TY - JOUR
AU - Berzofsky, Jay A.
AB - Abstract The ability of cytokines to steer CD4+ Th cell responses toward a Th1 or Th2 phenotype and enhance the magnitude of both CD8+ cytotoxic T lymphocytes (CTL) and antibody responses has clearly been demonstrated by our lab and others, but the influence of cytokines on protective immune responses is much less clear. Here we show an essential role for CD4+ Th1 helper cell induction and IFN-γ production in protection from viral challenge with a recombinant vaccinia virus expressing HIV-1MN viral envelope glycoprotein gp160. Complete protection from viral challenge is achieved only when the triple combination of exogenous cytokines granulocyte macrophage colony stimulating factor (GM-CSF), IL-12 and tumor necrosis factor (TNF)-α are co-administered with the peptide vaccine. In vivo depletion of CD4+ cells or immunization of IFN-γ-deficient mice abrogates protection. GM-CSF, IL-12 and TNF-α also synergize for the enhanced induction of CTL; however, adoptive transfer of a CD8+ CTL line afforded only partial protection in this viral challenge model. As a possible mechanism of in vivo protection we show that GM-CSF increases the percentage and activity of antigen-presenting dendritic cells in draining lymph nodes where the immune response is initiated. We further demonstrate synergy between IL-12 and the proinflammatory cytokine TNF-α in driving IFN-γ production. Thus, a combination of IL-12 and TNF-α is essential for the optimal development of Th1 responses and help for CTL induction in BALB/c mice, and is complemented by a third cytokine, GM-CSF, which enhances antigen presentation. cytokines, cytotoxic T lymphocyte, Th1 cells, viral immunity APC antigen-presenting cell, CD40L CD40 ligand, CTL cytotoxic T lymphocyte, GM-CSF granulocyte macrophage colony stimulating factor, ISA incomplete Seppic adjuvant, TNF tumor necrosis factor Introduction Previously we have shown that granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α each synergize with IL-12 in the induction of cytotoxic T lymphocytes (CTL) when included with a Th–CTL peptide vaccine construct delivered in an emulsion adjuvant, and that TNF-α and IL-12 synergize for IFN-γ production, skewing the CD4+ cell response to Th1 in BALB/c mice (1). However, we did not study the protective efficacy of this combination of cytokines or the mechanisms of synergy or protection. It is clear from this study and those of other groups that exogenous cytokines are a valuable adjunct to present vaccine modalities aimed at eliciting both optimum cellular and humoral immune responses and protection (1–10). More recently, the expression of cytokine combinations with co-stimulatory molecules, CD40 ligand (CD40L) or CTLA-4 blockage have proven effective in eliciting anti-tumor CTL responses (11–14). The potential effectiveness of cytokine combinations and co-stimulatory molecules or dendritic cell vaccines has mostly been determined empirically based upon mechanisms determined in vitro for Th cell differentiation and CTL induction. The mechanisms of cytokine combinations in protective studies in vivo, however, have not been clearly defined. Here, we address the ability of cytokines in vaccine formulations to protect against viral infection, and the mechanisms of protection and synergy. Previous work with our Th–CTL peptide constructs has shown a strict requirement for CD4+ T cell help in the production of neutralizing antibody and the induction of CTL (15–17). In vitro studies suggest that an optimum CTL response requires the concerted, reciprocal interaction of CD4+ Th cells, dendritic cells and CD8+ CTL (18–23). Dendritic cells play a central role in induction of cellular immune responses (24) and maturation of dendritic cells to a fully functional state requires activation via CD40–CD40L interaction (25–27). The mature dendritic cell possesses the requisite co-stimulatory molecules and secretes IL-12 and IL-15 for differentiation and proliferation of both Th1 CD4+ cells and CD8+ CTL (28–30). CD4+ help for CTL can be bypassed by antigen-presenting cells (APC) infected by viruses which subserve these functions (31) or immunization with `mature' dendritic cells (26,32–34). Although the role of CD4+ T cells versus CD8+ CTL in viral protection models is complex and depends on the nature of viral infection (35–38), recent findings suggest the survival and efficacy of CD8+ CTL may be inextricably linked to CD4+ cell function (37,39–41). CD4+ Th cells have been shown to play a critical role in clearance of a number of viral infections by providing either help for neutralizing antibody production (40,42,43), IFN-γ mediated antiviral effects (44–47) or help for CTL (16,17). Recently a role for CD4+ cells in maintaining CD8+ CTL effector function and memory in persistent viral infections has also been shown (48–51). It is generally assumed that Th1 differentiation favors CTL induction (52,53), whereas Th2 cells are inhibitory, possibly due to the suppressive effects of IL-10 on APC function (54,55). Th cell differentiation is highly regulated by cytokines present during initiation of the immune response (56). IL-12 acts directly on CD4+ cells to enhance priming for IFN-γ production (57,58); however, Th1 development appears to require both IL-12 and IFN-γ (59,60). IL-4 selects for acquisition of a Th2 phenotype (61), and most importantly IL-4 and IFN-γ exert reciprocal inhibition of Th1 and Th2 phenotypes respectively (58,62–64). In this study, we have now investigated the ability of cytokines and combinations of cytokines not only to induce both CD4+ and CD8+ cell-mediated immunity but also to protect against viral challenge, as well as examined the mechanism of synergy and protection. We have optimized our immunization protocol by including cytokines which localize and mature dendritic cells at the inductive site, and skew the immune response toward a CD4+ Th1 phenotype and CTL induction. We show that appropriate inflammatory