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Kuroda, Marcelo J.; Schmitz, Jörn E.; Barouch, Dan H.; Craiu, Abie; Allen, Todd M.; Sette, Alessandro; Watkins, David I.; Forman, Meryl A.; Letvin, Norman L.
doi: 10.1084/jem.187.9.1373pmid: 9565630
A tetrameric recombinant major histocompatibility complex (MHC) class I–peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and β 2 -microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C–M) representing the optimal nine–amino acid peptide of Mamu-A*01–restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C–M complex. Tetrameric Mamu-A*01/p11C, C–M complex bound to T cells of SIVmac-infected, Mamu-A*01 + , but not uninfected, Mamu-A*01 + , or infected, Mamu-A*01 − rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01 + rhesus monkeys was only found in the cluster of differentiation (CD)8α/β + T lymphocyte subset and the percentage of CD8α/β + T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01 + rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C–M complex–binding, but not nonbinding, CD8α/β + T cells. Furthermore, the percentage of CD8α/β + T cells binding this tetrameric Mamu-A*01/p11C, C–M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings. Footnotes Abbreviations used in this paper: β 2 m β 2 -microglobulin 1-D IEF one-dimensional isoelectric focusing APC allophycocyanin B-LCL B lymphoblastoid cell line CD cluster of differentiation LDA limiting dilution assay mac macaque p11C 12–amino acid fragment of SIVmac 251 Gag (amino acids 179–190) p11C C–M, 9–amino acid fragment of SIVmac 251 Gag (amino acids 181–189) pCTL precursor frequency of CTLs SIV simian immunodeficiency virus Submitted: 26 November 1997 Revision received 2 February 1997
Gallimore, Awen; Glithero, Ann; Godkin, Andrew; Tissot, Alain C.; Plückthun, Andreas; Elliott, Tim; Hengartner, Hans; Zinkernagel, Rolf
doi: 10.1084/jem.187.9.1383pmid: 9565631
This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2D b , chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by ( a ) staining CD8 + cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2D b in association with peptide GP33–41, and ( b ) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8 + T cells were GP33 tetramer + during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8 + T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin. Footnotes The authors would like to acknowledge Alana Althage, Kevin Maloy, and Karin Brduscha-Reim for helpful assistance and discussion. Awen Gallimore, Ann Glithero, and Tim Elliott are supported by The Wellcome Trust Foundation, Great Britain. A. Godkin is supported by The Medical Research Council, Great Britain. This work was also supported by grants from the Swiss National Science Foundation (grants 31-50900.97 and 31-50884.97) and Emily Dorothy Lagemann Stiftung. 1 Abbreviations used in this paper: β 2 M, β2 microglobulin; CD, cluster of differentiation; DTT, dithiothreitol; GP, glycoprotein; LCMV, lymphocytic choriomeningitis virus; NP, nucleoprotein; VV, vaccinia virus. A. Gallimore and A. Glithero contributed equally to this work. Submitted: 5 January 1998 Revision received 27 February 1998
Callan, M.F.C.; Tan, L.; Annels, N.; Ogg, G.S.; Wilson, J.D.K.; O'Callaghan, C.A.; Steven, N.; McMichael, A.J.; Rickinson, A.B.
doi: 10.1084/jem.187.9.1395pmid: 9565632
Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex–peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8 + T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8 + T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr. Footnotes Abbreviations used in this paper: AIM acute infectious mononucleosis CD cluster of differentiation EBNA Epstein-Barr nuclear antigen IM infectious mononucleosis Submitted: 5 February 1998 Revision received 5 March 1998
Barlow, Avlin K.; He, Xin; Janeway, Charles
doi: 10.1084/jem.187.9.1403pmid: 9565633
Major histocompatibility complex (MHC) class II molecules can present peptides derived from two different sources. The predominant source of peptide in uninfected antigen presenting cells (APCs) is from self-proteins that are synthesized within the cell and traffic through the MHC class II compartment. The other source of antigen is endocytosed proteins, which includes both self- and foreign proteins. Foreign protein antigens generate adaptive immune responses, whereas self-peptides stabilize the MHC class II heterodimer on the cell surface, allowing positive and negative selection of thymocytes. Therefore, self-antigens play an important normal role in shaping the T cell receptor repertoire as well as a pathological role in autoimmunity. To determine whether processing and presentation of self-antigens by MHC class II molecules differs depending on whether the antigen is supplied through synthesis within the cell or by endocytosis, we used a T cell clone against an Eα peptide presented by I-A b to show that processing through these two routes can differ. We also show that mice can be tolerant to the epitope formed through the endogenous route, but responsive to the epitope that can be formed through endocytosis. This suggests that negative selection occurs primarily against antigens that are synthesized within the APC, and that endocytosed self-antigens could serve as autoantigens. Finally, we also demonstrate that lipopolysaccharide-activated B cells are defective for uptake, processing, and presentation of this self-antigen, and that this correlates with the increased expression of the costimulatory molecules B7.1 and B7.2. This may provide a model for studying the onset of an autoimmune response. Footnotes A.K. Barlow's present address is Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. 2 Viret, C., H. Ramaswamy, D.B. Sant'Angelo, and C.A. Janeway, Jr., manuscript submitted for publication. Abbreviations used in this paper: CIIV class II loading vesicle Ii invariant chain MIIC MHC class II compartment Submitted: 9 June 1997 Revision received 4 February 1998
Frearson, Julie A.; Alexander, Denis R.
