MHC class I allele dosage alters CD8 expression by intestinal intraepithelial lymphocytes

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.     

On the self-referential nature of naive MHC class II-restricted T cells

The use of mutant mice expressing a normal MHC class II molecule surface level but a severely restricted self-peptide diversity (H-2Malpha(-/-)) previously revealed that T cells carrying the Ealpha(52-68)-I-A(b) complex-specific 1H3.1 TCR rely on self-peptide(s) recognition for both their peripheral persistence in irradiated hosts and their intrathymic positive selection. Here, we identify Ealpha(52-68) structurally related self-peptide(s) as a major contributor to in vivo positive selection of 1H3.1 TCR-transgenic thymocytes in I-A(b+)/I-Ealpha(-) mice. This is demonstrated by the drastic and specific reduction of the TCR high thymocyte population in 1H3.1 TCR-transgenic (Tg) mice treated with the Ealpha(52-68)-I-A(b) complex-specific Y-Ae mAb. Self-peptide(s) recognition is also driving the maturation of T cells carrying a distinct MHC class II-restricted specificity (the Ealpha(6) alphass TCR), since positive selection was also deficient in Ealpha(6) TCR Tg H-2Malpha(-/-) thymi. Such a requirement for recognition of self-determinants was mirrored in the periphery; Ealpha(6) TCR Tg naive T cells showed an impaired persistence in both H-2Malpha(-/-) and I-A(b)ss(-/-) irradiated hosts, whereas they persisted and slowly cycled in wild-type recipients. This moderate self-peptide(s)-dependent proliferation was associated with a surface phenotype intermediate between those of naive and activated/memory T cells; CD44 expression was up-regulated, but surface expression of other markers such as CD62L remained unaltered. Collectively, these observations indicate that maturation and maintenance of naive MHC class II-restricted T cells are self-oriented processes.     

The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes

The MHC class Ib molecule Qa-1 is the primary ligand for mouse CD94/NKG2A inhibitory receptors expressed on NK cells, in addition to presenting Ags to a subpopulation of T cells. CD94/NKG2A receptors specifically recognize Qa-1 bound to the MHC class Ia leader sequence-derived peptide Qdm. Qdm is the dominant peptide loaded onto Qa-1 under physiological conditions and this peptide has an optimal sequence for binding to Qa-1. Peptide dissociation experiments demonstrated that Qdm dissociates from soluble or cell surface Qa-1(b) molecules with a t(1/2) of approximately 1.5 h at 37 degrees C. In comparison, complexes of an optimal peptide (SIINFEKL) bound to the MHC class Ia molecule H-2K(b) dissociated with a t(1/2) in the range from 11 to 31 h. In contrast to K(b), the stability of cell surface Qa-1(b) molecules was independent of bound peptides, and several observations suggested that empty cell surface Qa-1(b) molecules might be unusually stable. Consistent with the rapid dissociation rate of Qdm from Qa-1(b), cells become susceptible to lysis by CD94/NKG2A(+) NK cells under conditions in which new Qa-1(b)/Qdm complexes cannot be continuously generated at the cell surface. These results support the hypothesis that Qa-1 has been selected as a specialized MHC molecule that is unable to form highly stable peptide complexes. We propose that the CD94/NKG2A-Qa-1/Qdm recognition system has evolved as a rapid sensor of the integrity of the MHC class I biosynthesis and Ag presentation pathway.     

