albicans [Ca]), or viral (cytomegalovirus Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted [CMV]) infections. We report that, in all cases, IL-7 enriched reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/infdis/jiy096 pathogen-specific CD4 T cells, enabling their detection and IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 1997 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 sensitizing them to Ag-specific recall responses. Furthermore, numbers of viable (0.1% Trypan blue-negative) cells were resus- IL-7 rescued chronically activated pathogen-specific effectors pended in complete media (Roswell Park Memorial Institute enhancing their Ag-recall responses. We believe that these data medium containing penicillin, streptomycin, glutamine, and open new avenues for diagnostic, prognostic, and therapeutic 10% FCS [all from Thermo Fisher Scientific] or autologous applications. serum) with or without human recombinant IL-7 (50  ng/mL, unless specified otherwise in individual figures; R&D Systems/ MATERIALS AND METHODS Biotechne, Minneapolis, MN) for 7 days. Where indicated, cells were stained with the fluorescent dye 5-(and-6)-carboxyu fl - Classification of Tuberculosis Patients orescein diacetate succinimidyl ester ([CFSE] Thermo Fisher Human immunodeficiency virus-seronegative patients with Scientific; 1 µM), in accordance with the manufacturer’s instruc - active tuberculosis ([TB] clinic and culture confirmed) were tions, using autologous serum or FCS to quench the labeling. recruited at the Clinic of Infectious Diseases, San Raffaele Where indicated, cells were first cultured in complete medium Hospital (Milan, Italy). They underwent tuberculin skin test - with or without bacterial SEB (1  μg/mL, Sigma/Merck). Aer ft ing (TST) administered by the Mantoux method with 0.1  mL 5 days, cells were harvested, washed and counted. Equal num- (5 tuberculin units) of Biocinetest-PPD tuberculin (Chiron ber of viable cells were finally seeded in culture with or with - Italia, Milan, Italy). The size of induration was evaluated aer ft out IL-7, for additional 7 days (day 12). Cyclosporine A ([CSA] 48–72 hours (an induration ≥10  mm was classified as posi - 0.5  µg/mL; Calbiochem/Merck) or anti-lymphocyte func- tive). Peripheral blood was drawn before starting any therapy tion-associated antigen (LFA)-1 blocking antibody (5  µg/mL; and following written informed consent. Healthy controls were a gift from Professor Ruggero Pardi, University Vita-Salute San selected among HIV-seronegative individuals with no history Raffaele, Milan) were added to the cultures. of TB exposure, no infection, and with negative reaction to the TST. Healthy controls were tested for the presence of Ca-Ag Mycobaterium tuberculosis Peptides and Candida albicans responses. Antigen-Specific Enzyme-Linked Immunospot Assay e enzy Th me-linked immunospot (ELISPOT) assay for interferon Ethics Statement (IFN)γ detection was performed as described previously [31]. Written informed consent or consultee approval to enroll was In brief, equal numbers of viable cells (5 × 10 cells/well) were secured for all study participants (patients and healthy donors). seeded in duplicate in 96-well plates (MAIPS4510; Millipore/ This study was approved by the Ethical Committee of the San Merck), precoated with anti-IFNγ capture monoclonal antibody Raffaele Scientific Institute, the UK National Research Ethics ([mAb] B-B1; Diaclone, Besançon, France), together with autol- Service (reference: 13/LO/1270), and the Brighton  and Sussex ogous irradiated PBMCs (5 × 10 cells/well), and Mycobacterium Medical School  (BSMS) Research Governance and Ethics tuberculosis peptides ([MTPs] a pool of 6 synthetic peptides, Committee (reference: 13/182/LLE) and carried out in accord- 2  µg/mL per peptide [Primm, Milan, Italy])  or Ca-Ag (25  µg/ ance with the approved guidelines. All data were anonymized. mL [Bio-Rad, Hercules, CA]) for 18 hours at 37°C, in 5% CO Cytomegalovirus Serology atmosphere. Biotinylated anti-IFNγ detection mAb (B-G1; Healthy donors were screened for the presence of CMV-specific Diaclone) was added (4 hours), followed by the streptavidin-al- antibody in serum. Cytomegalovirus immunoglobulin (Ig)G kaline phosphatase conjugate (1 hour) (Amersham Pharmacia serology (Architect CMV IgG; Abbot, Maidenhead, UK) was Biotech Europe GmbH, Freiburg, Germany). Aer ft a wash - performed at the Brighton and Sussex University Hospital Trust ing step, the nitroblue tetrazolium-5-bromo-4-chloro-3-in- virology laboratory. Cytomegalovirus-seropositive and -seroneg- dolylphosphate (Sigma) chromogenic substrate was added. + − ative individuals are referred to as CMV and CMV , respectively. Individual spot-forming cells were counted using an auto- mated image analysis system ELISPOT reader (AID-GmbH, Human Samples and T-Cell Cultures Strassberg, Germany). Mycobacterium tuberculosis peptides (20 Patients and healthy donors used for MTB- and Candida- amino acids) were derived from the ESAT-6 and CFP-10 secre- specific responses were part of a previously published tory proteins of MTB, purified (>70%), and previously validated cohort [31]. Donors (male/female, 10:9) used for CMV and [31]. Peripheral blood mononuclear cells in medium alone or Staphylococcus