Specificity of cytotoxicity T cells directed to influenza virus hemagglutinin.

Purified type A influenza viral hemagglutinin stimulates an in vitro cell-mediated cytotoxic cell response that exhibits a high degree of specificity for the immunizing hemagglutinin. The response magnitude is proportional to the hemagglutinin dose used for stimulation. The lytic activity of the effector cells is H-2 restricted. Analysis of the specificity of the response indicated that these cytotoxic T cells readily distinguish target cells expressing serologically unrelated hemagglutinin from target cells bearing hemagglutinins serologically related to the stimulating hemagglutinin. Further analysis of the fine specificity of cytotoxic T-cell recognition with serologically cross-reactive type A influenza hemagglutinins revealed a hierarchy of cross-reactivity among these hemagglutinins that was the converse of the serologic hierarchy. These results are discussed in terms of possible differences and similarities in the specificity repertoire of cytotoxic T cells and antibodies. Possible implications of these findings from the standpoint of cytotoxic T-cell induction are also discussed.

Immunologic recognition of influenza virus-infected cells. II. Expression of influenza A matrix protein on the infected cell surface and its role in recognition by cross-reactive cytotoxic T cells.

Two distinct subpopulations of cytotoxic T cells are generated in the primary or secondary response of mice to type A influenza viruses. One subpopulation is specific for the immunizing virus strain. The other subpopulation shows a high degree of cross-reactivity for heterologous type A virus of a different subtype. This report examines the possibility that distinct influenza virus antigens, expressed on the surface of the infected cell, are recognized by the different subpopulations of influenza-specific cytotoxic T cells. Data are presented which demonstrate that influenza A matrix protein, an internal virion antigen, is detectable on the surface of target cells infected with influenza A viruses of different subtypes. Since this viral antigen is type specific, i.e., serologically cross-reactive among all type A influenza viruses, it could serve as the target for cross-reactive cytotoxic T cells. To further examine the specificity of the two cytotoxic T-cell subpopulations, experiments were carried out by using the inhibitor of glycoprotein synthesis - 2-Deoxy-D-Glucose 2-DG. These experiments examine first the effect of 2-DG on the expression of influenza matrix protein and viral glycoprotein on the infected cell surface and second, the susceptibility of 2-DG-treated target cells to lysis by cytotoxic T cells. 2-DG inhibits the expression of the viral hemagglutinin glycoprotein on the cell surface but does not inhibit the expression of the nonglycosylated matrix protein. Furthermore, inhibition of glycoprotein synthesis in infected target cells abrogates the reactivity of infected target cells to lysis by virus strain-specific but not cross- reactive cytotoxic T cells. These findings suggest that the influenza glycoproteins (hemagglutinin and/or neuraminidase) and the nonglycosylated matrix protein are the targets for the virus strain- specific and cross-reactive cytotoxic T cells, respectively. These results are discussed in the light of available information on influenza virus structure and the biology of influenza infection and in terms of current models for cytotoxic T-cell recognition of virus-infected cells.

Virus-specific CD8+ T lymphocytes downregulate T helper cell type 2 cytokine secretion and pulmonary eosinophilia during experimental murine respiratory syncytial virus infection.

T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8+ T cytolytic T cells on the differentiation and activation of functionally distinct CD4+ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I-restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8+ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8+ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8+ T cells in this process was further supported by the results in CD8+ T cell deficient, beta 2 microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8+ T cell compartment. These results indicate that CD8+ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4+ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.

Incomplete CD8(+) T lymphocyte differentiation as a mechanism for subdominant cytotoxic T lymphocyte responses to a viral antigen.