Coreceptor function of CD4 in response to MHC class I molecule

Specificity of T cell receptor (TCR) and its interaction with coreceptor molecules play decisive role in successful passing of T lymphocytes via check-points during their development and finally determine the efficiency of adaptive immunity. Genes encoding alpha- and beta-chains of TCR hybridoma 1D1 have been cloned. The hybridoma 1D1 was established by the fusion of BWZ.36CD8alpha cell line with CD8+ memory cells specific to MHC class I H-2Kb molecule. Exploiting retroviral transduction of thymoma 4G4 cells with TCR genes and coreceptors CD4 and CD8, variants of this cell line expressing on the surface CD3/TCR complex and coreceptors, separately or simultaneously have been obtained. The main function of CD4 is stabilization of interaction between TCR and MHC class II molecule. Nevertheless, we have found that CD4 could successfully participate in the activation of transfectants via TCR specific to MHC class I molecule H-2Kb. Moreover, coreceptor CD4 dominates CDS, because the response of transfectants CD4+CD8+ is blocked by antibodies to CD4 and MHC Class II Ab molecule but not to coreceptor CD8. The response of CD4+ cells was not due to cross-reaction between TCR 1D1 with MHC class II molecules, because transfectants do not respond to splenocytes of H-2b knockouted mice with impaired assembly of TCR/beta2-microglobulin/peptide complexes resulting in their absence on the cell surphace. The effect of domination was not due to sequestration of kinase p56lck, because truncated CD4 with the loss of binding motif for p56lck remained functional in 4G4 cells. Results obtained can explain the number of features of intrathymic selection and represent experimental basis for development of new methods of cancer gene therapy.     

Homeostatic proliferation of a Qa-1b-restricted T cell: a distinction between the ligands required for positive selection and for proliferation in lymphopenic hosts

Naive T cells proliferate in response to self MHC molecules after transfer into lymphopenic hosts, a process that has been termed homeostatic proliferation (HP). Previous studies have demonstrated that HP is driven by low level signaling induced by interactions with the same MHC molecules responsible for positive selection in the thymus. Little is known about the homeostatic regulation of T cells specific for class Ib molecules, including Qa-1 and H2-M3, though it has been suggested that their capacity to undergo homeostatic expansion may be inherently limited. In this study, we demonstrate that naive 6C5 TCR transgenic T cells with specificity for Qa-1(b) have a capacity similar to conventional T cells to undergo HP after transfer into sublethally irradiated mice. Proliferation was largely dependent on the expression of beta(2)-microglobulin, and experiments with congenic recipients expressing Qa-1(a) instead of Qa-1(b) demonstrated that HP is specifically driven by Qa-1(b) and not through cross-recognition of classical class I molecules. Thus, the same MHC molecule that mediates positive selection of 6C5 T cells is also required for HP. Homeostatic expansion, like positive selection, occurs in the absence of a Qa-1 determinant modifier, the dominant self-peptide bound to Qa-1 molecules. However, experiments with TAP(-/-) recipients demonstrate a clear distinction between the ligand requirements for thymic selection and HP. Positive selection of 6C5 T cells is dependent on TAP function, thus selection is presumably mediated by TAP-dependent peptides. By contrast, HP occurs in TAP(-/-) recipients, providing an example where the ligand requirements for HP are less stringent than for thymic selection.     

Modulation of CD8+ T cell response to antigen by the levels of self MHC class I

The response of H-Y-specific TCR-transgenic CD8(+) T cells to Ag is characterized by poor proliferation, cytolytic activity, and IFN-gamma secretion. IFN-gamma secretion, but not cytotoxic function, can be rescued by the B7.1 molecule, suggesting that costimulation can selectively enhance some, but not all, effector CD8(+) T cell responses. Although the H-Y epitope binds H-2D(b) relatively less well than some other epitopes, it can induce potent CTL responses in nontransgenic mice, suggesting that the observed poor responsiveness of transgenic CD8(+) T cells cannot be ascribed to the epitope itself. Previously reported reactivity of this TCR to H-2A(b) is also not the cause of the poor responsiveness of the H-Y-specific CD8(+) T cells, as H-Y-specific CD8(+) T cells obtained from genetic backgrounds lacking H-2A(b) also responded poorly. Rather, reducing the levels of H-2(b) class I molecules by breeding the mice to (C57BL/6 x B10.D2)F(1) or TAP1(+/-) backgrounds partially restored cytotoxic activity and enhanced proliferative responses. These findings demonstrate that the self MHC class I gene dosage may regulate the extent of CD8(+) T cell responsiveness to Ag.     

Blockade of transgenic gamma delta T cell development in beta 2-microglobulin deficient mice.