cytokines are essential for the induction of a protective cellular immune response in this viral challenge model. Optimum induction of CTL requires the addition of exogenous cytokines GM-CSF, IL-12 and TNF-α, although the avidity of CTL elicited by peptide vaccine is not directly affected by exogenous cytokines delivered during immunization. Furthermore, we have shown that complete protection against challenge with a recombinant vaccinia virus expressing the HIV1-MN envelope protein gp160 is dependent upon the synergy of cytokines with TNF-α during immunization that skew the response toward Th1 cell IFN-γ production. Methods Animals Female BALB/c mice were used at 6–8 weeks of age (Animal Production Colonies, Frederick Cancer Research Facility, National Institutes of Health, Frederick, MD) for immunizations. All procedures with animals were carried out in accordance with institutionally approved protocols. IFN-γ knockout mice on the BALB/c background were obtained from Jackson Laboratory (Bar Harbor, ME). Immunizations and viral challenge Mice were immunized with 20 nmol of the Th–CTL vaccine construct PCLUS 6.1-18MN (DRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN), comprised of Th determinants from HIV-1 IIIB env sequence 827–853 linked to the CTL determinant P18MN shown in bold, amino acid residues 308–322), s.c. at the base of the tail in incomplete Seppic adjuvant (montanide ISA-51; a kind gift of Jean-Pierre Planchot, Seppic, Fairfield, NJ). Cytokines were included with peptide in the aqueous phase before mixing with the adjuvant to form a water in oil emulsion. Cytokines used in immunizations included GM-CSF (Peprotech, Rocky Hill, NJ) (3–5 μg/mouse), IL-12 (Genetics Institute, Cambridge, MA) (3–5 μg/mouse) and TNF-α (R & D Systems, Minneapolis, MN) (0.5 μg/mouse). Mice were boosted 2 weeks after the primary immunization and challenged i.p. 12 days later with recombinant vaccinia virus (5×106 p.f.u./mouse) expressing the HIV-1MN envelope protein gp160 (Vaccinia-MN was a kind gift of Bernard Moss, NIAID). Five days later virus titers in the ovaries of individual mice were determined on BSC-1 indicator cells as previously described (83). In vivo depletion of CD4+ and/or CD8+ cells was achieved by four consecutive i.p. doses of 0.5 mg/mouse/day of anti-CD4 (clone GK1.5) or anti-CD8 antibody (clone 2.43) beginning 2 days prior to viral challenge. Depleted cells were not detected, by FACS analysis, in spleens of treated mice (<0.5%) up to 6 days after treatment. Lymph node cultures Medium for incubation of mouse cell cultures consisted of RPMI:EHAA 50:50, supplemented with 2 mM l-glutamine, 100 μg/ml streptomycin,100 IU/ml penicillin G and 5×10–5 M 2-mercaptoethanol. Triplicate cultures of inguinal and subaortic lymph node cells from BALB/c mice injected 2 days previously with GM-CSF in adjuvant montanide ISA-51 or adjuvant alone were depleted of T and B cells using magnetic-coated beads (Dynal, Lake Success, NY) and plated in 96- well flat-bottom wells at 5×105 APC/well with and without antigen. The two different APC populations were assessed for their ability to support proliferation of a Th cell clone by adding 3×104 CD4+ Th1 cells, derived from BALB/c mice specific for the HIV-1bru peptide nef (182–198) and cloned by limiting dilution. Cells were harvested 72 h later after an 8 h pulse with 1 μCi [3H]thymidine. Geometric mean c.p.m. ± SEM were plotted versus peptide dose. As a readout of CD8+ CTL activation, IFN-γ production was measured in culture supernatants 60 h after in vitro stimulation of 5×104 CD8+ CTL added to 5×105 lymph node APC/well pulsed for 3 h with 0.5 or 0.05 μM P18MN Semiquantitative RT-PCR Total RNA was isolated from CD4+ T cells positively selected by magnetic beads (Dynal) followed by immediate lysis in guanidinum thiocyanate followed by acid phenol–chloroform extraction and precipitated overnight at –20°C with isopropanol. mRNA was reversed transcribed from 2–5 μg of total RNA using Superscript II first-strand cDNA synthesis kit (Life Technologies, Grand Island, NY). The following primer pairs were constructed using complete coding sequences for the housekeeping gene: HPRT forward GTTGGATACAGGCCAGACTTTGTTG; HPRT reverse TCGGTATCCGGTCGGATGGGAG generated a single PCR product of 450 bp. Murine cytokine primers and controls IL-2, IL-4, IL-10 and IFN-γ (Clontech Laboratories, Palo Alto, CA) were used at a concentration of 0.4 μM/30 μl reaction. Approximately 1 μg of cDNA was added to 30 μl Superscript PCR buffer (Life Technologies) with an additional 1 mM MgCl plus primers (0.4 μM/reaction) plus primers. PCR was performed for 30 cycles using the following parameters: 94°C, 30 s; 60°C, 1 min; 72°C, 1 min extended to 5 min during the last cycle. PCR products were run on 10% TBE gels for 1.5 h, stained for 10 min with Vistra Green (Amersham, Arlington Heights, IL) and fluorescent bands scanned using a phosphofluroimager (Molecular Dynamics, Sunnyvale CA). Semiquantitative analysis for expression of the gene of interest was made by normalization to HPRT, housekeeping gene expression. ELISA assay Aliquots of 100 μl of 48–72 h culture supernatants from lymph node or spleen cell cultures stimulated in vitro with helper peptide PCLUS 6.1 or CTL epitope P18MN were tested in an IFN-γ ELISA assay (Life Technologies) according to the manufacturer's instructions. Standards were curve-fit to a four-parameter logistic function and sample concentrations (ng/ml) interpolated from the standard curve. CTL assay Bulk spleen cell populations from immunized mice were re-stimulated in vitro with peptide-pulsed (0.5 μM P18MN for 2 h) spleen cells in 12-well plates (Costar). To 1×107 spleen cells from immunized mice was added 1×107 irradiated (3000 rad) peptide-pulsed syngeneic spleen cells. After 20 h Rat T Stim (Collaborative Biomedical, Bedford, MA) was added as a source of IL-2 to a final concentration of 7.5%. Four days later non-adherent cells were spun over mouse Lympholyte M (Cedarlane, Hornby, Ontario, Canada), replated and 2.5 U/ml recombinant murine IL-2 added. Cells were harvested on day 7–8 to test for CTL activity. Triplicate cultures of effector cells were diluted in 96-well round-bottom plates and 5000 target cells were added. Effector cells were tested against target cells, 18Neo (3T3 fibroblasts transfected with the neomycin gene) and P18MN-pulsed 18Neo cells in a 5-h assay. Spontaneous lysis of 51Cr-labeled target cells did not exceed 10%. Percent specific lysis was calculated as (c.p.m. of peptide-pulsed targets – c.p.m. of 18Neo without peptide)/(total c.p.m. – spontaneous c.p.m.)