aureus enterotoxin B (SEB)-specific responses stimulated with phytohemagglutinin ([PHA-P] Sigma; 5 µg/mL) were 68  ±  17  years old. Peripheral blood mononuclear cells were used to assess assay background and functionality. (PBMCs) were isolated by blood centrifugation over Ficoll- Hypaque (Sigma/Merck, Darmstadt, Germany) density gradi- Flow Cytometry Analysis and Intracellular Cytokine Staining ent. Cultures were derived either from freshly isolated (CMV/ Cells were harvested, washed with staining buffer (phos - SEB responses) or cryopreserved (90% fetal calf serum [FCS] and phate-buffered saline supplemented with 0.5% FCS and 10% dimethyl sulfoxide; MTB/Ca-Ag responses) PBMCs. Equal 0.02% NaN ), and incubated with mAbs directed against 1998 • JID 2018:217 (15 June) • Terrazzini et al Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 surface Ags for 15 minutes at room temperature. e Th follow - Patients with detectable (patient [Pt]#1 and Pt#2; Figure  1) or ing fluorescence-conjugated mAbs were used: anti-CD3-v500, undetectable (Pt#3; Figure 1) MTB-specific IFN γ-producing T anti-CD8-allophyocyanine-H7, anti-CD27-phycoerythrine cells were analyzed. Cryopreserved cells were tested in MTP- (PE), IL-2-fluorescein isothiocyanate, TNF α Alexa700 (all recall assays immediately aer t ft hawing (day [d]0; Figure  1A from BD Biosciences, Franklin Lakes, NJ); anti-CD4-peridi- and B) or at the end of a culture (d7) in IL-7 compared with nin chlorophyll, anti-IFNγ PE-Cy7 (Cyanine-7), anti-CD154 complete media (med). Not all patients with detectable MTP- Pacific-Blue (BioLegend, Cambridge, UK); anti-CD45RA ECD specific T-cell responses on fresh PBMCs [ 31] had a detect- (Beckman Coulter, UK); and Yellow live-dead stain (Thermo able MTP response aer t ft hawing, as seen in the case of Pt#2, Fisher Scientific). er Th eaer ft , cells were washed in staining compared with Pt#1. Nonetheless, we found that IL-7 selec- buffer before acquisition. tively enriched cultures for MTP-specific IFN γ T cells by 4- to Intracellular cytokine staining was used to measure MTP- 10-fold in all cases (Figure 1B). Although MTP-specific T cells specific cytokine release at single-cell level. Equal numbers of could also expand in control cultures (as seen for Pt#1, med), 6 + + CFSE-labeled cytokine-cultured cells (0.6  ×  10 ) were stimu- this was best explained by the increase of total CD3 CD4 T lated (6 hours) with unpulsed (nil) or MTP-pulsed (4  µg/mL) cells in some patients (data not shown). It is important to note autologous irradiated (5000 rad) PBMCs (3 × 10 cells), in the that the frequency of MTP-specific cells increased in IL-7 cul - presence of human anti-CD28-stimulating mAb (2  µg/mL; tures more than the levels found in control cultures, even if the + + BD Biosciences). In the last 5 hours of stimulation, Brefeldin percentage of total CD3 CD4 T cells remained similar. Of note, A  (10  µg/mL; Sigma) was added. To measure CMV-/SEB- IL-7 also allowed us to detect MTP-specific T cells in samples specific cytokine release, equal numbers of cells were stimu - derived from immunosuppressed patients (ie, anergic; Pt#3). lated with CMV lysate (2  µg/mL; Advanced Biotechnologies, Sensitization with IL-7 significantly increased absolute numbers Eldersburg, MD) or SEB (1  µg/mL) for 2 hours followed by of MTP-specific T cells in all the TB patients analyzed (n = 5; 14 hours in the presence of Brefeldin A.  er Th eaer ft , cells were Figure  1C) compared with non-Bacillus Calmette-Guérin- washed, surface-stained as described above, fixed, permeabi - vaccinated healthy donors (non-MTB-infected controls, n = 8). lized, and stained with anti-CD4, anti-IL-2, anti-IFNγ, and/or To independently confirm the presence of MTP-specific T anti-TNFα mAbs. Events were acquired on a BD FACSCalibur cells, we additionally performed ICS after MTP stimulation. or LSRII, and data were analyzed using the FlowJo-v9.x soft - Mycobacterium tuberculosis peptide-pulsed irradiated autol- ware (TreeStar Inc., Ashland, OR). ogous PBMCs were used as Ag-presenting cells (APCs). As with ELISPOT assays, in ICS we found that the frequency Statistical Analysis + + of MTP-specific, IL-2 IFNγ CD4 T cells increased in IL-7 GraphPad Prism 7.03 was used for statistical analyses. The cultures, compared with freshly thawed PMBCs (d0) and D’Agostino-Pearson and Shapiro-Wilk tests were used to deter- control cultures (med; Supplementary Figure  1). Thus, mine normality of data distribution. For normally distributed MTB-specific T-cell recall responses are enhanced after sen- data that passed both tests, means and standard deviation are sitization with IL-7. shown, and paired t tests are used to compare 2 groups. For non-normally distributed data, non-parametric paired tests Interleukin-7 Supports Fungal and Viral-Specific CD4 T-Cell Accumulation (Wilcoxon test) were used to compare 2 groups. For multi- in Peripheral Blood Mononuclear Cells From Infected Individuals ple-group comparisons, 2-way ANOVA with Sidak’s or Tukey’s We then asked whether exposure to IL-7 would also drive the multiple comparison corrections were used, as indicated in accumulation of CD4 T cells specific for fungal Ags derived the figures. Levels of significance are as follows: *, P  ≤  .05; **, from