T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen

Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self- reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area that mimics a structure presented on HLA B*4402. Thus, tolerance to a background major histocompatibility antigen can effectively diversify the TCR repertoire for a foreign epitope by deflecting the response away from an immunodominant combination of TCR-binding residues.     

Differential influence on cytotoxic T lymphocyte epitope presentation by controlled expression of either proteasome immunosubunits or PA28

The proteasome is the principal provider of major histocompatibility complex (MHC) class I-presented peptides. Interferon (IFN)-gamma induces expression of three catalytically active proteasome subunits (LMP2, LMP7, and MECL-1) and the proteasome-associated activator PA28. These molecules are thought to optimize the generation of MHC class I-presented peptides. However, known information on their contribution in vivo is very limited. Here, we examined the antigen processing of two murine leukemia virus-encoded cytotoxic T lymphocyte (CTL) epitopes in murine cell lines equipped with a tetracycline-controlled, IFN-gamma-independent expression system. We thus were able to segregate the role of the immunosubunits from the role of PA28. The presence of either immunosubunits or PA28 did not alter the presentation of a subdominant murine leukemia virus (MuLV)-derived CTL epitope. However, the presentation of the immunodominant MuLV-derived epitope was markedly enhanced upon induction of each of these two sets of genes. Thus, the IFN-gamma-inducible proteasome subunits and PA28 can independently enhance antigen presentation of some CTL epitopes. Our data show that tetracycline-regulated expression of PA28 increases CTL epitope generation without affecting the 20S proteasome composition or half-life. The differential effect of these IFN-gamma-inducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response.     

The heme moiety of cytochrome c is an autoreactive Ir gene-restricted T cell epitope

In these studies, we have shown that the heme moiety of cyt c is a dominant T cell epitope that induces a large proliferative response in lymph node T cells derived from SJL and B10.A mice when presented on either unfixed or fixed syngeneic APCs. Not only is this vigorous response observed for cyt c-primed T cell populations but also for populations obtained from naive SJL or B10.A mice. The reactivity to the heme moiety falls under strict MHC restriction, in that it is present only in murine strains bearing either the I-Ak or I-As molecule and can be blocked by antibodies specific for these class II molecules. Therefore, these findings require that the current models describing the nature of T cell epitopes be extended to include nonpeptide molecules. Furthermore, as the heme moiety is ubiquitous throughout the organism, although sequestered within proteins, the existence of heme-reactive T cell populations in unprimed animals provides another example of the existence of self-reactive T cell clones.     

Identification of viral molecules recognized by influenza-specific human cytotoxic T lymphocytes

Human cytotoxic T cells specific for influenza A virus were tested for recognition of each of the ten influenza A virus proteins expressed in target cells using recombinant vaccinia viruses. They recognized the matrix M1, polymerase PB2, and nucleoproteins of influenza virus in association with MHC class I antigens. These internal viral proteins were seen by CTL in conjunction with one or more of the available dependent HLA gene products. There was no detectable recognition of influenza virus surface glycoproteins in target cells.     

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope.

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202-221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site.     

The insulin A-chain epitope recognized by human T cells is posttranslationally modified

The autoimmune process that destroys the insulin-producing pancreatic beta cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4(+) T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4-restricted epitope within the first 13 amino acids of the A-chain (A1-13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4(+) T cell clones that recognized this epitope were isolated from an HLA DR4(+) child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4(+) donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D.     