×100. Statistical analysis Means were plotted as geometric or arithmetic based on the distribution of data within an experimental set, as determined by the Shapiro–Wilk test. ▵ means represent the experimental – no antigen control mean of triplicate samples. A two-way analysis of variance was applied to CTL data, thereby averaging the respective values for each curve over all the E:T ratios. Means for each treatment were compared using Tukey's multiple comparison test or Dunnett's method and significance determined at α = 0.05. IFN-γ data was treated similarly or by the Student's t-test at a P value of.01. Departures from additivity (synergism or antagonism) were assessed using the construct AB + control – A – B or for triple synergy ABC + control – A – B – C, where A, B and C denote individual treatment factors, and AB and ABC denotes the combination. Statistical analysis was performed using the JMP Statistical Software Program (SAS Institute, Cary, NC) on a Macintosh computer. Results Triple cytokine combination enhances CTL induction Previously we have shown that GM-CSF and TNF-α each synergize with IL-12 in the induction of CTL when included in the immunization with a Th–CTL vaccine construct in an emulsion adjuvant. Since we hypothesized that GM-CSF and TNF-α each synergize with IL-12 for induction of CTL by different mechanisms, we tested whether the combination of all three cytokines would enhance CTL induction. The combination of exogenous cytokines, GM-CSF, TNF-α and IL-12, leads to enhanced CTL induction following a single s.c. immunization and further increased the magnitude of the CTL response after two immunizations (Fig. 1A and B). Note that although with the large number of curves they seem to form a continuum, the horizontal shift in the E:T ratio curve between the `no cytokine' group (open circles) and the `triple cytokine' group (inverted triangles) ranged from 8- to 16-fold (Fig. 1A and B), indicating an 8- and 16-fold increase in lytic units. Synergism for CTL was observed with the triple cytokine combination after two immunizations at the two highest E:T ratios, and with TNF-α and IL-12 at all E:T ratios. Synergism was seen with all cytokine combinations, GM-CSF and IL-12, TNF-α and IL-12, and the triple cytokine combination GM-CSF, IL-12, and TNF-α at 0.05 μM peptide-pulsed targets in Fig. 1(C). From our experiments it appears that synergism for CTL is best achieved using suboptimal antigen concentrations and represents a fine balance between peptide and different cytokine combinations. Increasing antigen dose, multiple doses of GM-CSF or an increase in IL-12 concentration leads to increased CTL and disrupted the synergistic relationship although the effect of cytokine combinations was still at least either additive or enhanced compared to peptide alone or single cytokine administration. TNF-α was only effective over a narrow dose (0.1–0.5 μg/dose), as higher doses were inhibitory or toxic. Since the avidity of CD8+ CTL may better define a protective immune response than the magnitude of the response in protection from viral challenge, we wanted to determine if the avidity of CTL elicited following immunization was affected by exogenous cytokines included in the immunization. We therefore titrated the CTL response versus peptide concentration. Although the magnitude of CTL elicited differed among immunization groups, the avidity of CTL was similar regardless of which cytokines were included in the immunization (Fig. 1C). Peak CTL responses from the curves occurred in all groups at a similar peptide concentration (0.05 μM P18MN), indicating in vivo induction of moderate- to high-avidity CTL. Thus, cytokines included in the immunization enhanced the magnitude of CD8+ CTL responses elicited, but had no effect on the avidity of the population of CTL elicited to a single epitope. These effects were observed up to 1 month after immunization. Protection from recombinant vaccinia virus gp160MN viral challenge requires the triple cytokine combination and is mediated by IFN-γ production by CD4 cells Since protection against viral challenge had not been measured in this system previously, we asked whether vaccination with peptide and any combination of cytokines would protect against challenge with a recombinant vaccinia virus expressing HIV-1 gp160. It was important to determine whether and by what mechanism cytokines or combinations of cytokines might render a non-protective cellular immune response into a protective one. Immunized animals were challenged i.p. with a recombinant vaccinia virus expressing the envelope protein gp160 of HIV-1MN. Strikingly, protection from high-dose viral challenge was achieved in only two groups of mice, those which received either the combination of exogenous cytokines IL-12 and TNF-α or the triple combination GM-CSF, IL-12 and TNF-α (Fig. 2A and B). Furthermore, complete protection was consistently achieved after two immunizations only in the group receiving the triple cytokine combination (23 of 27 animals compared to five of 13 receiving IL-12 and TNF-α) (Fig. 2B). Complete protection was defined as the reduction in viral titer below the limit of detection (102 p.f.u./ovary). Despite enhanced CTL responses in animals immunized with the peptide vaccine plus GM-CSF and IL-12, little or no protection was seen with this cytokine combination in this viral