TAP1-deficient mice select a CD8+ T cell repertoire that displays both diversity and peptide specificity

Mice deficient in the gene encoding the transporter associated with antigen processing 1 (TAP1) are defective in providing major histocompatibility complex (MHC) class I molecules with cytosolic peptides. Consequently, these mice express reduced levels of MHC class I glycoproteins on the cell surface, and have reduced numbers of CD8⁺ T cells in the periphery. In the present study, we have addressed the diversity and specificity of the peripheral CD8⁺ T cell population in TAP1 -/- mice. CD8⁺ T cells were polyclonal with regard to T cell receptor (TCR) Vβ expression. Overall, Vβ usage in TAP1 -/- mice appeared to be very similar to that in wild-type mice, with significantly reduced levels of Vβ5.1/5.2-expressing CD8⁺ T cells as the only clear exception. This polyclonal population of CD8⁺ T cells readily mounted epitope-specific CTL responses against four out of five well-defined MHC class I-restricted peptides. In contrast to allospecific CTL, peptide-specific CTL from TAP1 -/- mice did not cross-react on cells expressing normal levels of H-2^b class I. The present results demonstrate that a polyclonal CD8⁺ T cell repertoire, displaying both diversity and peptide specificity, is positively selected in mice devoid of a functional peptide transporter. These observations imply that TAP-dependent peptides are not absolutely required for positive selection of a functionally diverse repertoire of CD8⁺ T cells.     

Antigen processing mutant T2 cells present viral antigen restricted through H-2K

Cytotoxic T lymphocytes (CTL) recognize foreign antigens as short peptides presented by class I molecules of the major histocompatibility complex (MHC). T2 cells are profoundly defective in the presentation of endogenously synthesized antigens to CTL due to a deletion of MHC class II-encoded genes for transporters associated with antigen presentation (TAP1/TAP2). Surprisingly, we here demonstrate that T2 cells, after infection with Sendai virus, are readily killed by H-2K^b restricted CD8⁺ T cells. In contrast to classical class I-mediated antigen presentation, the presentation of Sendai virus antigen inT2K^b cells is brefeldin A (BFA) insensitive. The present findings may suggest the presence of an alternative pathway for MHC class I-mediated antigen presentation in T2 cells.     

Clearance of Sendai virus by CD8+ T cells requires direct targeting to virus-infected epithelium

Minimal numbers of CD8⁺ T cells are found in bronchoalveolar lavage (BAL) populations recovered from Sendai virus-infected mice that are homozygous (?/?) for β2-microglobulin (β2-m) gene disruption. The prevalence of the CD8⁺ set was substantially increased in the pneumonic lungs of 8?12-week radiation chimeras made using substantially class I major histocompatibility complex (MHC) glycoprotein-negative β2-m (?/?) recipients and normal β2-m (+/+) bone marrow. Even so, the CD8⁺ (but not the CD4⁺) lymphocyte counts were still much lower than in the (+/+)→(+/+) controls. The (+/+)→(+/+) and (+/+)→(?/?) chimeras cleared Sendai virus and potent virus-immune CD8⁺ cytotoxic T lymphocytes (CTL) specific for H-2K^b + viral nucleoprotein peptide were found in the BAL from both groups. However, following in vivo depletion of the CD4⁺ population, only the (+/+)→(+/+) mice were able to deal with the infection. Similarly, adoptively transferred, H-2K^b-restricted CD8⁺ T cells from previously-primed (+/+) mice also failed to clear virus from the lungs of (+/+)→(?/?) chimeras infected within 2 weeks of reconstitution with bone marrow, though they were effective in the (+/+)→(+/+) controls. Sendai virus-immune CD8⁺ T cells are thus unable to eliminate virus-infected β2-m (?/?) lung epithelial cells that might be thought to be expressing very small amounts of either isolated class I heavy chain, or class I MHC glycoprotein that has bound β2-m derived from β2-m (+/+) T cells or macrophages present in the pneumonic lung. Furthermore, the CD8⁺ CTL that are being exposed to β2-m (+/+) stimulators in the BAL population cannot operate in some bystander mode to clear virus from respiratory epithelium.     

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2^b or H-2^d Tgβ-gal mice) expressed the mHAg like β-galactosidase (β-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tgβ-gal skin by a β-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous β-gal shed by keratinocytes. Indeed, our results showed that 1) H-2^b Tgβ-gal skin was rejected by H-2^bxs and H-2^bxd recipients; 2) rejection was mediated by β-gal-specific CD8⁺ CTL effectors; and 3) H-2^bxd mice having rejected H-2^b Tgβ-gal skin generated β-gal-specific CTL restricted by H-2^b and H-2^d class I molecules and rejected subsequently grafted H-2^d Tgβ-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous β-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.     