CD8(+) cytotoxic T lymphocytes (CTLs) recognize antigen in the context of major histocompatibility complex (MHC) class I molecules. Class I epitopes have been classified as dominant or subdominant depending on the magnitude of the CTL response to the epitope. In this report, we have examined the in vitro memory CTL response of H-2(d) haplotype murine CD8(+) T lymphocytes specific for a dominant and subdominant epitope of influenza hemagglutinin using activation marker expression and staining with soluble tetrameric MHC-peptide complexes. Immune CD8(+) T lymphocytes specific for the dominant HA204-210 epitope give rise to CTL effectors that display activation markers, stain with the HA204 tetramer, and exhibit effector functions (i.e., cytolytic activity and cytokine synthesis). In contrast, stimulation of memory CD8(+) T lymphocytes directed to the subdominant HA210-219 epitope results in the generation of a large population of activated CD8(+) T cells that exhibit weak cytolytic activity and fail to stain with the HA210 tetramer. After additional rounds of restimulation with antigen, the HA210-219-specific subdominant CD8(+) T lymphocytes give rise to daughter cells that acquire antigen-specific CTL effector activity and transition from a HA210 tetramer-negative to a tetramer-positive phenotype. These results suggest a novel mechanism to account for weak CD8(+) CTL responses to subdominant epitopes at the level of CD8(+) T lymphocyte differentiation into effector CTL. The implications of these findings for CD8(+) T lymphocyte activation are discussed.

Role of viral infectivity in the induction of influenza virus-specific cytotoxic T cells.

This report examines the requirement for infectious virus in the induction of influenza virus-specific cytotoxic T cells. Infectious influenza virus was found to be highly efficient at generating both primary and secondary cytotoxic T-cell response in vivo. Inactivated influenza virus however, failed to stimulate a detectable cytotoxic T-cell response in vivo even at immunizing doses 10(5)-10(6)-fold higher than the minimum stimulatory dose of infectious virus. Likewise inactivated virus failed to sensitize target cells for T cell-mediated lysis in vitro but could stimulate a specific cytotoxic response from primed cells in vitro. Possible requirements for the induction of virus-specific cytotoxic T-cell responses are discussed in light of these observations and those of other investigators.

Resistance to and recovery from lethal influenza virus infection in B lymphocyte-deficient mice.

In the adaptive immune response to most viruses, both the cellular and humoral arms of the immune system play complementary roles in eliminating virus and virus-infected cells and in promoting recovery. To evaluate the relative contribution of CD4+ and CD8+ effector T lymphocytes in virus clearance and recovery, we have examined the host response to lethal type A influenza virus infection in B lymphocyte-deficient mice with a targeted disruption in the immunoglobulin mu heavy chain. Our results indicate that naive B cell-deficient mice have a 50- 100-fold greater susceptibility to lethal type A influenza virus infection than do wild type mice. However, after priming with sublethal doses of influenza, immune B cell-deficient animals show an enhanced resistance to lethal virus infection. This finding indicates that an antibody-independent immune-mediated antiviral mechanism accounts for the increased resistance to lethal virus challenge. To assess the contribution of influenza-specific CD4+ and CD8+ effector T cells in this process, defined clonal populations of influenza-specific CD4+ and CD8+ effector T cells were adoptively transferred into lethally infected B cell-deficient mice. Cloned CD8+ effectors efficiently promoted recovery from lethal infection, whereas cloned CD4+ T cells conferred only partial protection. These results suggest that memory T lymphocytes can act independently of a humoral immune response in order to confer resistance to influenza infection in immune individuals. The potential implications of these results for vaccination against human influenza infection are discussed.

Recognition of an immunoglobulin VH epitope by influenza virus-specific class I major histocompatibility complex-restricted cytolytic T lymphocytes.

There are two immunogenic sites on the type A influenza A/Japan/57 (H2N2) hemagglutinin (HA) that can be recognized by class I major histocompatibility complex (MHC), H-2Kd-restricted cytolytic T lymphocytes (CTLs). One of these sites encompasses two distinct partially overlapping epitopes, which span HA residues 204-212 and 210-219. During the analysis of the fine specificity of CTL clones directed to the HA 210-219 epitope, we found that one clone 40-2 also recognized the myeloma cell line P3x63-Ag8. P3x63-Ag8 is derived from the MOPC 21 myeloma and expresses an immunoglobulin (Ig) heavy chain variable region (VH) gene which is a member of the murine 7183 VH gene family. Recognition was specific for the endogenously processed MOPC 21 heavy chain in association with the Kd molecules, since the SP2/0 derivative of P3x63-Ag8, which does not make a functional Ig H chain, is not recognized. The VH epitope recognized by clone 40-2 could be mapped to a 10 amino acid peptide spanning MOPC 21 VH residues 49-58. Cross-reactivity for the VH gene product was also demonstrable in some heterogeneous populations of CTL generated in response to influenza virus infection. These results represent the first demonstration of cross-reactivity for an endogenously processed product of a self-Ig by the CTL directed to a foreign antigen and raise the possibility that the Ig VH expression may regulate the CD8+ T cell response to foreign antigens.