Antiviral CD8+ T cell responses in neonatal mice: susceptibility to polyoma virus-induced tumors is associated with lack of cytotoxic function by viral antigen-specific T cells

Polyoma virus is a potent oncogenic pathogen when inoculated into newborn mice of particular H-2(k) strains. Using D(k) tetramers containing the dominant antipolyoma CD8(+) T cell epitope, middle T protein (MT)389-397, and intracellular interferon gamma staining, we enumerated MT389-specific CD8(+) T cells in infected neonates having opposite susceptibilities to polyoma virus-induced tumors. In resistant mice, MT389-specific CD8(+) T cells dramatically expanded during acute infection in neonates to a frequency rivaling that in adults; furthermore, in both neonatal and adult mice, this antipolyoma CD8(+) T cell response exhibited nearly identical T cell receptor (TCR) functional avidities and TCR functional fingerprints. Susceptible mice mounted an MT389-specific CD8(+) T cell response of only fourfold lower magnitude than resistant mice; but, in clear contrast to resistant mice, these CD8(+) T cells lacked ex vivo MT389-specific cytotoxic activity. However, MT389-specific CD8(+) T cells in resistant and susceptible mice expressed similar TCR avidities, perforin levels, and surface type O-glycan levels indicative of mature CD8(+) T cell effectors. Upon in vitro restimulation with infected antigen-presenting cells, CD8(+) T cells from acutely infected susceptible neonates acquired strong MT389-specific cytotoxicity. These findings indicate that polyoma-specific CD8(+) T cells are armed with, but restrained from deploying, their cytotoxic effector function in mice susceptible to polyoma virus tumorigenesis.     

Antivirally protective cytotoxic T cell memory to lymphocytic choriomeningitis virus is governed by persisting antigen

The basis of antiviral protection by memory cytotoxic T lymphocytes (CTL) was investigated in vivo and in vitro using lymphocytic choriomeningitis virus (LCMV) and recombinant vaccinia viruses expressing the LCMV-glycoprotein (vacc-GP) or -nucleoprotein (vacc-NP). The widely replicating LCMV with a tendency to persist induced solid long-term protective memory. The poorly replicating vaccinia recombinant viruses revealed in the vaccinated host that the antiviral capacity of the secondary immune T cell response and the protection against lethal LCM was dependent upon the immunizing antigen and its dose. Protection against lethal choriomeningitis is less sensitive to assess memory because it depends upon high levels of CTL precursors (p) and/or on an activated state of memory CTL. In contrast, antiviral protection measured as the capacity of the primed host to reduce virus titers after challenge infection correlated with elevated CTLp frequencies after immunization with live LCMV or recombinant vaccinia virus-expressing the major LCMV epitope. CTLp frequencies were constantly increased up to 70 d for LCMV immune mice, but rapidly decreased a few weeks after immunization with low dose vaccinia recombinant virus. For example, mice primed with 2 x 10(6) plaque-forming units (PFU) of vacc-NP, or 2 x 10(2) PFU, or 2 x 10(6) PFU of vacc-GP were antivirally protected on day 7 but not after day 30 when CTLp could not be measured any longer in vitro. However, greater priming doses of vacc-NP (10(4) or 2 x 10(6) PFU) as well as LCMV (2 x 10(2) PFU) induced elevated levels of CTLp and antiviral protection for 60 d or longer. Adoptive transfer experiments of immune spleen cells into syngeneic recipients without addition of antigen demonstrated that maintenance of the antiviral protective capacity of the transferred cells depended on the presence of viral antigen. Thus, antiviral protection by memory CTL may be rather short-lived since it is based on activated T cells continuously stimulated by persisting antigen. This is best achieved by high immunizing antigen doses yielded either by widely replicating viruses or high doses of poorly replicating recombinant vaccines.     

HLA B37 determines an influenza A virus nucleoprotein epitope recognized by cytotoxic T lymphocytes

Human influenza A virus-specific, cytotoxic T cells have been shown previously to recognize the virus nucleoprotein on infected cells. CTL preparations from four HLA B37-positive donors were shown to recognize a synthetic peptide that corresponded to amino acids 335-349 of the nucleoprotein sequence. Influenza-specific CTL from 10 donors of other HLA types failed to recognize this epitope. CD8+ CTL lines were derived from lymphocytes of two HLA B37-positive donors and used to show that the peptide was represented on virus-infected cells and to determine the probable boundaries of the epitope.     

