Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope-specific manner.

Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively. Silencing the K(b) /S190 epitope (K(b) /S190V194F ) in antigen-expressing vectors rescued the presentation of the K(b) /S208 epitope in stable transfectants and significantly enhanced priming of K(b) /S208-specific T cells in C57BL/6 mice. A K(b) /S190-mediated immunodominance operating in surface antigen-expressing cells, but not in rSP-pulsed cells, led to an efficient suppression in the presentation of the K(b) /S208 epitope and a consequent decrease in the priming of K(b) /S208-specific T cells. This K(b) /S190-mediated immunodominance also operated in 1.4HBV-S(mut) transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K(b) /S208- but not K(b) /S190-specific T cells in 1.4HBV-S(mut) tg mice. However, IFN-γ(+) K(b) /S208-specific T cells could not inhibit HBV replication in the liver of 1.4HBV-S(mut) tg mice. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines.

MHC-dependent inhibition of uterine NK cells impedes fetal growth and decidual vascular remodelling.

NK cells express variable receptors that engage polymorphic MHC class I molecules and regulate their function. Maternal NK cells accumulate at the maternal-fetal interface and can interact with MHC class I molecules from both parents. The relative contribution of the two sets of parental MHC molecules to uterine NK cell function is unknown. Here we show that, in mice, maternal and not paternal MHC educates uterine NK cells to mature and acquire functional competence. The presence of an additional MHC allele that binds more inhibitory than activating NK cell receptors results in suppressed NK cell function, compromised uterine arterial remodelling and reduced fetal growth. Notably, reduced fetal growth occurs irrespectively of the parental origin of the inhibitory MHC. This provides biological evidence for the impact of MHC-dependent NK inhibition as a risk factor for human pregnancy-related complications associated with impaired arterial remodelling.

T lymphocytes restrain spontaneous metastases in permanent dormancy.

Tumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting. The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models. Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immunomediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11 mouse fibrosarcoma clone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimental endpoints (3-8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearing mice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminated metastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at both local and systemic levels, with results supporting the evidence that the immune system was able to restrain spontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHC class I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I-positive. This model system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunities for immunotherapeutic management of metastatic disease.

Macrophage MHC and T-cell receptors essential for rejection of allografted skin and lymphoma.

BACKGROUND: Skin or organ allograft rejection is dependent on noncytotoxic CD4(+) T cells, but the mechanisms of recognition and rejection remain elusive. Previously, we demonstrated C57BL/6 (H-2D(b)K(b)) macrophage-mediated, cell-to-cell contact-dependent, d haplotype-specific lysis of allografts (e.g., BALB/c skin and Meth A cells; H-2D(d)K(d)) in the rejection site and isolated two cDNA clones encoding receptors on macrophages for H-2D(d) and H-2K(d), macrophage major histocompatibility complex receptor (MMR) 1 and 2, respectively. METHODS: To elucidate the role of MMR2 and T-cell receptors (TCRs) in graft rejection, we generated MMR2 knockout (KO) mice on a C57BL/6 background and transplanted D(d), K(d), or D(d)K(d) transgenic C57BL/6 skin or EL-4 lymphoma cells onto or into these KO mice. RESULTS: MMR2 KO mice lacking MMR2 mRNA or protein expression in their monocytes had no obvious abnormalities in terms of cell number in or composition of their lymphoid tissues or in T lymphocyte responses to alloantigen or nonalloantigen, whereas they failed to reject K(d) transgenic skin grafts. Surprisingly, they also lacked MMR1 mRNA and protein expression in their monocytes and failed to reject D(d) or D(d)K(d) transgenic skin grafts. However, they did reject skin grafts from mice expressing H-2I(d), minor H(d), or third-party major histocompatibility complex. On the contrary, D(d)-, K(d)-, or D(d)K(d)-EL-4 cells injected intradermally or intraperitoneally into MMR2 KO mice were rejected by TCR(αß)(+)/CD8(+) T cells in a transgene number-dependent and MMR-independent manner. CONCLUSIONS: These results demonstrate that MMRs on monocytes/macrophages and TCRs on cytotoxic T lymphocytes in mice were essential for recognition and rejection of allografted skin and lymphoma, respectively.

Cutting edge: innate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimuli.

Innate memory phenotype (IMP) CD8(+) T cells are nonconventional αß T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8(+) T cells in MHCI(-/-) recipients, resembling those in Itk(-/-) mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.

Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice.