The gamma delta T cell receptor (TCR) of the hybridoma KN6 recognizes the self molecule encoded by a class I gene which maps within the TL region of the major histocompatibility complex (MHC) of H-2b mice. Mice transgenic (Tg) for this TCR were crossed with mice genetically deficient in beta 2-microglobulin (beta 2m). No mature Tg gamma delta T cells were detected in the thymus or the spleen of the beta 2m- gamma delta Tg mice. We conclude that interaction between the Tg gamma delta TCR and a beta 2m-associated molecule (probably an MHC class I molecule) is required for the generation of mature Tg gamma delta T cells.     

In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.     

Coreceptor function of CD4 in response to the MHC class I molecule

The specificity of the T-cell receptor (TCR) and its interaction with coreceptors play a crucial role in T-cell passing through developmental checkpoints and, eventually, determine the efficiency of adaptive immunity. The genes for the α and β chains of TCR were cloned from T-cell hybridoma 1D1, which was obtained by fusion of BWZ.36CD8α cells with CD8⁺ memory cells specific for the H-2K^b MHC class I molecule. Retroviral transduction of the 1D1 TCR genes and the CD4 and CD8 coreceptor genes was used to obtain 4G4 thymoma variants that exposed the CD3/TCR complex together with CD4, CD8, or both of the coreceptors on their surface. Although the main function of CD4 is to stabilize the interaction of TCR with MHC class II molecules, CD4 was found to mediate the activation of transfected cells via TCR specific for the H-2K^b MHC class I molecule. Moreover, CD4 proved to dominate over CD8, since the response of CD4⁺CD8⁺ transfectants was suppressed by antibodies against CD4 and the A^b MHC class II molecule but not to CD8. The response of CD4⁺ transfectants was not due to a cross-reaction of 1D1 TCR with MHC class II molecules, because the transfectants did not respond to splenocytes of H-2^b knockout mice, which were defective in the assembly of the MHC class I molecule/β2 microglobulin/peptide complex and did not expose the complex on cell surface. The domination was not due to sequestration of p56^lck kinase, since CD4 devoid of the kinase-binding site was functional in 4G4 thymoma cells. The results were used to explain some features of intrathymic cell selection and assumed to provide an experimental basis for developing new methods of anticancer gene therapy.     

MHC class II molecules play a role in the selection of autoreactive class I-restricted CD8 T cells that are essential contributors to type 1 diabetes development in nonobese diabetic mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2(g7) molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2(b)) or CD8 expression (H2(q)). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2(nb1) MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.     

Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection

The present report provides the first extensive characterization of the OT-I TCR transgenic line, which produces MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). These cells are shown to be positively selected in vivo in H-2b C57BL/6 mice and in bm5 mice, which express the Kbm5 mutant molecule. In contrast, OT-I cells were not selected by mutant Kb molecules in bm1, bm3, bm8, bm10, bm11 or bm23 mice. Interestingly, however, when positive selection was examined in vitro in foetal thymic organ culture (FTOC), bm1 and bm8 were still poorly selective, but the bm3 haplotype now selected as efficiently as B6. The ability to select in vitro correlated with the capacity to present the ovalbumin (OVA) peptide to OT-I cells, as measured by induction of an OVA-specific proliferative response. These results suggest that a lower affinity TCR:MHC interaction may be necessary for positive selection in FTOC compared with selection in situ.     

MHC class II A alpha and E alpha molecules determine the clonal deletion of V beta 6+ T cells. Studies with recombinant and transgenic mice