The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I,but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2a^b suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igh^a haplotype, when neonatally transferred into histocompatible Igh^a/b heterozygotes, are able to induce a specific and total suppression of the IgG2a^b allotype. Nevertheless, only transfer of IgG2a^b-primed Igh^a T splenocytes induces this suppression in Igh^b/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8⁺ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4⁺CD8⁺ T cell cooperation and operated via the recognition by the involved TCR of Cγ2a^b-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Igh^b mice genetically deficient for MHC class I (β2-microglobulin^%, or β2m^%) or class II (I-Aβ^%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2a^b T cells transferred into Igh^b H-2^b I-Aβ^% mice carry out the suppression process normally, while in Igh^b H-2^b β2m^% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2a^b 103–118 peptide, identified as the sole Cγ2a^b-derived peptide able to amplify the anti-IgG2a^b T cell reactivity in Igh^a H-2^b mice, is also able to stabilize the H-2D^b, but not the H-2K^b class I molecules at the surface of RMA-S (TAP2^?, H-2^b) cells. These results indicate that, despite the CD4⁺/CD8⁺ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2a^b+ B cells for suppression establishment.     

Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell-mediated rejection response declines with advanced age

The present investigation explored age-related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H-2^b and H-2^bxH-2^k mice were engrafted with major histocompatibility complex (MHC) class II-disparate bm12 skin, rejection of which requires CD4⁺ T cells, and MHC class I-disparate bm1 skin, rejection of which requires CD8⁺ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Ia^bm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of K^bm1 CTL from T cells of aged mice. The alterations in Ia^bm12 allospecific responses were not attributable to quantitative changes in CD4⁺ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Ia^bm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4⁺ T cell-mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.     

Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2-microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild-type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I-deficient mice were tolerant to autologous MHC class I-deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon-inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self-tolerant state of MHC class I-deficient NK cells was broken by in vitro stimulation with IL-2 for 4 days. Under these conditions, NK cells from the MHC class I-deficient mice killed autologous MHC class I-deficient cells while MHC class I-positive targets were spared. The C-type lectin inhibitory receptor Ly49C has a specificity for H-2K^b and is expressed on a subset of NK1.1⁺ cells in B6 mice. Wild-type and all MHC class I-deficient mice had similar numbers of Ly49C-positive NK1.1⁺ cells. However, Ly49C expression was markedly down-regulated on NK1.1⁺ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL-2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.     

CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

Allogeneic recognition of class I molecules: Anti-H-2Ld repertoire of H-2b mice includes T cells recognizing mutant class II H-2b (Abm12) molecules

Two major histocompatibility complex (MHC) class I-reactive T cell clones derived from H-2^b mice, generated against the allogeneic L^d molecule, were found to recognize the H-2^b class II mutant A^bm12 molecule as well. In addition, these clones also recognize the class II A^s molecule, and display a class II-dependent reactivity to staphylococcal enterotoxin B. Neither the class I nor the class II alloreactivities of the clones were found to be dependent on other MHC molecules. Both clones express CD4+CD8^? phenotypes. The CD4 molecule appears to be involved in their class II reactivity, while little or no role for CD4 could be detected in the class I reactivity. This is the first report of a class I/class II cross-reactivity being mediated by CD4⁺ T cells. The structural basis for this cross-reactivity is discussed.     

Evaluation of functional heterogeneity in the CD8 subset with T cells from T cell receptor-transgenic mice

The question of functional differentiation within the CD8 subset has been addressed in a model of TcR-transgenic (TcR-tg) mice expressing a TcR specific for H-2K^b (Ti). CD8⁺ Ti⁺ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H-2K^b-expressing cells. In contrast to T cells from normal H-2^k littermates, alloantigen induction of CTL from TcR-tg mice is independent of CD4⁺ T helper (T_h) cells and is accompanied by high level secretion of interleukin-(IL)-2 by Ti⁺ CD8⁺ T cells. Precursor frequency analysis performed on CD8⁺ cells from TcR-tg mice revealed a high frequency of T_h as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8⁺ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR-tg mice) demonstrated a heterogeneous expression of Ly-6C on CD8⁺ cells with a large enrichment of Ly-6C^? cells among the Ti⁺ cells which persisted after stimulation with H-2^b cells in conditions that led to a homogeneous expression of the activation markers pgp-1 and CD69. The possibility that Ly-6C expression could mark functionally different subpopulations in CD8⁺ T cells was investigated. Stimulation of sorted populations of Ly-6C^? and Ly-6C⁺ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL-2 secretion. Thus, for CD8⁺ T cells expressing the same TcR, differentiation led to acquisition of both IL-2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper-dependent CTL precursors.     