doi: 10.1084/jem.187.9.1417pmid: 9565634
Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca 2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process. Footnotes Abbreviations used in this paper: AEBSF 4-(2-Aminoethyl)-benzenesulfonylfluoride.HCl csw corkscrew ERK extracellular regulated kinase GST glutathione s -transferase MAPK mitogen-activated protein kinase MBP myelin basic protein PI phosphatidylinositol PTPase phosphotyrosine phosphatase SH2 Src homology 2 SHP phosphotyrosine phosphatase Submitted: 12 July 1997 Revision received 17 February 1998
Kishimoto, Hidehiro; Surh, Charles D.; Sprent, Jonathan
doi: 10.1084/jem.187.9.1427pmid: 9565635
To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1–100 μg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vβ8 + cells at the level of the semi-mature population of HSA hi CD4 + 8 − cells found in the thymic medulla; deletion of CD4 + 8 + cells was minimal. SEB injection also caused marked elimination of Vβ8 + HSA hi CD4 + 8 − thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (≤1 μg) but not by high doses (100 μg). Similar findings applied when T cell receptor transgenic mice were injected with specific peptide. These findings suggest that clonal elimination of semi-mature medullary T cells is Fas independent at low doses of antigen but Fas dependent at high doses. Previous reports documenting that negative selection is not obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level. Footnotes This is publication no. 10869-IMM from the Scripps Research Institute. Abbreviations used in this paper: AICD activation-induced cell death BrdU bromodeoxyuridine DP double positive guinea pig C guinea pig complement HSA heat-stable antigen ova ovalbumin SEB staphylococcal enterotoxin B SP single positive TUNEL TdT-mediated dUTP-biotin nick end labeling Submitted: 11 August 1997 Revision received 2 March 1998
Sawada, Shinichiro; Gowrishankar, Kavitha; Kitamura, Rui; Suzuki, Misao; Suzuki, Gen; Tahara, Satoko; Koito, Atsushi
doi: 10.1084/jem.187.9.1439pmid: 9565636
T cell line–tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4 + T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4 + T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4 + T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4 + T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4 + T cell decline in patients. Footnotes Abbreviations used in this paper: CD4 (and CD8) SP CD4 (and CD8) single positive h human m murine M-tropic macrophage-tropic MIP macrophage inflammatory protein RANTES regulated on activation, normal T cell expressed and secreted SDF-1 stromal cell–derived factor 1 T-tropic T cell line–tropic Submitted: 19 September 1997 Revision received 2 March 1998
Tajima, Youichi; Huang, Eric J.; Vosseller, Keith; Ono, Masao; Moore, Malcolm A.S.; Besmer, Peter
doi: 10.1084/jem.187.9.1451pmid: 9565637
The Kit ligand (KL)/Kit receptor pair functions in hematopoiesis, gametogenesis, and melanogenesis. KL is encoded at the murine steel ( Sl ) locus and encodes a membrane growth factor which may be proteolytically processed to produce soluble KL. The membrane-associated form of KL is critical in mediating Kit function in vivo. Evidence for a role of cytoplasmic domain sequences of KL comes from the Sl 17H mutation, a splice site mutation that replaces the cytoplasmic domain with extraneous amino acids. Using deletion mutants and the Sl 17H allele, we have investigated the role of the cytoplasmic domain sequences of KL in biosynthetic processing and cell surface presentation. The normal KL protein products are processed for cell surface expression, where they form dimers. Both Sl 17H and the cytoplasmic deletion mutants of KL were processed to the cell surface; however, the rate of transport and protein stability were affected by the mutations. Deletion of cytoplasmic domain sequences of KL did not affect dimerization of KL. In contrast, dimerization of the Sl 17H protein was reduced substantially. In addition, we have characterized the hematopoietic cell compartment in Sl 17H mutant mice. The Sl 17H mutation has only minor effects on hematopoiesis. Tissue and peritoneal mast cell numbers were reduced in mutant mice as well as in myeloid progenitors. Interestingly, long-term bone marrow cultures from Sl 17H mice did not sustain the long-term production of hematopoietic cells. In addition, homing of normal hematopoietic progenitors to the spleen of irradiated Sl 17H / Sl 17H recipient mice was diminished in transplantation experiments, providing evidence for a role of Kit in homing or lodging. These results demonstrate that the membrane forms of KL exist as homodimers on the cell surface and that dimerization may play an important role in KL/Kit-mediated juxtacrine signaling. Footnotes Support by grants from the American Cancer Society and the National Institutes of Health is acknowledged. Abbreviations used in this paper: BFU-E burst-forming unit–erythroid BMMC bone marrow–derived mast cell CFU-GEMM CFU-granulocyte erythroid megakaryocyte macrophage CFU-S CFU-spleen endo endoglycosidase ER endoplasmic reticulum KL Kit ligand Sl steel VLA very late antigen W white-spotting WBC white blood cell Submitted: 21 November 1997 Revision received 9 February 1998
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