Interactions between MHC class II genes and minor lymphocyte stimulating (Mls) associated products are responsible for clonally deleting self-reactive T cells in mice. Here we demonstrate the role of the intact I-A and I-E molecules as well as the individual A alpha and E alpha chains in the deletion of cells bearing the V beta 6 TCR. DBA/1 (H-2q, Mls-1a) mice were crossed with various inbred congenic, recombinant, and transgenic strains and the F1's were screened for V beta 6 expression. All I-E+ strains were fully permissive in deleting V beta 6+ T cells. I-E- strains expressing I-A b,f,s,k,p permitted only partial deletion, while I-Aq strains showed no deletion. Recombinant I-Aq and I-Af strains which expressed E kappa alpha chain in the absence of E beta chain showed a decrease in V beta 6+ T cells as compared to their H-2q and H-2f counterparts. Furthermore, transgenic mice expressing E kappa alpha Aq beta gene in an H-2q haplotype (E kappa alpha Aq beta?) gave similar results to that of the recombinants in deleting V beta 6 T-cells. The role of the 1-A molecule was also shown by the partial deletion of V beta 6+ T cells in H-2q mice expressing transgenic I-Ak molecules. These results demonstrate that the E alpha chain is important in the deletion of V beta 6 T-cells in Mls-1a mice. The role of A alpha chain is also implied by the permissiveness of E kappa alpha Aq beta but not Aq alpha Aq beta molecules in the deletion of V beta 6+ T cells.     

The nonclassical major histocompatibility complex molecule Qa-2 protects tumor cells from NK cell- and lymphokine-activated killer cell-mediated cytolysis

The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2K(b)-expressing B78H1 targets also reduced LAK cell activity, while H-2D(b) offered no protection. Importantly, blocking with F(ab')(2) specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2(b) SCID and B6 mice established that NK1.1(+)TCR(-) NK and NK1.1(+)TCR(+) LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1(+) NK/T cells.     

Tolerance to noninherited maternal MHC antigens in mice

The phenomenon of tolerance to noninherited maternal Ags (NIMA) is poorly understood. To analyze the NIMA effect C57BL/6 (H-2(b/b)) males were mated with B6D2F(1) (H-2(b/d)) females, whereby 50% of the offspring are H-2(b/b) mice that have been exposed to maternal H-2(d) alloantigens. Controls were H-2(b/b) offspring of C57BL/6 mothers, either inbred C57BL/6 mice or F(1) backcross mice from breedings with H-2(b/d) fathers. We found that 57% of the H-2(b/b) offspring of semiallogeneic (H-2(b/d)) mothers accepted fully allogeneic DBA/2 (H-2(d/d)) heart grafts for >180 days, while similar transplants were all rejected by day 11 in controls (p < 0.0004). Foster nursing studies showed that both oral and in utero exposure to NIMA are required for this tolerogenic effect. An effect of NIMA was also found to extend the survival of skin grafts from a semiallogeneic donor (p < 0.02). Pretransplant analysis of splenocytes showed a 40-90% reduction of IL-2-, IL-5-, and IFN-gamma-producing T cells responding to H-2(d)-expressing APC in NIMA(d)-exposed vs control mice. Injection of pregnant BALB/c-dm2 (H-2L(d)-negative) female mice i.v. with H-2L(d)(61-80) peptide profoundly suppressed the offspring's indirect pathway alloreactive CD4(+) T cell response to H-2L(d). These results suggest that the natural exposure of the fetus and newborn to maternal cells and/or soluble MHC Ags suppresses NIMA-allospecific T cells of the offspring, predisposing to organ transplant tolerance in adult mice.     

NK cell inhibitory receptor Ly-49C residues involved in MHC class I binding.

Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2D(d), as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2K(b), H-2D(b), and H-2D(d). The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2D(d) in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.     

T cell subsets in (responder x nonresponder)F1 mice regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine (GAT)

Immune responses to GAT are controlled by H-2-linked Ir genes; soluble GAT stimulates antibody responses in responder mice (H-2b) but not in nonresponder mice (H-2q). In nonresponder mice, soluble GAT stimulates suppressor T cells that preempt function of helper T cells. After immunization with soluble GAT, spleen cells from (responder x nonresponder: H-2b X H-2q)F1 mice develop antibody responses to responder H-2b GAT-M phi but not to nonresponder H-2q GAT-M phi. This failure of immune F1 spleen cells to respond is due to an active suppressor T cell mechanism that is activated by H-2q, but not H-2b, GAT-M phi and involves two regulatory T cell subsets. Suppressor-inducer T cells are immune radiosensitive Lyt-1 +2-, I-A-, I-J+, Qa-1+ cells. Suppressor-effector T cells can be derived from virgin or immune spleens and are radiosensitive Lyt-1-2+, I-A-, I-J+, Qa-1+ cells. This suppressor mechanism can suppress responses of virgin or immune F1 helper T cells and B cells. Helper T cells specific for H-2b GAT-M phi are easily detected in F1 mice after immunization with soluble GAT; helper T cells specific for H-2q GAT-M phi are demonstrated after elimination of the suppressor-inducer and -effector cells. These helper T cells are radioresistant Lyt-1+2-, I-A+, I-J-, Qa-1- cells. These data indicate that the Ir gene defect in responses to GAT is not due to a failure of nonresponder M phi to present GAT and most likely is not due to a defective T cell repertoire, because the relevant helper T cells are primed in F1 mice by soluble GAT and can be demonstrated when suppressor cells are removed. These data are discussed in the context of mechanisms for expression of Ir gene function in responses to GAT, especially the balance between stimulation of helper vs suppressor T cells.     