Leishmania major infection in mice primes for specific major histocompatibility complex class I-restricted CD8+ cytotoxic T cell responses

This report shows that lymphoid tissues of mice which have resolved a primary infection with Leishmania major contain parasite-specific major histocompatibility complex (MHC) class I-restricted cytolytic CD8⁺ T cell precusors that can be expanded after specific restimulation in vitro with syngeneic antigen-presenting cells pulsed with a cyanogen bromide digest of L. major. In H-2^b mice, two distinct populations of CD8⁺ T cells were identified which both lysed target cells pulsed with L. major-derived peptides but were restricted by a different H-2^b class I gene product. Interestingly, these two populations appear to recognize different parasite-derived peptides. It is noteworthy that one K°-restricted CD8⁺ T cell line was able to specifically lyse syngeneic macrophages infected with viable L. major, indicating that some L. major-derived peptides may reach the MHC class I pathway of presentation from the phagolysosomal compartment where the parasites are confined in infected macrophages. The importance of these parasite-specific MHC class I restricted cytolytic CD8⁺ T cells for the elimination of L. major by the infected host remains to be determined.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

The degree of CD8 dependence of cytolytic T cell precursors is determined by the nature of the T cell receptor (TCR) and influences negative selection in TCR-transgenic mice

Although much has been learned about CD8 structure-function properties, it has so far not been tested whether the nature of the TCR is sufficient to transfer the property of CD8 dependence versus non-dependence to CD8⁺ cytotoxic T lymphocytes (CTL) and their precursors differentiating in T cell receptor (TCR)-transgenic (Tg) mice. In the present study, we compared the characteristics of dependence on CD8 for stimulation of CTL precursors and antigen-specific cytolysis by CD8⁺ T cells from two TCR-Tg mice expressing respectively the TCR (Tg) from a “CD8-dependent” and from a “CD8-independent” CTL clone, which were both reactive against the H-2K^b alloantigen and originated from H-2^k mice. The results indicate that the property of the Tg⁺CD8⁺ cells from H-2^k TCR-Tg mice corresponds to that of the CTL clone of origin, demonstrating that it is linked to the nature of the TCR. Consistent with this property, Tg⁺CD4⁺ cells could also differentiate into H-2K^b-specific CTL when originating from the “CD8-independent”, but not from the “CD8-dependent” Tg-TCR. The influence of the property of “CD8 dependence” on negative selection occurring in TCR-Tg H-2^klb mice was apparent at two levels: (i) in the thymus, the extent of deletion was much more pronounced for the “CD8-independent” TCR-Tg mice; (ii) in the periphery, Tg^+(hi) cells with low to negative CD8 expression were present for the “CD8-dependent” Tg-TCR, whereas only Tg⁺CD4^?CD8^? cells with low surface Tg-TCR and CD3 expression were found for the “CD8-independent” Tg-TCR, indicating that Tg⁺CD4^?CD8^? cells are susceptible to tolerance induction involving TCR/CD3 surface down-modulation. Furthermore, different in vitro conditions led to H-2K^b-induced stimulation of Tg⁺CD4^?CD8^? cells to differentiate into CTL detected in an anti-TCR clonotypic monoclonal antibody redirected cytolysis assay. Culture in interleukin-2 of H-2^klb Tg⁺CD4^?CD8^? cells was sufficient to induce CTL activity in the “CD8-independent” model, whereas stimulation with cells which overexpressed H-2K^b was required in addition to interleukin-2 to induce CTL differentiation in the “CD8-dependent” model. These data suggest that peripheral Tg⁺CD4^?CD8^? cells present in a situation of in vivo tolerance to H-2K^b can still be triggered by H-2K^b with a sensitivity correlated with the degree of CD8 dependence.     

TAP1-/- mice present oligoclonal BV-BJ expansions following the rejection of grafts bearing self antigens

Our previous work showed that transporter associated with antigen processing 1 (TAP1)^–/– (H‐2^b) mice rejected grafts from H‐2^b mice which display a normal density of class I major histocompatibility complex (MHC) molecules at the cell surface. Our results indicated that H‐2^b molecules themselves may be a target in this kind of rejection and that CD4⁺ T cells play a major role in this autoreactive process. Our data also suggested that TAP1^–/– mice, in addition to the well‐recognized phenotype of class I and CD8⁺ T‐cell deficiency, present a functional alteration in their autoreactive CD4⁺ T‐cell repertoires. In this model of inflammatory autoreactivity to modified self, we have analysed T‐cell receptor (TCR) V‐beta–J‐beta (BV‐BJ) usage by complementarity determining region 3 (CDR3) length spectratyping in splenocytes from naïve TAP1^–/– mice and transplanted TAP1^–/– mice that rejected B6 heart grafts or responded to synthetic self H‐2K^b peptides. Importantly, oligoclonal T‐cell expansions shared by different animals were detected in the peripheral T‐cell repertoire of transplanted TAP1^–/– mice. Such public expansions were also induced in vitro by H‐2K^b peptides, suggesting that dominant class I peptides can induce preferential expansions of restricted T‐cell populations during rejection. Some of these public T‐cell expansions were also detected in transplanted mice even before in vitro stimulation with peptides, indicating that post‐transplantation expansion of these populations had occurred in vivo. The functional activity of these T‐cell populations awaits elucidation, as do the underlying mechanisms involved in the inflammatory autoreactive process, in TAP1^–/– mice.     