The inhibitory programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) pathway contributes to the functional down-regulation of T cell responses during persistent systemic and local virus infections. The blockade of PD-1-PD-L1-mediated inhibition is considered as a therapeutic approach to reinvigorate antiviral T cell responses. Yet previous studies reported that PD-L1-deficient mice develop fatal pathology during early systemic lymphocytic choriomeningitis virus (LCMV) infection, suggesting a host protective role of T cell down-regulation. As the exact mechanisms of pathology development remained unclear, we set out to delineate in detail the underlying pathogenesis. Mice deficient in PD-1-PD-L1 signaling or lacking PD-1 signaling in CD8 T cells succumbed to fatal CD8 T cell-mediated immunopathology early after systemic LCMV infection. In the absence of regulation via PD-1, CD8 T cells killed infected vascular endothelial cells via perforin-mediated cytolysis, thereby severely compromising vascular integrity. This resulted in systemic vascular leakage and a consequential collapse of the circulatory system. Our results indicate that the PD-1-PD-L1 pathway protects the vascular system from severe CD8 T cell-mediated damage during early systemic LCMV infection, highlighting a pivotal physiological role of T cell down-regulation and suggesting the potential development of immunopathological side effects when interfering with the PD-1-PD-L1 pathway during systemic virus infections.

H2 control of natural T regulatory cell frequency in the lymph node correlates with susceptibility to day 3 thymectomy-induced autoimmune disease.

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by natural regulatory T cells (nTregs) and development of autoimmune ovarian dysgenesis (AOD) and autoimmune dacryoadenitis (ADA) in A/J and (C57BL/6J × A/J) F(1) hybrids (B6A), but not in C57BL/6J (B6) mice. Previously, using quantitative trait locus (QTL) linkage analysis, we showed that D3Tx-AOD is controlled by five unlinked QTL (Aod1-Aod5) and H2. In this study, using D3Tx B6-Chr(A/J)/NaJ chromosome (Chr) substitution strains, we confirm that QTL on Chr16 (Aod1a/Aod1b), Chr3 (Aod2), Chr1 (Aod3), Chr2 (Aod4), Chr7 (Aod5), and Chr17 (H2) control D3Tx-AOD susceptibility. In addition, we also present data mapping QTL controlling D3Tx-ADA to Chr17 (Ada1/H2), Chr1 (Ada2), and Chr3 (Ada3). Importantly, B6-ChrX(A/J) mice were as resistant to D3Tx-AOD and D3Tx-ADA as B6 mice, thereby excluding Foxp3 as a susceptibility gene in these models. Moreover, we report quantitative differences in the frequency of nTregs in the lymph nodes (LNs), but not spleen or thymus, of AOD/ADA-resistant B6 and AOD/ADA-susceptible A/J, B6A, and B6-Chr17(A/J) mice. Similar results correlating with experimental allergic encephalomyelitis and orchitis susceptibility were seen with B10.S and SJL/J mice. Using H2-congenic mice, we show that the observed difference in frequency of LN nTregs is controlled by Ada1/H2. These data support the existence of an LN-specific, H2-controlled mechanism regulating the prevalence of nTregs in autoimmune disease susceptibility.

MHC class I and TCR avidity control the CD8 T cell response to IL-15/IL-15Rα complex.

IL-15 operates via a unique mechanism termed transpresentation. In this system, IL-15 produced by one cell type is bound to IL-15Rα expressed by the same cell and is presented to apposing cells expressing the IL-15Rß/γC complex. We have shown that administering soluble IL-15Rα complexed with IL-15 can greatly enhance IL-15 activity. We now show that the naive CD8 T cell response to exogenous IL-15/IL-15Rα complex is MHC class I dependent. In the absence of ß2 microglobulin, naive CD8 T cells scarcely proliferated in response to IL-15/IL-15Rα complex, whereas memory cells proliferated, although to a lesser extent, compared with levels in control mice. The loss of ß2m or FcRn slightly reduced the extended half-life of IL-15/IL-15Rα complex, whereas FcRn deficiency only partially reduced the naive CD8 T cell proliferative response to IL-15/IL-15Rα complex. In addition, we demonstrated a link between TCR avidity and the ability of a T cell to respond to IL-15/IL-15Rα complex. Thus, T cells expressing low-avidity TCR responded poorly to IL-15/IL-15Rα complex, which correlated with a poor homeostatic proliferative response to lymphopenia. The inclusion of cognate peptide along with complex resulted in enhanced proliferation, even when TCR avidity was low. IL-15/IL-15Rα complex treatment, along with peptide immunization, also enhanced activation and the migratory ability of responding T cells. These data suggest that IL-15/IL-15Rα complex has selective effects on Ag-activated CD8 T cells. Our findings have important implications for directing IL-15/IL-15Rα complex-based therapy to specific Ag targets and illustrate the possible adjuvant uses of IL-15/IL-15Rα complex.