The induction of virus-specific cytotoxic T lymphocytes with solubilized viral and membrane proteins

Reconstituted membranes were prepared from detergent solubilized P815 (H-2d) tumor cell membranes and solubilized Sendai virus protein. These reconstituted membranes stimulated a virus-specific H-2-restricted secondary CTL response. Stimulating activity was dependent upon the presence of both viral and P815 protein in the same membrane and was restricted to the H-2 specificity present in the reconstituted membrane. Liposomes prepared from solubilized Sendai virus proteins and partially purified H-2 alloantigen also had activity for CTL induction. The results demonstrate the feasibility of using detergent solubilized membrane proteins to study antigen recognition by virus-specific, H-2 restricted cytolytic T lymphocytes.     

Synthetic phospholipid vesicles containing a purified viral antigen and cell membrane proteins stimulate the development of cytotoxic T lymphocytes

Synthetic phospholipid vesicles (liposomes) containing the purified glycoprotein (G) of vesicular stomatitis virus (VSV) and solubilized membrane proteins from cells of the appropriate H-2 haplotype elicited H-2-restricted cytotoxic T lymphocytes (CTL) that lysed VSV-infected target cells. The CTL were elicited by intact liposomes, not by released components. Thus, when spleen cells from VSV-primed H-2d X H-2b hybrid mice were stimulated with liposomes having G protein + membrane proteins from cells with one of the parental H-2 haplotypes, the resulting CTL lysed only VSV-infected target cells with that parent's H-2 type. This result argues against the view that T cells in general recognize only processed antigenic fragments on macrophages. Moreover, liposomes were only effective when G protein and cell membrane proteins were included in the same vesicles. This result suggests that for effective interaction with CTL precursors the antigen (G protein) and products of the H-2 complex must be closer to each other than 600--1,000 angstrom, the diameter of the lipid vesicles used in this study.     

T cell receptor variable gene usage in a specific cytotoxic T cell response. Primary structure of the antigen-MHC receptor of four hapten-specific cytotoxic T cell clones

The primary structure of the alpha and beta chains of the T cell antigen receptor in four cytotoxic T cell clones specific for N-iodoacetyl-sulfonic-naphthyl-ethylene-diamine (AED)-haptenated target cells displaying a particular class I MHC molecule has been determined. Two of the T cell clones, 8/10-2 and 5/10-20K, recognize AED-modified targets in association with H-2Kb, while the other two clones 5/10-20D and C9 react with AED-modified cells in the context of H-2Db. Comparison of the nucleotide sequences of both the alpha and beta chain cDNAs and their deduced protein sequences indicates that a specific variable gene segment was not used to recognize the hapten and/or class I gene products. Furthermore, there does not appear to be any conserved amino acid residues used in the AED-specific response other than the framework amino acids. However, when the two clones 8/10-2 and 5/10-20D were compared, a striking similarity was seen in the J segments. These two clones that recognize AED in the context of different MHC epitopes used identical J alpha (J alpha 810) and J beta (J beta 2.6) gene segments. C9, specific for AED-Db, shared identical V beta (V beta 6) and J beta gene segments (J beta 1.1) as those of a cytotoxic T cell that recognizes allogeneic targets expressing Db. These data indicate that a simple rule governing the usage of the variable regions of either the alpha or beta T cell receptor (TcR) genes in the recognition of antigen and MHC gene products cannot be formulated. However, subtle similarities can be detected in some situations between the primary structures of the TcR and the targets they recognize.     