Nonconventional (TL-encoded) major histocompatibility complex molecules present processed viral antigen to cytotoxic T lymphocytes.

A large number of class I-like genes are located distal to the K and D regions of the murine major histocompatibility complex (MHC) within the Q and TL region. The function of the molecules encoded within this region is obscure since unlike conventional MHC gene products, these molecules have not been reported to present processed environmental antigens to T cells. In the present report, we demonstrate that a peptide corresponding to processed influenza virus hemagglutinin can be recognized by CD8+ T cell receptor alpha/beta-positive cytotoxic T lymphocytes (CTL) in association with a MHC class I-like product encoded within the TL region. Thus, nonconventional class I MHC molecules can bind and present processed environmental antigens, and TCR-alpha/beta CTL directed to such peptide MHC complexes are represented in the mature T cell pool. Our data imply that Q/TL region products may be charged by peptides generated through an antigen processing and presentation pathway distinct from the pathway used by conventional MHC molecules and not normally available to environmental antigens.

Presentation of viral antigen to class I major histocompatibility complex-restricted cytotoxic T lymphocyte. Recognition of an immunodominant influenza hemagglutinin site by cytotoxic T lymphocyte is independent of the position of the site in the hemagglutinin translation product.

Class I major histocompatibility complex (MHC) restricted T lymphocytes preferentially recognize fragments of polypeptides processed through a nonendosomal presentation pathway. At present the intracellular compartment(s) in which polypeptide fragmentation occurs and factors which influence the formation of an antigenic epitope are not well understood. To assess the role of residues flanking an antigenic site in the generation of the antigenic moiety recognized by class I MHC restricted T lymphocytes we have moved the coding sequence for an immunodominant H-2Kd restricted site on the influenza A/JAPAN/57 hemagglutinin (residues 202-221) by site-directed mutagenesis to six different positions along the coding sequence of the hemagglutinin gene. We have found that all six classes of mutants are recognized by MHC class I restricted T cells as efficiently as the wild type hemagglutinin gene product. Thus neither N-terminal to C-terminal position within the translation product nor sequences flanking the antigenic site influence processing.

Class I major histocompatibility complex-restricted T lymphocyte recognition of the influenza hemagglutinin. Overlap between class I cytotoxic T lymphocytes and antibody sites.

The influenza hemagglutinin is a critical regulator of disease expression during influenza virus infection and serves as a major target for the host immune response to this pathogen. In this report, we have analyzed an immunodominant site on the hemagglutinin (residues 202-221) recognized by murine class I MHC-restricted T lymphocytes. This analysis has revealed evidence for the duplication of a T cell recognition site within the region 202-221. We have also identified critical amino acids necessary for class I-restricted T cell recognition within these two epitopes. In addition, we provide evidence that a site on the influenza hemagglutinin recognized by neutralizing antibody directly overlaps with an epitope recognized by class I MHC-restricted CTL.

Regulation of T lymphocyte proliferation. Interleukin 2-mediated induction of c-myb gene expression is dependent on T lymphocyte activation state.

We previously reported that with time, after antigenic stimulation of antigen-regulated murine T lymphocyte clones, total IL-2-R expression decayed 10-50-fold, commensurate with a decline in the ability of the cells to proliferate to IL-2. However, late after antigenic stimulation, when the cells were refractory to the IL-2-proliferative stimulus, high levels of high affinity IL-2-R remained. In this report we further explore the basis of unresponsiveness to IL-2 in the quiescent clones. We show that the proto-oncogene c-myc is induced in the late cell population by IL-2 to comparable levels observed early after antigen stimulation. IL-2-dependent c-myb induction, however, is seen only early after activation but not in the late-activated population. Analysis of the IL-2-dependent expression of c-myb mRNA with time after antigenic stimulation showed that steadystate c-myb expression declines dramatically with kinetics closely paralleling a decay in IL-2-dependent proliferative ability. In contrast, steadystate c-myc expression remains high throughout this period. Expression of c-myb is critical for proliferation of these cells since antisense oligodeoxy-nucleotide to c-myb can inhibit their IL-2-dependent proliferation. We present evidence for a pathway of c-myb induction via the TCR that is independent of the IL-2/IL-2-R interaction. In addition, the inhibition of IL-2-R-induced c-myb expression by 2-aminopurine and enhanced induction of c-myb via the TCR demonstrate that TCR activation and IL-2-R activation lead to induction of c-myb by different mechanisms.