Ovalbumin-derived precursor peptides are transferred sequentially from gp96 and calreticulin to MHC class I in the endoplasmic reticulum.

Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC class I (MHC-I) are associated with several chaperones. Heat shock proteins 70, 90, and the TCP-1 ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study, we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly, we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC-I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC-I and hence presentation of the MHC-I-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC-I H chains and beta2-microglobulin as a trimolecular complex.

SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation.

Previously, we showed that 2B4 is a dominant inhibitory receptor in SHIP-deficient NK cells that prevents efficient cytolysis of complex targets. We show in this study that 2B4 deficiency restores homeostatic control and cytolytic function to SHIP-deficient NK cells. However, 2B4(-/-)SHIP(-/-) NK cells still exhibit a profound disruption of their NK receptor repertoire and are compromised for induction of IFN-gamma by several NK-activating receptors, including NKp46, NK.1.1, and NKG2D. In addition, we find that 2B4(-/-) NK cells have an extensively disrupted repertoire, including a supernormal frequency of NKp46(+) NK cells. Consequently IFN-gamma is induced on a much higher percentage of 2B4(-/-) NK cells following engagement of NKp46. We also find that both SHIP and 2B4 are required to prevent expression of Ly49B, a myeloid lineage MHC class I receptor not normally expressed by the NK lineage. Finally, when SHIP-deficient NK cells are on an H-2(d) background, they exhibit supernormal levels of Ly49A and possess normal cytolytic function against MHC-matched tumor targets and enhanced cytolysis of MHC mismatched tumor targets. However, despite normal or elevated cytolytic function, H2d SHIP(-/-) NK cells exhibit poor induction of IFN-gamma like their H2b(+) or 2B4(-/-) counterparts, demonstrating a uniform requirement for SHIP in induction of IFN-gamma downstream of key NK activating receptors. These findings reveal a complex interplay of SHIP, 2B4, and MHC in the regulation of homeostasis, effector function, and repertoire formation in the NK cell lineage.

Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity.

Major histocompatibility complex class I (MHCI) genes were discovered unexpectedly in healthy CNS neurons in a screen for genes regulated by neural activity. In mice lacking just 2 of the 50+ MHCI genes H2-K(b) and H2-D(b), ocular dominance (OD) plasticity is enhanced. Mice lacking PirB, an MHCI receptor, have a similar phenotype. H2-K(b) and H2-D(b) are expressed not only in visual cortex, but also in lateral geniculate nucleus (LGN), where protein localization correlates strongly with synaptic markers and complement protein C1q. In K(b)D(b-/-) mice, developmental refinement of retinogeniculate projections is impaired, similar to C1q(-/-) mice. These phenotypes in K(b)D(b-/-) mice are strikingly similar to those in beta2 m(-/-)TAP1(-/-) mice, which lack cell surface expression of all MHCIs, implying that H2-K(b) and H2-D(b) can account for observed changes in synapse plasticity. H2-K(b) and H2-D(b) ligands, signaling via neuronal MHCI receptors, may enable activity-dependent remodeling of brain circuits during developmental critical periods.

Microchimerism is strongly correlated with tolerance to noninherited maternal antigens in mice.

In mice and humans, the immunologic effects of developmental exposure to noninherited maternal antigens (NIMAs) are quite variable. This heterogeneity likely reflects differences in the relative levels of NIMA-specific T regulatory (T(R)) versus T effector (T(E)) cells. We hypothesized that maintenance of NIMA-specific T(R) cells in the adult requires continuous exposure to maternal cells and antigens (eg, maternal microchimerism [MMc]). To test this idea, we used 2 sensitive quantitative polymerase chain reaction (qPCR) tests to detect MMc in different organs of NIMA(d)-exposed H2(b) mice. MMc was detected in 100% of neonates and a majority (61%) of adults; nursing by a NIMA+ mother was essential for preserving MMc into adulthood. MMc was most prevalent in heart, lungs, liver, and blood, but was rarely detected in unfractionated lymphoid tissues. However, MMc was detectable in isolated CD4+, CD11b+, and CD11c+ cell subsets of spleen, and in lineage-positive cells in heart. Suppression of delayed type hypersensitivity (DTH) and in vivo lymphoproliferation correlated with MMc levels, suggesting a link between T(R) and maternal cell engraftment. In the absence of neonatal exposure to NIMA via breastfeeding, MMc was lost, which was accompanied by sensitization to NIMA in some offspring, indicating a role of oral exposure in maintaining a favorable T(R) > T(E) balance.