Reduction of otherwise remarkably stable virus-specific cytotoxic T lymphocyte memory by heterologous viral infections

Experimental analyses of the acute cytotoxic T lymphocyte (CTL) response to viruses have focused on studying these infections in immunologically naive hosts. In the natural environment, however, viral CTL responses occur in hosts that are already immune to other infectious agents. To address which factors contribute to the maintenance and waning of immunological memory, the following study examined the frequencies of virus-specific CTL precursor cells (pCTL) not only using the usual experimental paradigm where mice undergo acute infections with a single virus, and in mice immune to a single virus, but also in immune mice after challenge with various heterologous viruses. As determined by limiting dilution assays, the pCTL frequency (p/f) per CD8+ T cell specific for lymphocytic choriomeningitis virus (LCMV), Pichinde virus (PV), or vaccinia virus (VV) increased during the acute infections, peaking at days 7-8 with frequencies as high as 1/27-1/74. Acute viral infections such as these elicit major expansions in the CD8+ T cell number, which has been reported to undergo apoptosis and decline after most of the viral antigen has been cleared. Although the decline in the total number of virus-specific pCTL after their peak in the acute infection was substantial, for all three viruses the virus- specific p/f per CD8+ T cell decreased only two- to fourfold and remained at these high levels with little fluctuation for well over a year. The ratios of the three immunodominant peptide-specific to total LCMV-specific clones remained unchanged between days 7 and 8 of acute infection and long-term memory, suggesting that the apoptotic events did not discriminate on the basis of T cell receptor specificity, but instead nonspecifically eliminated a large proportion of the activated T cells. However, when one to five heterologous viruses (LCMV, PV, VV, murine cytomegalovirus, and vesicular stomatitis virus) were sequentially introduced into this otherwise stable memory pool, the stability of the memory pool was disrupted. With each successive infection, after the immune system had returned to homeostasis, the memory p/f specific to viruses from earlier infections declined. Reductions in memory p/f were observed in all tested immunological compartments (spleen, peripheral blood, lymph nodes, and peritoneal cavity), and on average in the spleen revealed a 3 +/- 0.4-fold decrease in p/f after one additional viral infection and an 8.4 +/- 3- fold decrease after two additional viral infections. Thus, subsequent challenges with heterologous antigens, which themselves induce memory CTL, may contribute to the waning of CTL memory pool to earlier viruses as the immune system accommodates ever-increasing numbers of new memory cells within a limited lymphoid population. This demonstrates that virus infections do not occur in immunological isolation, and that CD8+ T cell responses are continually being modulated by other infectious agents.     

The role of L3T4 in recognition of Ia by a cytotoxic, H-2Dd-specific T cell hybridoma

The expression of T4/T8 surface markers on human T cells and of L3T4/Lyt-2 on murine T cells has lead to the association of these surface markers with recognition of either class II or class I major histocompatibility complex (MHC) antigens. It has been suggested that these T cell surface antigens interact with MHC antigens. We have examined the role of L3T4 in the recognition of Dd by the T cell hybridoma, 3DT52.5. This T cell hybridoma was found to be specific for the N/Cl domain of Dd. The recognition of a class I antigen by an Lyt-2-, L3T4+ T cell hybridoma allowed the separate evaluation of interactions between L3T4/Ia and the T cell antigen receptor, Dd. Recognition by this hybridoma resulted in the production of interleukin 2 (IL-2) and cytolytic activity. Antibody blocking experiments have demonstrated that L3T4 was involved in triggering the effector function of 3DT52.5 only on Ia+ stimulator or target cells. We have demonstrated that an L3T4+, Dd-specific T cell hybridoma, 3DT52.5, uses the L3T4 molecule to directly interact with nonpolymorphic Ia determinants.     