endemic pathogens, such as Ca. Freshly thawed PBMCs P ≤ .005; and ***, P ≤ .0005, unless specified differently in indi - derived from individuals with recurrent Ca infections were vidual figures. immediately analyzed for IFNγ expression in Ca-Ag-recall ELISPOT, in the presence of Ag-pulsed irradiated autologous RESULTS PBMCs as APCs (d0; Figure  2), or seeded in culture with or without IL-7 (d7 and med, respectively; Figure 2), and analyzed Interleukin-7 Enhances Recall Responses of Mycobaterium tuberculo- aer 7  ft days. As in the case of TB-specific T cells, Ca-Ag-specific sis-Specific CD4 T Cells IFNγ spots were also remarkably increased in IL-7-driven cul- To investigate putative effects of IL-7 on pathogen-specific T tures, compared with controls (d0 and med; Figure  2A). In cells, we first analyzed chronically infected TB patients. These multiple individuals (n = 4), Ca-Ag-specific effectors were sig - were chosen based on clinical history and manifestation of acute nificantly increased by ~10-fold over the levels found at d0 aer ft MTB infection (clinic and culture confirmed), positive reaction IL-7 exposure compared with control medium (Figure 2B). to the TST, and ability of PBMCs to respond to stimulation with Because CMV-specific CD4 T cells have been recently shown major histocompatibility complex-II-restricted MTB-specific to significantly expand in elderly individuals chronically infected promiscuous peptides (MTPs) in IFNγ ELISPOT assays [31]. IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 1999 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 ex vivo (d0 equivalent) Cryopreserved PBMCs ELISPOT (18 + 4 hrs) unstimulated (nil) IL-7 vs control medium (med) MTP Ag-restimulation d0 d7 d7 ** d0 med IL-7 C nil Pt.#1 Pt.#2 Pt.#3 d0 IL-7 d0 IL-7 healthy MTB donors infected Figure  1. Interleukin (IL)-7 enhances Mycobacterium tuberculosis (MTB)-specific T-cell responses. (A) Cryopreserved peripheral blood mononuclear cells (PBMCs) from tuberculosis (TB) patients were analyzed for interferon (IFN)γ release by MTB-specific peptide (MTP) enzyme-linked immunospot (ELISPOT) assay at the time of thawing (day [d]0) and after a 7-day culture in the absence (control medium [med], dashed line) or presence of human recombinant IL-7 (solid line), in parallel to unstimulated controls (nil). For each patient, 2 duplicate vials were sequentially thawed, staggered in time (dash dotted lines): the first vial was used to set up the cultures at d0 (left dashdotted arrow); the second vial was thawed 7 days later (right dash dotted arrow) to provide for the following: (1) ex vivo control cells to be analyzed in parallel to cultured cells and (2) autologous feeder cells for the restimulation assays (refer to Materials and Methods for details). (B) Background IFNγ release was measured for any condition in antigen (Ag)-unpulsed control wells (nil), as representatively depicted for patient (Pt.)#1 (top row). The IFNγ-spots after restimulation with MTP promiscuous peptides were detected in 3 TB patients (Pt. #1, #2, and #3) after thawing (d0) and/or a 7-day culture (d7) in IL-7 or med. (C) The effect of the IL-7 treatment (d0 vs d7, IL-7) on MTP-specific IFN γ-spot accumulation in PBMCs derived from healthy donors (open circles, n = 8) and MTB-infected patients (open triangles, n = 5) was analyzed by the ELISPOT assay in multiple individuals. Statistical significance was determined using a 2-way analysis of variance with Sidak’s multiple comparison correction to evaluate (1) the effect of IL-7 treatment in the 2 different groups over time and (2) the diagnostic power of the discrimination of the 2 groups before and after IL-7 treatment, as indicated: *, P ≤ .05; **, P ≤ .005. A B d7 ** d0 med IL-7 10 IL-7 med nil Ca-Ag med IL-7 vs d0 vs d0 Figure 2. Interleukin (IL)-7 supports the expansion and effector function of Candida albicans (Ca)-specific T cells. (A) After thawing, peripheral blood mononuclear cells from a donor with recurrent Ca infection were analyzed for the release of the effector cytokine, interferon (IFN)γ, using a Ca-antigen (Ag)-specific enzyme-linked immunospot assay at the time of thawing (day [d]0) and after a 7-day culture in the absence (d7, control medium [med]) or presence of human recombinant IL-7 (d7, IL-7). Background IFNγ release was measured in Ag-unpulsed control wells (nil, top wells) in the presence of irradiated autologous feeder cells. (B) The range and average fold increase of Ca-Ag- specific IFN γ spots in control medium (med vs d0, white bar) and IL-7 (IL-7 vs d0, dotted bar) cultures over the levels found after thawing (d0) is shown. Statistically significant accumulation of Ca-Ag-specific IFN γ-spots was evaluated in multiple donors (n = 4) using a paired t test. *P ≤ .05; **, P ≤ .005; ***, P ≤ .0005. 