NK cell receptors and their MHC class I ligands in host response to cytomegalovirus: insights from the mouse genome.

The complex interaction between natural killer (NK) cells and cytomegalovirus is a paradigm of the co-evolution between genomes of large DNA viruses and their host immune systems. Both human and mouse cytomegalovirus posses numerous mechanisms to avoid NK cell detection. Linkage studies, positional cloning and functional studies in mice and cells, have led to the identification of key genes governing resistance to cytomegalovirus, including various NK cell activating receptors of major histocompatibility complex (MHC) class I. These receptors, however, seem to require either viral or host MHC class I molecules to operate recognition and elimination of the cytomegalovirus-infected cell leading to host resistance. Here we will review the genes and molecules involved in these mechanisms while contrasting their function with that of other NK cell receptors. Activating receptors of MHC class I may represent a window of therapeutic intervention during human infection with viruses, of which cytomegalovirus remains an important health threat.

H-2Kb-restricted CTL epitopes from mouse heparanase elicit an antitumor immune response in vivo.

The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancer-specific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumor-associated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K(b)-restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptide-binding assay to determine the affinity of the predicted epitopes with the H-2K(b) molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K(b), and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K(b) (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumor-bearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K(b)-restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies.

H-2g, a glucose analog of blood group H antigen, mediates mononuclear cell recruitment via Src and phosphatidylinositol 3-kinase pathways.

OBJECTIVE: Monocyte recruitment by proinflammatory cytokines is a hallmark of rheumatoid arthritis (RA). Lewis(y-6) and H (Le(y)/H) are blood group antigens up-regulated on RA synovial endothelium. We have previously shown that both soluble Le(y)/H and a glucose analog of H, H-2g, are angiogenic and mediateleukocyte-endothelial adhesion via induction of intercellular adhesion molecule 1. We hypothesized that soluble Le(y)/H plays an important role in monocyte recruitment in RA. METHODS: We examined the role of H-2g in monocyte chemotaxis in vitro. We used an RA synovial tissue (ST)-SCID mouse chimera model to evaluate the role of H-2g in monocyte recruitment in vivo. We used Western blots to examine signaling molecules activated by H-2g in monocytes. RESULTS: H-2g induced human monocyte migration in vitro, which was mediated by Src and phosphatidylinositol 3-kinase (PI 3-kinase), since inhibitors and antisense oligodeoxynucleotides (ODNs) of Src and PI 3-kinase significantly decreased H-2g-induced monocyte migration (P < 0.05). H-2g significantly increased mononuclear cell (MNC) homing in vivo into an RA ST-SCID mouse chimera (P < 0.05). Transfection of MNCs with Src antisense ODNs blocked H-2g-induced MNC recruitment into the RA ST-SCID mouse chimera. Additionally, H-2g induced marked phosphorylation of protein kinase CalphaI/betaII (PKCalphaI/betaII), Src, IkappaBalpha, and Akt in monocytes. Src, Akt, and NF-kappaB were shown to be downstream targets of PKCalphaI/betaII, since an inhibitor of PKCalphaI/betaII reduced H-2g-mediated phosphorylation of Src, Akt, and NF-kappaB in monocytes. CONCLUSION: These data suggest that H-2g may be a novel mediator of monocyte recruitment in chronic inflammatory diseases like RA.

The spectrum of HLA-DQ and HLA-DR alleles, 2006: a listing correlating sequence and structure with function.