Inducible expression of insulin receptors on T lymphocyte clones.

This study demonstrates that the insulin receptors are expressed on the surface of some T cell clones after specific antigenic stimulation. The insulin receptors on these lymphocytes are physicochemically similar to insulin receptors present on cells which express the receptors constitutively (adipocytes, hepatocytes, etc.). The kinetics of expression of insulin receptors on cloned, noncytotoxic T cells after specific antigenic stimulation closely parallels that of [3H]thymidine incorporation in such cultures.

Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection.

T lymphocytes play a primary role in recovery from viral infections and in antiviral immunity. Although viral-specific CD8+ and CD4+ T cells have been shown to be able to lyse virally infected targets in vitro and promote recovery from lethal infection in vivo, the role of CD4+ T lymphocytes and their mechanism(s) of action in viral immunity are not well understood. The ability to further dissect the role that CD4+ T cells play in the immune response to a number of pathogens has been greatly enhanced by evidence for more extensive heterogeneity among the CD4+ T lymphocytes. To further examine the role of CD4+ T cells in the immune response to influenza infection, we have generated influenza virus-specific CD4+ T cell clones from influenza-primed BALB/c mice with differential cytokine secretion profiles that are defined as T helper type 1 (Th1) clones by the production of interleukin 2 (IL-2) and interferon gamma (IFN-gamma), or as Th2 clones by the production of IL-4, IL-5, and IL-10. Our studies have revealed that Th1 clones are cytolytic in vitro and protective against lethal challenge with virus in vivo, whereas Th2 clones are noncytolytic and not protective. Upon further evaluation of these clonal populations we have shown that not only are the Th2 clones nonprotective, but that pulmonary pathology is exacerbated as compared with control mice as evidenced by delayed viral clearance and massive pulmonary eosinophilia. These data suggest that virus-specific CD4+ T cells of the Th2 subset may not play a primary role in virus clearance and recovery and may lead to immune mediated potentiation of injury.

In vivo effector function of influenza virus-specific cytotoxic T lymphocyte clones is highly specific.

Cloned lines of murine cytotoxic T lymphocytes (CTL) directed to type A influenza virus confer complete protection upon adoptive transfer to syngeneic mice lethally infected by influenza virus. The exquisite specificity exhibited by a subtype-specific cloned CTL in culture is reflected in its capacity to eliminate pulmonary virus and mediate recovery only in those mice infected by the virus subtype recognized by this cloned line in vitro. A cross-reactive CTL cloned line protects mice infected by either of two influenza virus subtypes. In mice dually infected with two virus subtypes, the subtype-specific CTL clone only reduces lung virus levels of the recognized virus subtype and cannot prevent these mice from dying. In contrast, adoptive transfer of the cross-reactive CTL clone into mice simultaneously infected with two virus subtypes results in reduction of pulmonary titers of both subtypes and promotes complete recovery. These results directly implicate CTL as an important antiviral defense mechanism in experimental influenza infection. In addition, these results indicate that both the induction and expression of antiviral effector activity by CTL in vivo is highly specific and therefore favor the concept that CTL express their antiviral effect in vivo by direct cytolysis of infected cells.

Heterogeneity and specificity of cloned lines of influenza-virus specific cytotoxic T lymphocytes.