2000 • JID 2018:217 (15 June) • Terrazzini et al MTP MTP-specific IFNγ spots/well Ca-specific IFNγ spot fold increase Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 with the endemic CMV [32, 33], we investigated CMV-specific their proliferation [29]. Thus, we asked whether IL-7-driven CD4 T-cell responsiveness to IL-7 in cultures derived from accumulation of human, pathogen-specific T cells also requires + − CMV-seropositive (CMV ) patients. CMV-seronegative (CMV ) cell division. To this aim, we set up CFSE-labeled PBMC cul- individuals were used as controls. Freshly derived PBMCs were tures (Figure  5A and B). We found that although a small frac- cultured in the presence of IL-7 or control medium (med) for tion of CD4 T cells derived from healthy donors (~1%–10%) 7 days, and then CMV-recall responses were tested in ICS, using underwent several rounds of “spontaneous” (ie, in the absence total CMV lysate as a source of viral Ags (Figure 3A, top). Again, of any introduced stimulation) cell division in complete cells cultured in IL-7 tended to show higher CMV-recall responses media (Figure  5A, top panel), dividing cells were dramatically compared with controls (Figure 3A). Of note, these CMV-specific increased upon IL-7 addition (Figure 5A, bottom panel). These + + T cells were mostly polyfunctional, TNFα IFNγ -producing cells cells expressed high levels of Bcl-2 (Supplementary Figure 3), in (Figure 3A) and remained undetectable in cultures derived from agreement with the prosurvival role of the cytokine [20]. More CMV individuals (Supplementary Figure 2A). Because we found importantly, IL-7 favored the expansion of 2 cell populations, that T cells express varying amounts of CD127 and upregulate it distinguishable for their proliferation potential (ie, either fast in control medium (Supplementary Figure 2B), as expected [34], or slow; F and S, respectively, in the figures) and with distinct we first rested freshly thawed PBMCs for 5 days, and then we sub - mechanistic requirements. Indeed, although both populations jected them to IL-7-driven cultures (Figure 3B, top). Under these were best detected in high-density cell cultures (Supplementary conditions, we found that IL-7 more potently and reproducibly Figure  4), they proved differentially sensitive to CSA. Fast- enabled the accumulation of CMV-specific T-cell responses from but not slow-dividing cells were sensitive to CSA inhibition + + CMV individuals (n = 10; Figure 3C). The IFN γ cells expressed (Figure  5B). In addition, fast-dividing cells were sensitive to low levels of CD127, indicative of IL-7-driven receptor down- anti-LFA-1 mAb blockade (Supplementary Figure 5), suggesting modulation [34], upon cytokine-driven activation (Figure  3D) that cell-to-cell contacts are also required for their proliferation. while maintaining high levels of LFA-1 (data not shown) and Of note, IL-7 elicited similar effects in autologous sera and FCS polyfunctional cytokine expression (Figure 3B). Thus, in addition (Supplementary Figure 4). We reckoned that, although slow-di- to bacterial responses, fungal and viral-specific responses are also viding cells represent cells undergoing homeostatic expan- enhanced by IL-7 signals. sion, known to occur via CSA-insensitive mechanisms [19] (Figure  5B, right panel in top row), fast-dividing cells may Interleukin-7 Promotes Responsiveness of Staphylococcus represent a distinct population of Ag-experienced memory aureus- Specific T Cells cells proliferating via a CSA-sensitive mechanism (Figure  5B, Bacterial superantigens, such as SEB, can lead to T-cell anergy middle-right panel in top row). Flow cytometry analysis indi- − + [35] or suppression [36]. We asked whether culturing the cells cated that although both T (CD45RA , CD62L ) and T CM EM − − in IL-7 might also help SEB-specific T-cell responses ( Figure 4). (CD45RA , CD62L ) cells accumulated in response to IL-7 The PBMCs from healthy donors were rested 5 days in control (Figure  5B, middle and bottom panel), only fast-dividing T CM − + medium and then left untreated or cultured in IL-7 for 7 days. (CD45RA , CD62L ) cells appeared sensitive to CSA (Figure 5B, The SEB-recall responses were then tested in ICS ( Figure  4A, bottom panel). It is interesting to note that the IL-7-driven top). We found that although SEB-specific T cells could be accumulation of CMV (Figure 5C, top), SEB (n = 15; Figure 5C, + + detected in control PBMC cultures (med), IFNγ or TNFα and bottom and right), and MTB-specific ( Supplementary Figure 6) + + TNFα IFNγ cells were all enriched for in IL-7-driven cultures polyfunctional memory T cells was also completely dependent from many individuals, significantly ( Figure 4A and B, n = 17). upon CSA-sensitive signaling. Taken together, our data indicate As seen for CMV-specific T cells ( Figure  3D), cytokine SEB- that IL-7-driven cultures might recapitulate the events account- specific cells expressed lower levels of CD127 ( Figure 4C) while ing for the maintenance of Ag-experienced memory T-cell sub- maintaining high levels of LFA-1 (data not shown). sets in vivo [15] and improve their identification and selection We also tested whether IL-7 is capable of supporting SEB- in vitro. responsiveness even after Ag-driven expansion, in vitro DISCUSSION (Figure  4D). We found that IL-7 significantly enhanced responses after SEB-restimulation (Figure 4D). Thus, cultur- In this study, we demonstrate that IL-7 supports the in vitro ing T cells in IL-7 either before or after Ag recall allows for a expansion of human pathogen-specific T cells, favoring and, higher frequency of Ag-specific cells to be identified. in some instances, enabling their enumeration and charac- terization. Interleukin-7-sensitive