challenge model. Thus, surprisingly, enhancement in protection did not strictly correlate with enhancement of CTL response. While several cytokine combinations enhanced CTL, protection in vivo was more discriminating and was consistently achieved only with the triple combination, showing clearly the synergy of all three cytokines. To determine which T cells were responsible for mediating protection, animals receiving PCLUS 6.1-18MN plus the triple cytokine combination GM-CSF, IL-12 and TNF-α were depleted of CD4+ or CD8+ cells for 4 consecutive days beginning 2 days prior to viral challenge. Eight out of 10 animals receiving peptide plus the triple cytokine combination were completely protected, whereas complete protection was not obtained in any of the animals depleted of CD4+ cells (Fig. 3A), although partial protection was achieved in four of these 10 mice treated with anti-CD4 antibody and a significant reduction (3 log, >99.9%) in viral titer was seen in three of these four animals. Since partial protection was obtained in a number of animals treated with anti-CD4 this suggested that CD8+ cells may also contribute to protection. Anti-CD8 treatment alone did not affect protection, although combined with anti-CD4 it seemed to make the inhibition of protection more complete, as no animals were protected following treatment with both anti-CD4 and anti-CD8 antibody. IFN-γ appeared to be necessary for protection since neutralization of IFN-γ by a single dose of antibody was sufficient to abrogate protection. Therefore, we tested IFN-γ knockout mice for protection following two immunizations with the triple cytokine combination. None of these animals were protected from a viral challenge, confirming the importance of IFN-γ (Fig. 3A). We conclude that the major mechanism of protection was mediated by CD4+ T cells and IFN-γ. To directly determine what role CD8+ CTL might also play, we adoptively transferred 7×106 cells of a P18MN-specific CD8+ CTL clone (i.v) and challenged i.p. with virus (Fig. 3B). Although a significant reduction (>2 log, 99%) was achieved with a single infusion at the time of challenge, none of the animals were completely protected. We as well as other groups have shown a role for CD8+ CTL in protection from recombinant vaccinia virus challenge (65–67); however, complete protection in this study, where animals were immunized s.c and challenged i.p. 12 days later, was found to correlate with IFN-γ production by CD4+ cells. CD8+ CTL lines derived from immunized mice were capable of producing high levels of IFN-γ and partial protection in unimmunized mice from viral challenge was achieved following adoptive transfer of a CD8+ CTL clone. We did not determine whether the protection achieved by adoptive transfer of the CD8+ CTL clone was attributable to the high levels of IFN-γ production following stimulation in vivo or direct killing of virus-infected cells. We have shown in Fig. 3(B) that adoptive transfer of a CD8+ CTL line reduces the viral titer by ~100-fold, but it is possible that more cells, or cells that trafficked more efficiently to the site of viral replication (the ovaries), would be more effective. We have not transferred bulk immune spleen cells. However, we have found, in unpublished results, that if we immunize s.c. and challenge i.p., the protection is dominated by CD4+ cells as shown in Fig. 3, but if we immunize i.p. and challenge i.p., then CD8+ cells play a more dominant role. Although we have not been able to establish the mechanism of this difference, we believe it is likely to depend on the trafficking of the CD8+ T cells, that may depend on the site of deposition of the antigen. Therefore, we believe that this finding supports the conclusion that both CD4+ and CD8+ cells contribute to the protection, and their relative importance depends on trafficking. To understand the mechanism of synergy among these cytokines for enhancing immune responses and also protection, we examined the mechanistic role of each cytokine. GM-CSF treatment enhances the cellular immune response by recruiting APC precursors to draining lymph nodes. Since GM-CSF when included with peptide immunization was effective in enhancing both proliferative and CTL responses, we performed a simple experiment to evaluate its mechanism of action. Although it has been presumed to act by enhancing antigen presentation, this assumption has never been tested directly, to our knowledge. Lymph node cells from mock-treated and GM-CSF-treated animals were tested for their functional ability to stimulate proliferation of a CD4+ T cell clone and IFN-γ production by a CD8+ CTL clone (Fig. 4). T cell-depleted lymph node cells from GM-CSF-treated animals elicited both a more potent and efficient proliferative response of a CD4+ T cell clone specific for HIVbru (nef 182–198) than lymph node cells from mock-treated animals (Fig. 4A). Approximately 30 times less peptide was required to elicit similar proliferation at low-dose peptide (compare curves at 50×103 c.p.m.) and the maximum proliferative response could not be obtained with lymph nodes from mock-treated animals at any peptide concentration. Similar results were obtained with a CD8+ CTL clone as an indicator. Lymph node cells from GM-CSF-treated animals elicited significantly greater IFN-γ production at both the optimum stimulating dose of 0.5 μM P18MN and 1 log lower dose peptide (P < 0.001) (Fig. 4B). This difference in the functional ability of lymph node cells from GM-CSF-treated animals could be explained by a 50% increase in CD11c+ cells (dendritic cells) (from 8.42% of positive MHC class II gated cells in mock-treated animals to 12.6% in GM-CSF-treated animals) as determined by FACS analysis (data not shown). Furthermore, like GM-CSF the triple combination of cytokines GM-CSF, IL-12 and TNF-α increases the number of CD11c+ dendritic cells. However, we find that only the triple combination, not the double, also results in a marked increase in CD11b+ macrophages, which may contribute to protection by making oxidative effector molecules in response to the IFN-γ. There was a proportional increase in the number of cells expressing B7-1 from ~8% in animals treated with GM-CSF or IL-12/TNF-α to 28% of class II+ cells in animals treated with the triple cytokine combination. Although we observed an increase in CD11C+ cells in draining lymph nodes when animals were treated with carrier-free GM-CSF in ISA-51, there was not an increase in co-stimulatory molecules B7-1, B7-1, ICAM or the class I Dd molecule, or CD40 expression on these cells in the absence of Th-dependent or Th-independent activating factors (data not shown). These data clearly demonstrate the role of GM-CSF in enhancing the magnitude of cellular immune responses through the recruitment of antigen-presenting dendritic cells to the inductive site of the immune response and Th-dependent maturation following antigen presentation as well. TNF-α synergizes with IL-12 in selecting a CD4+ cell Th1 phenotype Because protection was dependent on CD4+ T cells and on IFN-γ, we investigated the role of the cytokine synergy in enhancing the response of CD4+ T cells that make this cytokine, Th1 cells. To determine Th cell phenotypes we immunized animals with peptide vaccine plus cytokines, and performed RT-PCR for cytokine message expression and IFN-γ production following 24 h in vitro stimulation of 7-day draining lymph node cells with the helper peptide PCLUS 6.1 (Fig. 5A and C). Peptide vaccine alone without cytokines elicited a Th2-like response: IL-4, low IFN-γ expression and a weak proliferative response (Fig. 5A and B). Individual cytokines, GM-CSF, IL-12 or TNF-α lead to a mixed response. GM-CSF was noteworthy in enhancing IL-4 expression (1.9-fold) and IL-10 (2.3-fold) compared to peptide alone, as well as enhancing IL-2 and proliferation. Significantly, GM-CSF alone did not enhance IFN-γ production by a population of bulk CD4+ cells. Only CD4+ cells from mice immunized with PCLUS 6.1-18MN plus the combination of cytokines TNF-α and IL-12 or TNF-α, GM-CSF and IL-12 showed evidence of skewing toward a Th1 response as determined by the up-regulation of IFN-γ message by CD4+ T cells (19- and 12-fold increase respectively, relative to peptide alone). Although all groups receiving single cytokines showed enhanced expression of IFN-γ compared to peptide alone (3.8- to 5.6-fold increase) only animals receiving IL-12 and TNF-α or GM-CSF, IL-12 and TNF-α showed significant increases in IFN-γ production after in vitro stimulation (Fig.5C). IL-4 was slightly enhanced in both groups (<2-fold), whereas IL-10 was enhanced 2.4-fold in animals receiving the triple cytokine combination. CD4+ cell proliferation was also higher in these two groups (Fig.5B). Similar results were seen in lymph nodes at day 10. Since message expression and in vitro production of IFN-γ were routinely highest in animals receiving the combination IL-12 plus TNF-α, yet protection from viral challenge was most consistently achieved with the triple cytokine combination in which CTL were the highest, this suggested a role for CD8+ cells not revealed in the depletion experiments. Therefore, we did a side by side comparison of IFN-γ production in bulk spleen cell cultures stimulated with helper peptide or the CTL epitope. Consistent with previous results, CD4+ cell IFN-γ production was significantly increased over control peptide, and all other groups after a single immunization and after boosting when TNF-α and IL-12 or GM-CSF, IL-12 and TNF-α were included in the immunization (Fig. 6A). A similar pattern was seen in the CD8+ cell response, although levels of IFN-γ were significantly lower than seen in the CD4+ cell response (Fig. 6B). Since complete protection was shown to be CD4+ T cell IFN-γ mediated, and only partial protection was achieved following adoptive transfer of CD8+ CTL, either more CD4+ T cells are activated to produce IFN-γ at the site of viral challenge than the number of CD8+ T cells adoptively transferred or the ability of the adoptively transferred CD8+ T cells to effectively migrate to the site during initial viral infection maybe limiting. Also a direct role of CD8+ CTL lysis of virus-infected cells, not revealed by in vivo depletion, may explain the difference in protection between those animals receiving IL-12 and TNF-α versus triple cytokine treatment since IFN-γ production was similar in both groups. We are further investigating this possibility. The only difference found between these two groups was a higher level of CTL induction in animals receiving peptide vaccine plus the triple cytokine combination. Discussion In this study we have shown an essential role for novel cytokine synergies in eliciting maximal CD8+ CTL responses and CD4+ Th1 effector cell responses capable of complete protection from a recombinant vaccinia viral challenge, and we have examined the mechanism of this synergy and protection. Previous studies by ourselves (1,88) and others (7,8,68,69) have shown enhancement of CTL responses by cytokines and have detected some bilateral synergies, but the mechanisms of CTL enhancement or of synergistic interactions was not determined. Further, synergistic effects on protection against viral infection have not been demonstrated previously. The current study focuses on these two issues: synergistic effects on protection from viral infection and the mechanisms of cytokine synergy and protection. We show here that by optimizing the combination of cytokines used in the adjuvant (GM-CSF, IL-12 and TNF-α) enhanced CTL induction occurs and protection is achieved. However, this correlation does not imply that CTL are the major mechanism of protection. Previously we have demonstrated a role for CD8+ CTL in protection from recombinant vaccinia viral challenge (65–67) and, in this study, adoptive transfer of a CD8+ CTL clone into unimmunized mice led to partial protection. However, we have not previously seen complete protection