An immunodominant epitope present in multiple class I MHC molecules and recognized by cytotoxic T lymphocytes

CTL derived from (C3H x B6.K1)F1 animals were sensitized against L cells that express the transfected gene product Q10d/Ld. These CTL were highly crossreactive against three other class I molecules, H-2Kbm1, H-2Ld, and H-2Kd. In an attempt to define this crossreactive epitope it was noted that between 25 and 39% of amino acids in the alpha helices and central beta strands of these three molecules vary from Q10d. These amino acids represent residues that have been proposed to potentially interact with a peptide antigen or TCR (21). However, all four molecules share the amino acid tyrosine at positions 155 and 156. Additionally, Q10d, H-2Kbm1, and H-2Ld share alanine at position 152, while H-2Kd has an aspartic acid. We showed that these residues were important in controlling this epitope by the finding that anti-Q10d CTL did not recognize H-2Kbm1 revertant molecules that had either the position 152 alanine changed back to the wild-type H-2Kb residue (glutamic acid) or position 155 and 156 tyrosines changed back to wild-type residues arginine and leucine. Further evidence that these molecules share a crossreactive epitope was noted by the failure of (C3H x H-2Kbm1)F1 animals to generate CTL that recognized H-2Ld or H-2Kd, and the inability of (C3H x BALB/c)F1 animals to generate CTL reactive against H-2Kbm1. CTL from these mice were still able to recognize Q10d/Ld indicating that other epitopes could be detected if natural tolerance prevented recognition of the crossreactive epitope. To further define the epitope, CTL clones were generated against Q10d/Ld and maintained on either H-2Kbm1 or BALB/c feeder cells. In addition to testing these clones on the target cells described above, mutant molecules derived from H-2Ld, which have amino acid substitutions in their alpha 1 domain, were analyzed. It was noted that some anti-Q10 clones that did not crossreact on H-2Ld did react against H-2Ld mutant antigens that had H-2Dd amino acid substitutions in the alpha 1 domain at positions 63, 65, 66, and 70. Other clones had differential reactivities on these H-2Ld mutants further substantiating that alpha 1 domain amino acids play a role in controlling the expression of the crossreactive epitope. Thus, four class I molecules with multiple amino acid differences in their alpha 1 and alpha 2 domains share a crossreactive epitope readily recognized by alloreactive CTL.(ABSTRACT TRUNCATED AT 250 WORDS)     

An alloresponse in humans is dominated by cytotoxic T lymphocytes (CTL) cross-reactive with a single Epstein-Barr virus CTL epitope: implications for graft-versus-host disease

The phenomenon of T cell allorecognition is difficult to accommodate within the framework of a T cell repertoire positively selected in the thymus, unless allorecognition results from the cross-reactions of self- major histocompatibility complex restricted T cells. Herein, we demonstrate the dual specificity of cytotoxic T lymphocyte (CTL) clones for the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, presented on HLA-B8, and the alloantigen HLA-B*4402. CTL which recognized peptide FLRGRAYGL in association with HLA-B8 could be reactivated in vitro from healthy individuals who had been exposed previously to EBV, using stimulator cells expressing the cross-reacting alloantigen HLA-B*4402. Limiting dilution analysis of the alloresponse to HLA-B*4402 in eight healthy individuals revealed that HLA-B8+, EBV- sero+ donors had higher CTL precursor frequencies for alloantigen HLA- B*4402 than EBV-sero- control donors. It is surprising that the majority (65-100%) of anti-HLA-B*4402 CTL, generated in limiting dilution mixed lymphocyte reactions between responder cells from HLA- B8+, EBV-sero+ individuals and HLA-B*4402+ stimulators, also recognized the EBV CTL epitope FLRGRAYGL/HLA-B8. In contrast to previous studies showing extensive diversity in the T cell repertoire against individual alloantigens, these data demonstrate that the response to an alloantigen can be dominated by CTL cross-reactive with a single viral epitope, thus illustrating a possible mechanism for the frequent clinical association between herpesvirus exposure and graft-versus-host disease after bone marrow transplants.     