pathogen-specific effectors Interleukin-7 Drives Cyclosporine A-Sensitive Central Memory T-Cell included cells specific for endemically diffused pathogens (eg, Division S aureus, C albicans, CMV, and MTB, in parts of the world), In a previous mouse study, we found that IL-7 favored the in infecting humans recurrently during the lifetime, persisting vitro expansion of tumor-Ag-experienced T cells, by promoting (MTB) and/or reactivating in the body (CMV). u Th s, our data IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 2001 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 A B d0 d7 d0 d5 d12 PBMCs (CMV+ donor) CMV (ICS) PBMCs wash CMV (ICS) IL-7 unstimulated (nil) IL-7 unstimulated (nil) med control medium (med) med CMV Ag-recall CMV Ag-recall 5 5 0.0133 10 10 0.137 4 0.177 8.85e-3 nil 3 med IL-7 5 5 0.027 0.21 0.14 1.82 10 10 2 2 10 10 4 4 10 10 99.8 0.0111 99.8 0.0155 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 3 3 10 10 5 0.0326 0 0 0.21 4 0.0802 4 0.128 3 3 10 med 99.7 0.11 96.6 1.40 –10 –10 3 3 4 5 3 3 4 5 +CMV 3 –10 01 10 10 10 –10 0 10 10 0 TNFα 2 2 10 10 0 80.4 0.15 5 0 5 4.91 0.088 99.7 0.0576 99.8 7.52e-3 10 2 3 4 5 2 3 4 5 (2815) (2033) (285) (316) 0 10 10 10 10 0 10 10 10 10 4 4 10 10 0.259 3 3 0.191 4 10 10 4 0.138 0.489 IL-7 0 0 +CMV 3 3 19.4 0.064 93.2 1.76 –10 –10 2 3 3 4 5 3 3 4 5 10 –10 01 10 10 10 –10 0 10 10 0 99.3 0.268 99.3 0.0236 IFNγ 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 TNFα C D 1 * –1 –2 med IL-7 med IL-7 Figure 3. Interleukin (IL)-7 supports the accumulation of antiviral cytomegalovirus (CMV)-specific T cells. (A) Freshly derived peripheral blood mononuclear cells (PBMCs) from CMV donors were analyzed for inflammatory cytokine release after CMV-lysate intracellular cytokine staining (ICS) assay after a 7-day culture in the absence (d7, con - trol medium [med], dashed black line) or presence of human recombinant IL-7 (d7, IL-7, black line). Left and right dot plots show the levels of IL-2 and tumor necrosis factor (TNF)α or interferon (IFN)γ and TNFα in gated CD4 T cells, respectively. Background levels of cytokine secretion were typically measured in unstimulated controls (nil). (B) Freshly isolated PBMCs from CMV donors were rested for 5 days in plain medium (dashed black line) before a 7-day culture in the absence (d12, med, dashed black line) or presence of human recombinant IL-7 (d12, IL-7, black line). At day 12 (d12), CD4 T cells were analyzed for IFNγ and TNFα release (top row) alongside expression of CD127 + + (bottom row), after CMV-lysate ICS assay. Cytomegalovirus-specific IFN γ CD4 T cells show high CD127 expression in the resting cultures, whereas they downregulated CD127 expression upon exposure to IL-7. Background levels of cytokine secretion were typically measured in ICS unstimulated (nil) controls. A representative of >10 inde- + + + + pendent experiments is shown. (C) After subtraction of individual background levels of IFNγ and TNFα and IFNγ TNFα CD4 T cells detected in unstimulated controls (nil), + + + + the percentage of CMV-specific cytokine (IFNγ and TNFα ) CD4 T cells was evaluated in 10 independent CMV donors in IL-7 (IL-7, open triangles) compared with control medium ([med], open circles) cultures. The graph shows a statistically significant (paired Wilcoxon test, * P = .03) increase of the frequency (Log ) of CMV-specific CD4 T + + cells after IL-7 culture. (D) CD127 expression (Log mean fluorescence intensity [MFI]) is significantly downregulated in CMV-specific IFN γ CD4 T cells exposed to IL-7 (IL-7) compared with control medium (med) at d12 in 6 biologically independent replicates (paired Wilcoxon test, *P = .03). 2002 • JID 2018:217 (15 June) • Terrazzini et al CMV + + CMV-specific cytokine CD4 IL-2 (Log10 %) IFNγ + + CD127 on IFNγ CD4 (Log10 MFI) CD127 IFNγ Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 + + A B Single IFNγ Single TNFα 1 *** d0 d5 d12 10 PBMCs wash SEB (ICS) IL-7 Unstimulated (nil) med med SEB Ag-recall –1 –1 med IL-7 5 5 0.024 0.043 0.024 5.32E–3 10 10 –2 –2 4 4 10 10 10 10 med IL-7 med IL-7 nil 3 3 10 10 + + + IFNγ TNFα Total cytokine 0 0 2 2 3 3 10 10 –10 99.8 0.11 –10 99.9 0.11 *** *** 3 3 4 5 3 3 4 5 –10 0 10 10 10 –10 0 10 10 10 1 1 10 10 5 5 1.34 8.90 0.41 2.41 10 10 4 4 10 10 0 0 10 10 SEB 3 3 10 10 0 0 –1 –1 10 10 3 3 92.1 5.11 77.5 12.3 –10 –10 3 3 4 5 3 3 4 5 –10 0 10 10 10 –10 0 10 10 10 –2 –2 10 10 TNFα med IL-7 med IL-7 C D SEB d0 d5 d12 med IL-7 PBMCs wash SEB (ICS) 5 54.6 1.25 5 1.59 0.079 10 10 IL-7 SEB (2220) (1585) (338) (211) 4 4 10 10 med SEB Ag-recall 3 3 10 10 SEB d12 0 0 3 3 42.6 1.54 88.2 10.1 –10 –10 4.81 6.59 3 3 4 5 3 3 4 5 5 11.1 –10 0 10 10 10 –10 0 10 10 10 40 IFNγ 4 IL-7 *** 10 –10 3 3 4 5 –10 0 10 10 10 0.80 0.68 2.68 med –10 3 3 4 5 –10 0 10 10 10 med IL-7 med IL-7 TNFα Figure  4. Interleukin (IL)-7 supports superantigen-specific responses. (A) Freshly isolated peripheral blood mononuclear cells (PBMCs) were rested for 5  days in plain medium (dashed black line) then incubated for 1 week in IL-7 or control medium ([med] respectively IL-7, black line and med, dashed black line). At day (d)12, cells were stimulated with Staphylococcus aureus enterotoxin B (SEB) followed by an intracellular cytokine staining (ICS) assay to test antigen (Ag)-specific