Control of thymus-independent intestinal intraepithelial lymphocytes by β2-microglobulin

Murine intestinal intraepithelial lymphocytes (i-IEL) comprise thymusdependent cells such as T cell receptor (TcR) α/β CD8α/β⁺ i-IEL, as well as thymus-independent ones such as TcRα/β CD8α/α⁺ and TcRγ/δ CD8α/α⁺ i-IEL. Whilst the development of the CD8α/β expressing i-IEL is strictly contingent on major histocompatibility complex (MHC) class I surface expression, that of CD8α/α i-IEL appears largely MHC class I independent. We have used β2-microglobulin (β2m)^?/? mutant mice lacking surface-expressed MHC class I and TcRα/β CD8α/β⁺ i-IEL to analyze the potential impact of MHC class I on regional activation of thymus-independent i-IEL. To analyze the role of TcRγ/δ i-IEL in regional cell interactions, these mice were treated with the anti-TcRγ/δ mAb, GL3. Whilst numbers of TcRα/β CD8α/α i-IEL were markedly reduced in βm^+/? mice, those of TcRγ/δ i-IEL were elevated. Administration of GL3 in vivo caused TcR down-modulation and functional inactivation of TcRγ/δ i-IEL in β2m^+/? mice. In contrast, TcR expression and functional activities of TcRγ/δ i-IEL from β2m^?/? mice were not impaired by GL3 treatment. The TcRα/β CD8β i-IEL from β2m^?/? mice were expanded and functionally activated as a consequence of TcRγ/δ engagement. The TcRγ/δ i-IEL and TcRα/β CD8α/α⁺ i-IEL from athymic nu/nu mice which express MHC class I, but lack TcRα/β CD8α/β⁺ i-IEL, responded to TcRγ/δ engagement as those from the β2m^+/? controls. In addition, the TcRγ/δ i-IEL from TcRβ^?/? and TcRβ^+/? mutants were equally affected by GL3. We conclude that the absence of β2m renders TcRγ/δ i-IEL resistant to TcR-mediated inactivation and promotes activation of TcRα/β CD8β^? i-IEL. The activation of TcRγ/δ i-IEL seems to be directly controlled by β2m/MHC class I expression and independent from TcRα/β CD8β⁺ i-IEL. Regulation of self-reactive thymus-independent i-IEL through β2m/MHC class I may contribute to control of autoreactive immune responses in the intestine.     

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL

We selected three peptides from the germ-line sequence of the Vβ8.2 and Jβ2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative K^d- and L^d-restricted epitopes. Immunization of BALB/c (H-2^d) mice with the Vβ8.2(67–90) 23-mer peptide 1 as well as the 15-mer Vβ8.2(95–108)-peptide 2 efficiently primed specific CD8⁺ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-Vβ8.2 epitopes. Vβ8.2⁺ T cells were not deleted in TCR peptide-immunized mice because the fractions of Vβ8.2⁺ CD4⁺ and Vβ8.2⁺ CD8⁺ T cells in spleen and lymph nodes were not altered. The proliferative response of Vβ8.2⁺ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60–80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the Jβ2.3-derived peptide 3 stimulated a CD8⁺ CTL response against a class I-restricted epitope within this Jβ segment that was also generated during natural “endogenous” processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8⁺ CTL recognize class I-restricted, natural Vβ/Jβ-TCR epitopes. Such anti-TCR CTL may, thus, operate in Vβ-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.     

Emergence in Cx knockout mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant x light chain region

Allotype- or idiotype-specific CD4⁺ T cells have been reported to recognize immunoglobulin (Ig) peptides presented by class II molecules. In contrast, few data are available concerning the generation of Ig peptide-specific CD8⁺ T cells. We have therefore investigated whether T-depleted spleen cells from Ig x light chain-expressing 129/Sv mice (129x^+/+) could induce, in Cx knockout mice (129 x^?/?), the generation of Ig constant x light chain region (Cx)-specific cytotoxic T lymphocytes (CTL). The determination of TCRβ chain expressed by nine CTL clones, together with the use of a library of overlapping peptides spanning the whole Cx sequence, show that the B cells from x^+/+ mice are able to elicit in Cx knockout mice, the emergence of a diverse CTL repertoire that recognizes one single Cx peptide presented by the H-2K^b class I molecule. In addition, these data support the notion that B cells are able to process and present on their class I molecules, peptides generated from their own x light chains.