by CD8+ CTL achieved in this challenge model. CD8+ CTL undergo a massive burst following a number of virus infections and have been suggested to play a major role in the control of many persistent viral infections (70–73). The subsequent fate of CD8+ CTL and the ability to control or eliminate infection may be dependent upon the initial viral load and the concerted interactions between T cells and APC, co-stimulation, and cytokine combinations which support maximum induction, and survival of activated CTL. Clearly when CD4+ cell function is compromised, as in disease progression toward AIDS, the CD8+ CTL response is inadequate for disease control (37,49). Since it appears that events occurring during a narrow window of time during the initial infection are critical in the development of persistent or latent infections, the challenge for prophylactic vaccine development is to insure a fully functional CD4+ and CD8+ CTL repertoire at the site of initial infection that could eliminate virus-infected cells before development of escape mutants (49,67,74–77) and dissemination of the virus systemically. Both CD8+ CTL and CD4+ cells are considered to play a significant role in the control of viremia and persistent infection with HIV. In this study, although cytokine combinations have been shown to quantitatively enhance T cell responses and vaccine efficacy, it is clear from this study and our previous studies that a protective CD8+ CTL response requires that activated CTL are present, in adequate numbers, locally at the site of viral challenge (66,67,78). In the recombinant HIV-1 gp160 vaccinia model system used in this study, adoptive transfer of a CD8+ CTL clone directed to the recombinant protein provided partial protection in a self-limiting infection, but was not robust enough to completely eliminate virus within the first few days following viral challenge. However, surprisingly, CD4+ cells play a more direct role here than just maintenance of CD8+ T cell activity. We show here that CD4+ T cells are the predominant effector cell mediating protection. The mechanism appears to be IFN-γ production, as shown by using both antibody blocking and knockout mice. This result is consistent with earlier studies showing an important role for IFN-γ in controlling vaccinia infection (79). In this study we further investigated the mechanisms of cytokine synergies responsible for eliciting a protective response. We demonstrated a sequential and concerted action of cytokine combinations in eliciting optimum CD4+ and CD8+ CTL responses. GM-CSF has been presumed to act as an adjuvant by mobilizing bone marrow myeloid-derived dendritic cell precursors to draining lymph nodes increasing the efficiency of antigen presentation for induction of cell-mediated immunity, but its mechanism of action as an adjuvant has never been directly tested to our knowledge. Although a number of studies have shown that GM-CSF enhances T cell responses, here we demonstrate experimentally that the mechanism does indeed involve enhancement of APC activity in the draining lymph nodes, enhancing stimulation of both a CD4+ Th1 cell and CD8+ CTL response. However, it is noteworthy to comment on the lack of protection achieved with the combination of cytokines GM-CSF and IL-12. Although GM-CSF enhances CD4 Th cell responses, neither GM-CSF or IL-12 alone or in combination were capable of skewing the response toward a Th1 phenotype and a population of IFN-γ-secreting cells mediating protection. TNF-α has been shown to have pleiotropic regulatory functions: enhancing APC function (80) and maturation of dendritic cells and IL-12 production (81,82). Furthermore, in vivo (1) and in vitro studies (83) have shown TNF-α to synergize with IL-12 for production of IFN-γ and skewing of the Th response to Th1. TNF-α may act directly on T cells via the TNF-R either to provide a co-stimulatory signal or, dependent upon the state of activation, to induce apoptosis in established T cell lines or memory cells following antigen stimulation with high-dose antigen (84), although in this study we did not see inhibition of proliferative responses in CD4+ or CD8+ cell cultures by the addition of exogenous TNF-α. These results may explain the necessity of both an appropriate inflammatory response in which TNF-α is produced, and IL12 production by APC stimulated through CD40–CD40L interaction, in initiating a Th1 response. TNF-α has also been shown to play a significant role in CTL induction in a graft versus leukemia mouse model (85). Since TNF-α may also be a contributing factor in apoptosis of activated T cells, the kinetics and level of TNF-α maybe a critical factor in the balance between proliferation, apoptosis and establishment of memory T cells. If TNF levels remain high during proliferation in the presence of high antigen levels, cell death may occur, diminishing the pool of memory T cells, whereas if TNF-α levels are controlled, maximum expansion of the precursor pool and memory will ensue. IL-12 also has been shown to have pleiotropic functions, as an obligatory cytokine for induction of Th1 responses and induction of IFN-γ, and as growth and survival factor for CD4+ and CD8+ CTL (29). Although IL-12 requires TNF-α as a cofactor in induction of IFN-γ in BALB/c mice (1,86), studies have shown that IL-12 alone may be sufficient to drive Th1 responses in other inbred and congenic strains of mice (83). This enhanced Th1 response led to enhanced effector function in vivo, as well as mediating the maturation and conditioning of dendritic cell APC which enhanced the CTL response. Here we have shown that TNF-α and IL-12 strikingly synergize for production of IFN-γ (1 and present data). We hypothesized that the mechanism may involve a synergistic up-regulation of mRNA for IL-12β2R by the combination of TNF-α and IL-12. In a follow-up study, we indeed found a direct synergistic effect of IL-12 and TNF-α on naive CD4+ cell IFN-γ and IL-12βR expression (J. D. Ahlers, I. M. Belyakov, S. Matsui and J. A. Berzofsky, submitted). Thus synergy for IFN-γ production may be mediated by an enhanced sensitivity of CD4+ T cells for IL-12 induced by up-regulation of the IL-12 receptor. This in turn contributes to synergy for protection. We conclude that protection against HIV gp160-expressing recombinant vaccinia virus in this system in which animals were immunized s.c. with peptide vaccine plus cytokines and challenged at a different site (i.p.) depends primarily on IFN-γ production, which in this case is mediated primarily by CD4+ Th1 cells although CD8+ CTL can also do so. Three cytokines can synergize to maximize IFN-γ production as well as CTL induction, and thus to provide the strongest and most robust protection against viral challenge. We have shown that these cytokines act by complementary mechanisms, GM-CSF to increase the antigen-presenting activity of dendritic cells, TNF-α to act with IL-12 in up-regulating IFN-γ production. We find that only the triple combination, not the double combination, not only gives an increase in the number of dendritic cells (CD11c+) in the draining lymph node, which can contribute to the magnitude of the immune response, but also gives an increase in the number of CD11b (Mac-1)+ macrophages, which might play a functional role in protection by making oxidative effector molecules in response to the IFN-γ. Thus, the combination of the high IFN-γ with the presence of macrophages that can carry out downstream effector functions in response to the IFN-γ, may provide an explanation for the difference in protection seen between the triple cytokine combination GM-CSF, IL-12 and TNF-α compared to immunization with IL-12 and TNF-α. Since incomplete Freund's adjuvant, like the emulsion adjuvant ISA-51 used in this study, has been shown to influence development of a Th2 phenotype following immunization with protein antigens (87), appropriate combinations of cytokines can exert a powerful selective influence on the developing immune response in vivo independent of the adjuvant used. Vaccines that take advantage of these cytokines may be able to maximize protection without the need for adjuvants that induce an inflammatory response with more severe side effects. Fig. 1. View largeDownload slide Triple cytokine combination elicits greater CTL induction after a single s.c. immunization (A) and boosting (B) with peptide vaccine PCLUS 6.1-18MN. Avidity of CTL induced with peptide vaccine is unaffected by exogenous cytokines (C). In (A) and (B) immune spleen cells stimulated for 7 days were titrated at E:T ratios shown on 18 Neo targets pulsed with 0.5 μM P18MN. (C) Targets were pulsed with the indicated concentrations of P18MN and the E:T ratio was 50:1. Significant differences and synergy between cytokine groups were calculated as described in Methods. Results were reproducible in five separate experiments. Fig. 1. View largeDownload slide Triple cytokine combination elicits greater CTL induction after a single s.c. immunization (A) and boosting (B) with peptide vaccine PCLUS 6.1-18MN. Avidity of CTL induced with peptide vaccine is unaffected by exogenous cytokines (C). In (A) and (B) immune spleen cells stimulated for 7 days were titrated at E:T ratios shown on 18 Neo targets pulsed with 0.5 μM P18MN. (C) Targets were pulsed with the indicated concentrations of P18MN and the E:T ratio was 50:1. Significant differences and synergy between cytokine groups were calculated as described in Methods. Results were reproducible in five separate experiments. Fig. 2. View largeDownload slide Complete protection (i.e. no detectable virus at a dilution of 10–2) from i.p. viral challenge with recombinant vaccinia gp160MN envelope protein is achieved only in animals that received exogenous cytokine combinations: TNF-α and IL-12 or GM-CSF, TNF-α and IL-12 (A). Cumulative mean viral ovary titer ± SEM per group are shown tested 12 days following two s.c. immunizations with PCLUS 6.1-18MN plus cytokines as indicated. The solid black bar indicates no prior immunization to viral challenge (B). The percent of animals with virus titers below the limit of detection (102 p.f.u./ovary) is shown. Fig. 2. View largeDownload slide Complete protection (i.e. no detectable virus at a dilution of 10–2) from i.p. viral challenge with recombinant vaccinia gp160MN envelope protein is achieved only in animals that received exogenous cytokine combinations: TNF-α and IL-12 or GM-CSF, TNF-α and IL-12 (A). Cumulative mean viral ovary titer ± SEM per group are shown tested 12 days following two s.c. immunizations with PCLUS 6.1-18MN plus cytokines as indicated. The solid black bar indicates no prior immunization to viral challenge (B). The percent of animals with virus titers below the limit of detection (102 p.f.u./ovary) is shown. Fig. 3. View largeDownload slide Anti-CD4+ cell depletion in vivo abrogates protection from viral challenge following s.c. immunization with peptide plus exogenous cytokines GM-CSF, TNF-α and IL-12. Viral titers in ovaries from individual mice 5 days post i.p. challenge with 5×106 p.f.u. recombinant vaccinia HIV-1MN gp160 are shown (A). Two separate experiments are represented by open and closed symbols. Complete protection is achieved in 80% of animals immunized with the Th–CTL vaccine construct plus triple cytokine combination, GM-CSF, TNF-α and IL-12. Anti-CD8+ cell depletion (0.5 mg/day i.p for 4 days) did not affect protective immunity, whereas anti-CD4+ (0.5 mg/day i.p for 4 days) or both anti-CD4+ and anti-CD8+ abolished protection. Protection is mediated through IFN-γ production since a single dose of anti-IFN-γ (0.5 mg i.p.) the day of immunization (open triangles) or 3-day treatment (closed triangles) abrogated complete protection. Protective immunity could not be achieved by immunization of IFN-γ knockout mice (crossed diamonds). (B) Adoptive transfer of 7×106 cells (i.v.) of P18MN CD8+ CTL clone afforded partial protection from recombinant vaccinia gp160MN