tumor necrosis factor (TNF) α and interferon (IFN)γ release, compared with unstimulated controls (nil). Dot plots show that the frequency of SEB-specific CD4 T cells producing TNFα and/or IFNγ increased + + + + upon exposure to IL-7 in 1 representative donor. (B) After subtraction of individual background levels of IFNγ and TNFα and IFNγ TNFα CD4 T cells detected in unstimulated + + + + + controls (nil), the percentage of SEB-specific cytokine (IFNγ , TNFα , IFNγ TNFα and total) CD4 T cells was evaluated in 17 independent donors in IL-7 (IL-7, open triangles) compared with control medium (med, open circles) cultures. The graphs show a statistically significant increase of the frequency (Log ) of SEB-specific cytokine CD4 T cells after IL-7 culture. All tests are paired Wilcoxon tests with the exception of the single TNFα analysis (paired t test): *, P ≤ .05; **, P ≤ .005; ***, P ≤ .0005. (C) At d12, cells cultured as in (A) were tested for CD127 expression in parallel to cytokine release in SEB-specific ICS assay. CD127 expression (Log mean fluorescence intensity [MFI]) is + + significantly decreased in SEB-specific IFN γ CD4 T cells exposed to IL-7 (IL-7, downward triangles) compared with control medium (med, upward triangles) in 12 independent biological replicates (paired Wilcoxon test; ***, P = .0005). (D) Freshly isolated PBMCs (d0) were stimulated with the SEB superantigen (dotted black line) for 5 days (d0–5). At d5, cells were washed and incubated for 1 week in IL-7 (IL-7, black line; d5–12) or control medium (med, dashed black line; d5–12). At d12, cells were stimulated with SEB (overnight) to test Ag-specific TNF α and IFNγ release, by ICS. Dot plots show that the frequency of SEB-specific CD4 T cells producing TNFα and/or IFNγ increased upon exposure to IL-7 (IL-7 d5–12, top row). The graph on the right shows data from the same cultures derived from 6 independent biological replicates. Statistically significant + + accumulation of SEB-specific, cytokine CD4 T cells was evaluated using a Wilcoxon matched-pairs signed-ranked test (P = .03). IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 2003 CD127 IFNγ + + CD127 on IFNγ CD4 (Log10 MFI) IFNγ + + + + % cytokine of CD4 % cytokine of CD4 (Log10) (Log10) + + %cytokine of CD4 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 A B med CSA IL-7 IL-7+CSA 4 4 4 4 4 10 10 10 10 10 0.01 0.25 0.32 0.46 44.6 0.003 0.36 56.9 2.67 10.2 31.1 0.48 9.18 34.9 3 3 3 3 3 10 10 10 10 10 FS 2 2 2 2 2 10 10 10 10 10 med FS 1 1 1 1 1 10 10 10 10 10 0.92 0.41 98 0 0 0 0 0 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CFSE 0.27 0.16 4 4 4 10 10 10 10 50 0.862 35.6 1.61 41.1 1.75 3 3 3 10 10 10 IL-7 2 2 2 10 10 10 S 1 1 1 18 26 55 10 10 10 0 1 2 3 4 47.7 1.44 60.5 2.31 55.5 1.66 0 0 0 10 10 10 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CFSE 4 4 4 10 10 10 55 1.25 39 1.93 8.33 0 3 3 3 10 10 10 2 2 2 10 10 10 1 1 1 10 10 10 0 43.3 0.417 0 56.2 2.89 0 89.9 1.72 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD45RA + + Single IFNγ Single TNFα 1 *** IL-7 IL-7+CSA 5 5 0.14 1.82 0.16 0.64 0 10 10 4 4 10 10 –1 10 –1 CMV 3 3 10 10 0 0 3 3 96.6 1.40 99.1 0.062 –10 –10 –2 –2 10 10 3 3 4 5 3 3 4 5 –10 0 10 10 10 –10 0 10 10 10 IL-7 IL-7+CSA IL-7 IL-7+CSA 5 1.72 24.2 5 1.99 0.42 10 10 + + + IFNγ TNFα Total cytokine 4 4 10 10 2 2 10 10 *** *** 3 3 SEB 10 10 1 1 10 10 0 0 3 3 –10 69.4 4.72 –10 96.8 0.98 3 3 4 5 3 3 4 5 0 0 –10 0 10 10 10 –10 0 10 10 10 10 10 TNFα –1 –1 10 10 –2 –2 10 10 IL-7 IL-7+CSA IL-7 IL-7+CSA Figure 5. Sensitization by interleukin (IL)-7 promotes pathogen-specific CD4 T cell proliferation in a cyclosporine A (CSA)-sensitive manner. 5-(and-6)-carboxyfluorescein diacetate succinimilyl ester (CFSE)-labeled peripheral blood mononuclear cells (PBMCs) (A–C) from healthy donors (A and B) were cultured for 7 days in the absence (med) or presence of human recombinant IL-7 (IL-7). (A) Proliferation of viable CD4 T cells in high-density cultures (5 × 10 cells/mL) was determined by flow cytometry after staining with anti-CD4 monoclonal antibody (mAb) and TO-PRO-3 (an intercalant agent of deoxyribonucleic acid entering necrotic and apoptotic cells). Contour plots depict the rela- − + 6 tive CFSE content within the same number of viable, TO-PRO-3 CD4 T cells. (B) Cells cultured (4 × 10 cells/mL) in the presence of IL-7 for 7 days in the absence or in the presence of CSA. At d7, cells were stained with anti-CD4, anti-CD45RA, and anti-CD62L mAb, and the relative CFSE content was analyzed by flow cytometry in total CD4 T + hi cells (top row). Fast- (F), slow- (S), and nondividing cells were determined based on CSA inhibition, as indicated. The percentage of naive (CD45RA CD62L ), central memory − hi − lo (CD45RA CD62L ), and effector memory (CD45RA CD62L ) cells were then investigated in slow- compared with fast-proliferating CD4 T cells. (C) Freshly isolated PBMCs were rested for 5 days then incubated for 1 week in IL-7 or IL-7 plus CSA (IL-7+CSA). At d12, cells were stimulated with Staphylococcus aureus enterotoxin B (SEB) during an intracellular cytokine staining (ICS) assay to test antigen-specific tumor necrosis factor (TNF) α and interferon (IFN)-γ release, compared with unstimulated controls (refer to Figures 3 and 4). Left dot