The list of alleles in the HLA-DRB, HLA-DQA, and HLA-DQB gene loci has grown enormously since the last listing in this journal 8 years ago. Crystal structure determination of several human and mouse HLA class II alleles, representative of two gene loci in each species, enables a direct comparison of ortholog and paralog loci. A new numbering system is suggested, extending earlier suggestions by [Fremont et al. in Immunity 8:305-317, (1998)], which will bring in line all the structural features of various gene loci, regardless of animal species. This system allows for structural equivalence of residues from different gene loci. The listing also highlights all amino acid residues participating in the various functions of these molecules, from antigenic peptide binding to homodimer formation, CD4 binding, membrane anchoring, and cytoplasmic signal transduction, indicative of the variety of functions of these molecules. It is remarkable that despite the enormous number of unique alleles listed thus far (DQA = 22, DQB = 54, DRA = 2, and DRB = 409), there is invariance at many specific positions in man, but slightly less so in mouse or rat, despite their much lower number of alleles at each gene locus in the latter two species. Certain key polymorphisms (from substitutions to an eight-residue insertion in the cytoplasmic tail of certain DQB alleles) that have thus far gone unnoticed are highly suggestive of differences or diversities in function and thus call for further investigation into the properties of these specific alleles. This listing is amenable to supplementation by future additions of new alleles and the highlighting of new functions to be discovered, providing thus a unifying platform of reference in all animal species for the MHC class II allelic counterparts, aiding research in the field and furthering our understanding of the functions of these molecules.

Potent T cell agonism mediated by a very rapid TCR/pMHC interaction.

The interaction between T cell receptors (TCR) and peptide-major histocompatibility complex (pMHC) antigens can lead to varying degrees of agonism (T cell activation), or antagonism. The P14 TCR recognises the lymphocytic choriomeningitis virus (LCMV)-derived peptide, gp33 residues 33-41 (KAVYNFATC), presented in the context of H-2D(b). The cellular responses to various related H-2D(b) peptide ligands are very well characterised, and P14 TCR-transgenic mice have been used extensively in models of virus infection, autoimmunity and tumour rejection. Here, we analyse the binding of the P14 soluble TCR to a broad panel of related H-2D(b)-peptide complexes by surface plasmon resonance, and compare this with their diverse cellular responses. P14 TCR binds H-2D(b)-gp33 with a KD of 3 microM (+/-0.5 microM), typical of an immunodominant antiviral TCR, but with unusually fast kinetics (k(off) = 1 s(-1)), corresponding to a half-life of 0.7 s at 25 degrees C, outside the range previously observed for murine agonist TCR/pMHC interactions. The most striking feature of these data is that a very short half-life does not preclude the ability of a TCR/pMHC interaction to induce antiviral immunity, autoimmune disease and tumour rejection.

The role of structurally conserved class I MHC in tumor rejection: contribution of the Q8 locus.

The mouse multimember family of Qa-2 oligomorphic class I MHC genes is continuously undergoing duplications and deletions that alter the number of the two "prototype" Qa-2 sequences, Q8 and Q9. The frequent recombination events within the Q region lead to strain-specific modulation of the cumulative Qa-2 expression levels. Q9 protects C57BL/6 hosts from multiple disparate tumors and functions as a major CTL restriction element for shared tumor-associated Ags. We have now analyzed functional and structural properties of Q8, a class I MHC that differs significantly from Q9 in the peptide-binding, CTL-interacting alpha(1) and alpha(2) regions. Unexpectedly, we find that the extracellular domains of Q8 and Q9 act similarly during primary and secondary rejection of tumors, are recognized by cross-reactive antitumor CTL, have overlapping peptide-binding motifs, and are both assembled via the transporter associated with the Ag processing pathway. These findings suggest that shared Ag-presenting functions of the "odd" and "even" Qa-2 loci may contribute to the selective pressures shaping the haplotype-dependent quantitative variation of Qa-2 protein expression.

TCR-alpha CDR3 loop audition regulates positive selection.

How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-beta-transgenic mice in which MHC shapes TCR-alpha use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of alphabeta pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the alpha CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.

Persistence of antigen is required to maintain transplantation tolerance induced by genetic modification of bone marrow stem cells.

Genetic modification of hematopoietic stem cells (HSCs) resulting in a state of molecular chimerism can be used to induce donor-specific tolerance to allografts. However, the requirements for maintaining tolerance in molecular chimeras remain unknown. Here, we examined whether long-term expression of a retrovirally encoded alloantigen in hematopoietic cells is required to maintain donor-specific tolerance in molecular chimeras. To this end, mice were reconstituted with syngeneic bone marrow transduced with retroviruses carrying the gene encoding the allogeneic MHC class I molecule Kb. Following induction of molecular chimerism, mice were depleted of cells expressing Kb by administration of the anti-Kb monoclonal antibody Y-3. Mice that were effectively depleted of cells expressing the retrovirally encoded MHC class I antigen rejected Kb disparate skin allografts. In contrast, control molecular chimeras accepted Kb disparate skin allografts indefinitely. These data suggest maintenance of tolerance in molecular chimeras requires long-term expression of retrovirally transduced alloantigen on the progeny of retrovirally transduced HSCs.