challenge. Individual animals and the mean (represented by a horizontal line) are shown for each group. Fig. 3. View largeDownload slide Anti-CD4+ cell depletion in vivo abrogates protection from viral challenge following s.c. immunization with peptide plus exogenous cytokines GM-CSF, TNF-α and IL-12. Viral titers in ovaries from individual mice 5 days post i.p. challenge with 5×106 p.f.u. recombinant vaccinia HIV-1MN gp160 are shown (A). Two separate experiments are represented by open and closed symbols. Complete protection is achieved in 80% of animals immunized with the Th–CTL vaccine construct plus triple cytokine combination, GM-CSF, TNF-α and IL-12. Anti-CD8+ cell depletion (0.5 mg/day i.p for 4 days) did not affect protective immunity, whereas anti-CD4+ (0.5 mg/day i.p for 4 days) or both anti-CD4+ and anti-CD8+ abolished protection. Protection is mediated through IFN-γ production since a single dose of anti-IFN-γ (0.5 mg i.p.) the day of immunization (open triangles) or 3-day treatment (closed triangles) abrogated complete protection. Protective immunity could not be achieved by immunization of IFN-γ knockout mice (crossed diamonds). (B) Adoptive transfer of 7×106 cells (i.v.) of P18MN CD8+ CTL clone afforded partial protection from recombinant vaccinia gp160MN challenge. Individual animals and the mean (represented by a horizontal line) are shown for each group. Fig. 4. View largeDownload slide Lymph node cells from GM-CSF-treated animals are better APC for stimulation of both CD4+ Th and CD8+ CTL clones. (A) Proliferative response of BALB/c nef (182–198) CD4+ Th cell using as APC T cell-depleted normal lymph node cells pooled from two mice treated s.c. with ISA only (open circles) or lymph nodes from GM-CSF-treated animals (closed circles). Individual symbols represent no antigen. (B) CD8+ CTL P18MN-specific IFN-γ production 48 h following in vitro stimulation with peptide presented by normal lymph node APC (open bars) and lymph node APC from GM-CSF-treated mice (closed bars). Differences between normal and GM-CSF-treated lymph node cells were significant in all cases tested at P < 0.001(Student's t-test). Fig. 4. View largeDownload slide Lymph node cells from GM-CSF-treated animals are better APC for stimulation of both CD4+ Th and CD8+ CTL clones. (A) Proliferative response of BALB/c nef (182–198) CD4+ Th cell using as APC T cell-depleted normal lymph node cells pooled from two mice treated s.c. with ISA only (open circles) or lymph nodes from GM-CSF-treated animals (closed circles). Individual symbols represent no antigen. (B) CD8+ CTL P18MN-specific IFN-γ production 48 h following in vitro stimulation with peptide presented by normal lymph node APC (open bars) and lymph node APC from GM-CSF-treated mice (closed bars). Differences between normal and GM-CSF-treated lymph node cells were significant in all cases tested at P < 0.001(Student's t-test). Fig. 5. View largeDownload slide TNF-α/IL-12 and GM-CSF/TNF-α/IL-12 skew Th cell response toward a Th1 phenotype. CD4+ cytokine message by semi-quantitative RT-PCR from draining lymph nodes 7 days following immunization with PCLUS 6.1-18MN and cytokines (A). Replicate samples (nine 96-well-plate cultures) of CD4+ selected cells following overnight in vitro stimulation were pooled and PCR performed for 25–30 cycles (not saturating). Similar experimental results were seen in three separate experiments. Proliferative response (B) and IFN-γ production (C) of draining lymph nodes following s.c. immunization and in vitro stimulation with 1 μM helper peptide PCLUS 6.1. Background proliferation without antigen was <3000 c.p.m. in all groups tested and no IFN-γ production was detected in the absence of antigen stimulation. Fig. 5. View largeDownload slide TNF-α/IL-12 and GM-CSF/TNF-α/IL-12 skew Th cell response toward a Th1 phenotype. CD4+ cytokine message by semi-quantitative RT-PCR from draining lymph nodes 7 days following immunization with PCLUS 6.1-18MN and cytokines (A). Replicate samples (nine 96-well-plate cultures) of CD4+ selected cells following overnight in vitro stimulation were pooled and PCR performed for 25–30 cycles (not saturating). Similar experimental results were seen in three separate experiments. Proliferative response (B) and IFN-γ production (C) of draining lymph nodes following s.c. immunization and in vitro stimulation with 1 μM helper peptide PCLUS 6.1. Background proliferation without antigen was <3000 c.p.m. in all groups tested and no IFN-γ production was detected in the absence of antigen stimulation. Fig. 6. View largeDownload slide CD4+ cells make more IFN-γ than CD8+ cells following peptide immunization in animals treated with TNF-α/IL-12 and GM-CSF/TNF-α/IL-12. IFN-γ production (A) in CD4+ cells following in vitro stimulation with the helper peptide, PCLUS 6.1, and (B) CD8+ cells stimulated in vitro with 1 μM P18MN after a single s.c. immunization (left) and boosting (right) with PCLUS 6.1-18MN and exogenous cytokines. Note difference in scales. Fig. 6. View largeDownload slide CD4+ cells make more IFN-γ than CD8+ cells following peptide immunization in animals treated with TNF-α/IL-12 and GM-CSF/TNF-α/IL-12. IFN-γ production (A) in CD4+ cells following in vitro stimulation with the helper peptide, PCLUS 6.1, and (B) CD8+ cells stimulated in vitro with 1 μM P18MN after a single s.c. immunization (left) and boosting (right) with PCLUS 6.1-18MN and exogenous cytokines. Note difference in scales. Transmitting editor: H. Wigzell We thank Drs Alan Sher and William E. Paul for critical reading of the manuscript and helpful suggestions. 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TI - Mechanisms of cytokine synergy essential for vaccine protection against viral challenge
JF - International Immunology
DO - 10.1093/intimm/13.7.897
DA - 2001-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mechanisms-of-cytokine-synergy-essential-for-vaccine-protection-IimYPlamBf
SP - 897
EP - 908
VL - 13
IS - 7
DP - DeepDyve
ER -