Continuous lines of murine cytotoxic T lymphocytes (CTL) directed to type A influenza viruses have been generated in vitro by stimulation of individual CTL precursors in the presence of T cell-growth factor TCGF and syngeneic virus-infected stimulator cells. The cloned CTL lines are H-2 restricted in their target cell recognition and exhibit distinct patterns of influenza virus recognition. All CTL lines appear to be restricted in target cell recognition to either the H-2K or the H-2D end of the appropriate H-2 haplotype. Likewise, CTL lines of F1 origin are restricted in recognition exclusively to one of the parental haplotypes. All CTL lines examined express the Thy-1.2 and the Lyt-2-surface antigen markers. 4 of 11 cytotoxic lines examined also expressed detectable levels of the Lyt-1- surface antigen. These findings confirm at the clonal level previous observations on the H-2K/D restriction of virus-specific CTL and also demonstrate heterogeneity among H-2 restricted CTL both from the standpoint of viral antigen recognition and cell surface phenotype.

Simultaneous expression of H-2-restricted and alloreactive recognition by a cloned line of influenza virus-specific cytotoxic T lymphocytes.

Based on theoretical considerations and several types of experimental evidence with heterogeneous cell populations it has been proposed that alloreactive T cells and major histocompatibility complex (MHC)-restricted T cells directed to foreign non-NHC antigens represent overlapping subsets. In this report we provide direct evidence for this hypothesis at the clonal level. We have isolated a cloned continuous influenza virus-specific cytotoxic T cell (CTL) line derived from a single (H-2b X H-2d)F1 CTL precursor which simultaneously exhibits H-2-restricted cytotoxicity of influenza A/Japan/305/57 virus in association with H-2Kd and alloreactive cytotoxicity for H-2Kk alloantigen. Cold target inhibition data demonstrate that both MHC-restricted and alloreactive recognition is mediated by the same cell population. In addition to cross-reactivity at the target cell level, we shown that this cloned CTL line can be specifically stimulated to proliferate either by A/Japan/305/57 virus-infected cells expressing H-2Kd or by uninfected cells of the H-2Kk haplotype.

The requirement for proteasome activity class I major histocompatibility complex antigen presentation is dictated by the length of preprocessed antigen.

Accumulating evidence has implicated the proteasome in the processing of protein along the major histocompatibility complex (MHC) class I presentation pathway. The availability of potent proteasome inhibitors provides an opportunity to examine the role of proteasome function in antigen presentation by MHC class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). We have investigated the processing and presenting of antigenic epitopes from influenza hemagglutinin in target cells treated with the inhibitor of proteasome activity MG132. In the absence of proteasome activity, the processing and presentation of the full-length hemagglutinin was abolished, suggesting the requirement for proteasome function in the processing and presentation of the hemagglutinin glycoprotein. Epitope-containing translation products as short as 21 amino acids when expressed in target cells required proteasome activity for processing and presentation of the hemagglutin epitope to CTLs. However, when endogenous peptides of 17 amino acids or shorter were expressed in target cells, the processing and presentation of epitopes contained in these peptides were insensitive to the proteasome inhibitor. Our results support the hypothesis that proteasome activity is required for the generation of peptides presented by MHC class I molecules and that the requirement for proteasome activity is dependent on the size of the translation product expressed in the target cell. The implications of these findings are discussed.

Differences in antigen presentation to MHC class I-and class II-restricted influenza virus-specific cytolytic T lymphocyte clones.

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA-specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II-restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets.

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.

Sendai virus-specific, H-2-restricted cytotoxic T lymphocyte responses of nude mice grafted with allogeneic or semi-allogeneic thymus glands.

The in vitro secondary cytotoxic T lymphocyte (CTL) response to Sendai virus-treated stimulator cells by primed spleen cells from thymus gland-grafted nude mice was examined. BALB/c (H-2d) nude mice grafted with allogeneic C57BL/10 (H-2b) thymus glands developed CTL responses directed exclusively to Sendai virus-infected H-2d target cells. (C57BL/6 X BALB/c)F1 nude mice grafted with thymus glands of either parent developed CTL responses preferentially against infected target cells expressing the MHC antigens present in the parental thymus graft, but also had detectable activity for infected target cells of the parental haplotype not expressed in the thymus. These results provide evidence against the concept that self recognition by MHC-restricted CTL is directed exclusively by the MCH type of the thymus.