MHC class I-Ly49 interactions shape the Ly49 repertoire on murine NK cells

This study aims to determine how the interaction of Ly49 receptors with MHC class I molecules shapes the development of the Ly49 repertoire. We have examined the percentage of NK cells that expressed Ly49A, Ly49G2, and Ly49D in single and double Ly49A/C-transgenic mice on four different MHC backgrounds, H-2(b), H-2(d), H-2(b/d), and beta(2)-microglobulin(-/-). The results show that the total numbers of NK cells were not different among the strains. The prior expression of a Ly49 receptor capable of binding to self MHC class I altered the percentage of NK cells expressing endogenous Ly49A, Ly49G2, and Ly49D even in mice in which no MHC ligand was present for the latter receptors. The NK cells in the Ly49-transgenic mice expressed the same level of endogenous Ly49 receptors as wild-type mice of a similar MHC background. In contrast, the number of NK T cells was reduced in mice in which the Ly49 transgene could bind to a MHC class I molecule. The onset of Ly49 receptor expression on NK cells during ontogeny was not altered in the presence of transgenic Ly49 receptors. These data support a sequential model and argue against a selection model for Ly49 repertoire development on NK cells.     

T cell recognition of an engineered MHC class I molecule: implications for peptide-independent alloreactivity

Previously, we described H-2K(bW9) (K(bW9)), an engineered variant of the murine MHC class I molecule H-2K(b) (K(b)), devoid of the central anchor ("C") pocket owing to a point mutation on the floor of the peptide binding site; this substitution drastically altered selection of bound peptides, such that the peptide repertoires of K(b) and K(bW9) are largely nonoverlapping in vivo. On the basis of these observations, we used K(bW9) and K(b) to revisit the role of peptides in alloreactive T cell recognition. We first compared Ab and TCR recognition of K(bW9) and K(b). Six of six K(b)-specific mAbs, directed against different parts of the molecule, recognized K(bW9) well, albeit at different levels than K(b). Furthermore, K(bW9) readily served as a restriction element for a peptide-specific syngeneic CTL response. Therefore, K(bW9) mutation did not result in gross distortions of the TCR-interacting surface of class I, which was comparable between K(b) and K(bW9). Interestingly, when K(bW9) was used to stimulate allogeneic T cells, it induced an infrequent CTL population that cross-reacted against K(b) and was specific for peptide-independent MHC epitopes. By contrast, K(b)-induced alloreactive CTLs recognized K(b) in a peptide-specific manner, did not cross-react on K(bW9), and were present at much higher frequencies than those induced by K(bW9). Thus, induction of rare peptide-independent CTLs depended on unique structural features of K(bW9), likely due to the elevated floor of the peptide-binding groove and the consequent protruding position of the peptide. These results shed new light on the relationship between TCR and peptide-MHC complex in peptide-independent allorecognition.     

A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.     

Induction of alloreactive CD4 T cell tolerance in molecular chimeras: a possible role for regulatory T cells

Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K(b) were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b), albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K(b)-transduced bone marrow permanently accepted K(b) disparate skin grafts. CD4+CD25+ T cells from mice reconstituted with H-2K(b)-transduced bone marrow prevented rejection of K(b) disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.