HLA-A Bw4表位与HIV-1病毒载量和CD+4T淋巴细胞计数关系的研究

目的 研究含Bw4表位的HLA-A抗原在HIV-1感染者疾病进展中的作用.方法 应用高分辨率人类白细胞抗原(HLA)分型方法对340例HIV-1感染者进行HLA-A和HLA-B位点的等位基因分型,确定Bw4表位的携带情况,分析HLA-A和HLA-B携带Bw4表位与感染者CD4+4T淋巴细胞计数和血浆病毒载量的关系.结果 与不携带Bw4者(0Bw4)(CD+4T淋巴细胞计数:中位数294个/μ1,病毒载量:中位数6.29×104拷贝/m1)相比,1Bw4-A基因型者和2Bw4-AA基因型者的CD+4T淋巴细胞计数水平(分别为307和308个/μ1)基本相当,而病毒载量(分别为1.53×105和2.68×105拷贝/ml)有增高趋势.与不携带Bw4表位的感染者相比,仅在HLA-B抗原中含Bw4表位的HIV-1感染者的CD+4T淋巴细胞计数(417:个/μ1)显著增高(P=0.013),而病毒载量(2.10×104拷贝/ml)显著降低(P=0.007).结论 含有Bw4表位的HLA-B抗原在HIV-1感染中具有延缓疾病进展的作用,而含有Bw4表位的HLA-A抗原对HIV-1感染者未见保护性作用.     

Negative regulation of T cell receptor-lipid raft interaction by cytotoxic T lymphocyte-associated antigen 4

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an essential negative regulator of T cell activation. Recent evidence suggests that CTLA-4 association with the immunological synapse during contact with antigen-presenting cells is important for its inhibitory function. In the present study, we observed a direct interaction of CTLA-4 with the phosphorylated form of T cell receptor (TCR)zeta within the glycolipid-enriched microdomains associated with the T cell signaling complex. In this setting, CTLA-4 regulated the accumulation/retention of TCRzeta in the signaling complex, as the lipid raft fractions from CTLA-4KO T cells contained significantly higher amounts of the TCR components when compared with wild-type littermates. In contrast, coligation of CTLA-4 with the TCR during T cell activation selectively decreased the amount of TCRzeta that accumulated in the rafts. These results suggest that CTLA-4 functions to regulate T cell signaling by controlling TCR accumulation and/or retention within this a critical component of the immunological synapse.     

Haplotype-specific suppression of cytotoxic T cell induction by antigen inappropriately presented on T cells

To detect a strong cytotoxic T lymphocyte (CTL) response to minor histocompatibility (H) antigens in a 5-d mixed lymphocyte culture, it is necessary to use a responder that has been primed in vivo with antigen-bearing cells. It has previously been shown that minor-H- specific CTL can be primed in vivo both directly by foreign spleen cells and by presentation of foreign minor H antigens on host antigen- presenting cells. This latter route is evident in the phenomenon of cross-priming, in which H-2 heterozygous (A x B)F1 mice injected 2 wk previously with minor H-different H-2A (A') spleen cells generate both H-2A- and H-2B-restricted minor-H-specific CTL. In a study of the kinetics of direct- vs. cross-priming to minors in F1 mice, we have found that minor H-different T cells actually suppress the induction of virgin CTL capable of recognizing them. CTL activity measured from F1 mice 3-6 d after injection with viable A' spleen cells is largely H-2B restricted. The H-2A-restricted response recovers such that roughly equal A- and B-restricted activity is detected in mice as early as 8-10 d postinjection. This temporary hyporeactivity does not result from generalized immunosuppression--it is specific for those CTL that recognize the foreign minor H antigen in the context of the H-2 antigens on the injected spleen cells. The injected spleen cells that mediate this suppression are radiosensitive T cells; Lyt-2+ T cells are highly efficient at suppressing the induction of CTL in vivo. No graft vs. host reaction by the injected T cells appears to be required, as suppression of direct primed CTL can be mediated by spleen cells that are wholly tolerant of both host H-2 and minor H antigens. Suppression cannot be demonstrated by in vitro mixing experiments. Several possible mechanisms for haplotype-specific suppression are discussed, including inactivation of responding CTL by veto cells and in vivo sequestration of responding CTL by the injected spleen cells.