plots show that the IL-7-driven accumulation of (top) cytomegalovirus (CMV)- and (bottom) SEB-specific CD4 T cells producing TNFα and/or IFNγ + + + + is reversed in the presence of CSA, by d12. After subtraction of individual background levels of IFNγ , TNFα , and IFNγ TNFα CD4 T cells detected in unstimulated controls + + + + + (nil), the percentage of SEB-specific cytokine (IFNγ , TNFα , IFNγ TNFα , and total) CD4 T cells was evaluated in 15 independent biological replicates in IL-7 (open circles) compared with IL-7+CSA cultures (open triangles). The right graphs show a statistically significant decrease in the frequency (Log ) of SEB-specific cytokine (except for IFNγ ) CD4 T cells in the presence of CSA at d12. All tests are paired Wilcoxon tests; *, P ≤ .05; **, P ≤ .005; ***, P ≤ .0005. 2004 • JID 2018:217 (15 June) • Terrazzini et al TO-PRO-3 IFNγ CD62L CD4 + + + + %cytokine of CD4 %cytokine of CD4 (Log10) (Log10) Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 will help the study and isolation of such pathogen-specific T developing systemic lupus erythematosus aer 3 dos ft es of IL-7 cells and others relevant to various clinical settings, includ- [45]. We speculate that the risk of developing autoimmunity ing harmful infections, especially in the case of immune-sup- aer IL ft -7 treatment may vary dependent on individual clin - pressed (HIV, transplantation, aged etc) or chronically infected ical history, genetic predisposition, and the administration individuals, in which pathogen-specific T cells might be low in regimen. Further studies are needed to define the long-term frequency and/or hyporesponsive. consequences of IL-7 administration. Previous studies showed that IL-7 can support immune- It is interesting to note that among memory cells, poly- + + cell reconstitution in lymphopenic conditions [22–25], restore functional T cells (double positive for IFNγ and IL-2 or sepsis-induced lymphocyte dysfunctions ([37, 38]), enhance TNFα ) were mostly enriched for by IL-7. Such cells were effector function of autoreactive T cells [ 8–13], and expand detected in subjects with chronic viral (including CMV [32]) tumor-reactive T cells [29, 30]. We then asked whether patho- infections and previously referred to as intermediate poly- gen-reactive T cells also benefited from IL-7 exposure. Our functional memory cells [46]. It is possible that IL-7 favors results show that IL-7 promotes the selective expansion of a differentiation of these cells in vitro (and possibly also in fraction of memory CD4 T cells containing pathogen-specific vivo), starting from IFNγ-producing cells. With respect to cells, best observed in high cell-density cultures. Although IL-7 maintaining polyfunctional T cells, IL-7 appears superior significantly enhanced overall cell recovery (by ~1.5-fold), cyto - to the cognate Ag by favoring (central) memory cell sur- kine-producing CD4 T cells were not enriched at a polyclonal vival [15–17], rather than terminal differentiation, acti- level (as detected by 12-O-tetradecanoyl-phorbol-13-acetate vation-induced cell death, and/or exhaustion [29]. Thus, and ionomycin stimulation; Supplementary Figure  7) unlike IL-7 may be useful for the expansion of human polyclonal that seen for pathogen-specific cells. These cells showed a fast and polyfunctional pathogen-specific CD4 (and to a lower + − + rate of proliferation, sensitive to CSA and LFA-1 inhibition, extent, CD3 CD4 or CD8 ; Supplementary Figure  8A–E) both in autologous sera and FCS. u Th s, neither homeostatic cell T cells that are hard to identify, even in the case of a rel- division [19, 39–42], known to be CSA insensitive [19, 42], nor atively well studied pathogen (such as CMV [47, 48]). food-related, bovine Ags seem to account for the accumulation Accordingly, exposing cells to IL-7 enabled better enumer- of fast-dividing cells. However, our results suggest the existence ation of in vivo-primed CMV-/SEB-specific cells, although of a cell-associated ligand capable of synergizing with IL-7 sig- these trends were less apparent in CD8 T cells, which did nals to promote the proliferation and responsiveness of patho- not undergo fast proliferation to the extent of CD4 T cells gen-specific T cells. We speculate that self-Ag/TCR-initiated (Supplementary Figure  8F). This was also the case when T signals may play a role in the IL-7 cultures. cells were Ag-restimulated in vitro before the IL-7 culture, Although further research is needed to identify such sig- opening the possibility that IL-7 (with or without Ag) might nals in IL-7 cultures, our data support a role for IL-7 in T cell- be superior to Ag alone in expanding T cells derived from in driven immunopathology in chronic and persistent infections, vivo-primed individuals. Our data also support the hypoth- coinfections, or autoimmunity. Accordingly, IL-7 is expressed esis that IL-7 sustains the preferential accumulation of poly- in inflamed tissues of patients with (rheumatic) autoimmune functional T-cell subsets within the repertoire of certain diseases, where it can be produced by several cell types [43] individuals, including perhaps the inflated responses of CD4 (including macrophages, dendritic cells, and fibroblasts) and T cells detected in elderly CMV individuals [32, 33]. Future favor pathogenic Th1- and Th17-associated cytokine secre - studies are needed to address this possibility. tion. Furthermore, dysregulated IL-7 expression or activation CONCLUSIONS of CD127 were found in patients with autoimmune conditions [8–13], suggesting that IL-7 supports the function of patho- We previously suggested that among other CD132-cytokines, genic effector cells in autoimmunity. In agreement  with this, IL-7 played nonredundant roles and outperformed IL-2 in blocking the IL-7R in experimental animal models amelio- driving Ag-experienced T-cell accumulation and mediating rated autoimmune disease manifestations [44]. Thus, together the expansion of less differentiated cells useful for gene therapy with available data, our results suggest that IL-7 might awaken [26, 30]. We now provide evidence supporting the use of IL-7 to auto-reactive T cells, or pathogen-specific effector T cells with reveal and expand in vivo-primed pathogen-specific lympho - cross-reactivity to self-Ag, hence contributing to autoimmu- cytes of clinical relevance, either as biomarkers of viral infection nity. However, this might not equally apply to patients with and disease activity [49] or as therapeutic tools. This may be rel - immune dysfunctions due to sepsis or chronic HIV/hepatitis evant for the treatment of chronic infectious diseases and can- C virus infection. Indeed, IL-7 administration mainly in HIV- cer, because adoptive immunotherapy with less differentiated T infected or immunosuppressed patients was generally well tol- cells is preferable over the transfer of terminally differentiated erated [22, 23, 25, 27, 28, 45], with a single report of a patient ee ff ctors [ 50]. IL-7 Sustains in Vivo-Primed T Cells • JID 2018:217 (15 June) • 2005 Downloaded from https://academic.oup.com/jid/article/217/12/1997/4913790 by DeepDyve user on 20 July 2022 Supplementary Data 6. Noguchi M, Nakamura Y, Russell SM, et  al. Interleukin-2 receptor gamma chain: a functional component of the Supplementary materials are available at The Journal of interleukin-7 receptor. Science 1993; 262:1877–80. Infectious Diseases online. Consisting of data provided by 7. Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective the authors to benefit the reader, the posted materials are not IL7R expression in T(-)B(+)NK(+) severe combined immu- copyedited and are the sole responsibility of the authors, so nodeficiency. Nat Genet 1998; 20:394–7. questions or comments should be addressed to the corre- 8. Nuro-Gyina PK, Rieser EL, Granitto MC, et al. Regulation of sponding author. effector function of CNS autoreactive CD4 T cells through inhibitory receptors and IL-7Rα. J Neuroinflammation Notes 2016; 13:302. Acknowledgments. We thank George Morrow and Dr. 9. Arbelaez CA, Glatigny S, Duhen R, Eberl G, Oukka M, Helen Stewart (University of Sussex) for technical support and Bettelli E. IL-7/IL-7 receptor signaling differentially ae ff cts reading the manuscript, respectively. We also thank Antonio effector CD4+ T cell subsets involved in experimental auto - Lanzavecchia and members of the San Raffaele Institute for use - immune encephalomyelitis. J Immunol 2015; 195:1974–83. ful discussions and suggestions. 10. Hartgring SA, Bijlsma JW, Lafeber FP, van Roon JA. Disclaimer. e Th funders had no role in study design, data Interleukin-7 induced immunopathology in arthritis. Ann collection and analysis, decision to publish or preparation of the Rheum Dis 2006; 65(Suppl 3):iii69–74. manuscript. 11. Penaranda C, Kuswanto W, Hofmann J, et al. IL-7 receptor Financial support. S.  C.  is supported by a Brighton and blockade reverses autoimmune diabetes by promoting inhi- Sussex Medical School Internal Fellowship (University of bition of effector/memory T cells. Proc Natl Acad Sci U S A Sussex). This work was funded by grants from the follow - 2012; 109:12668–73. ing: “Associazione Italiana Ricerca sul Cancro (AIRC)”; the 12. Watanabe M, Yamazaki M, Okamoto R, et  al. Therapeutic “Compagnia San Paolo IMI”; the Italian “Ministero dell’Istruz- approaches to chronic intestinal inflammation by specific ione, dell’Università e della Ricerca, Fondo per gli Investimenti targeting of mucosal IL-7/IL-7R signal pathway. Curr Drug della Ricerca di Base” (grant number RBNE017B4C_006; to Targets Inflamm Allergy 2003; 2:119–23. A. M.); the University of Brighton PaBS Output Enhancement 13. Mazzucchelli RI, Riva A, Durum SK. The human IL-7 Fund to N. T.;  and the University of Sussex Research receptor gene: deletions, polymorphisms and mutations. Development Fund (grant number RDF 3-021; to S. C.).  Semin Immunol 2012; 24:225–30. Potential conflicts of interest. S. C. and A. M. are inven- 14. Kondrack RM, Harbertson J, Tan JT, McBreen ME, Surh CD, tors in a patent application for the use of common-ɣ chain Bradley LM. Interleukin 7 regulates the survival and gener- cytokines for the visualization, isolation and genetic modi- ation of memory CD4 cells. 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Journal

The Journal of Infectious DiseasesOxford University Press

Published: May 25, 2018

Keywords: interleukin-7; t-lymphocytes; cytokine; candida albicans; peripheral blood mononuclear cell

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