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Effective CD8(+) T cell responses depend on presentation of a stable peptide repertoire by MHC class I (MHC I) molecules on the cell surface. The overall quality of peptide-MHC I complexes (pMHC I) is determined by poorly understood mechanisms that generate and load peptides with appropriate consensus motifs onto MHC I. In this article, we show that both tapasin (Tpn), a key component of the peptide loading complex, and the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP) are quintessential editors of distinct structural features of the peptide repertoire. We carried out reciprocal immunization of wild-type mice with cells from Tpn- or ERAAP-deficient mice. Specificity analysis of T cell responses showed that absence of Tpn or ERAAP independently altered the peptide repertoire by causing loss as well as gain of new pMHC I. Changes in amino acid sequences of MHC-bound peptides revealed that ERAAP and Tpn, respectively, defined the characteristic amino and carboxy termini of canonical MHC I peptides. Thus, the optimal pMHC I repertoire is produced by two distinct peptide editing steps in the endoplasmic reticulum.
Cytotoxic T lymphocytes (CTLs) are critical for control of respiratory syncytial virus (RSV) infection in humans and mice. To investigate cellular immune responses to infection, it is important to identify major histocompatibility complex (MHC) class I-restricted CTL epitopes. In this study, we identified a new RSV-specific, H-2K(d)-restricted subdominant epitope in the M2 protein, M2(127-135) (amino acids 127 to 135). This finding allowed us to study the frequency of T lymphocytes responding to two H-2K(d)-presented epitopes in the same protein following RSV infection by enzyme-linked immunospot (ELISPOT) and intracellular cytokine assays for both lymphoid and nonlymphoid tissues. For the subdominant epitope, we identified an optimal nine-amino-acid peptide, VYNTVISYI, which contained an H-2K(d) consensus sequence with Y at position 2 and I at position 9. In addition, an MHC class I stabilization assay using TAP-2-deficient RMA-S cells transfected with K(d) or L(d) indicated that the epitope was presented by K(d). The ratios of T lymphocytes during the peak CTL response to RSV infection that were specific for M2(82-90) (dominant) to T lymphocytes specific for M2(127-135) (subdominant) were approximately 3:1 in the spleen and 10:1 in the lung. These ratios were observed consistently in primary or secondary infection by the ELISPOT assay and in secondary infection by MHC/peptide tetramer staining. The number of antigen-specific T lymphocytes dropped in the 6 weeks after infection; however, the proportions of T lymphocytes specific for the immunodominant and subdominant epitopes were maintained to a remarkable degree in a tissue-specific manner. These studies will facilitate investigation of the regulation of immunodominance of RSV-specific CTL epitopes.
Unlike T cells restricted by major histocompatibility complex (MHC) class Ia or class II molecules, T cells restricted by MHC class I-like molecules demonstrate properties of both innate and adaptive immunity and are therefore considered innate-like lymphocytes (ILLs). ILLs are believed to have immunoregulatory functions, but their roles in autoimmunity and defense against infections remain elusive. To study the properties of ILLs, we generated mice expressing only MHC class I-like molecules by crossing CIITA-/- with Kb-/-Db-/- mice. Surprisingly, these mice developed a lymphoproliferative syndrome and autoimmunity, most notably inflammatory bowel disease (IBD) and insulitis. The CD8+ ILLs in these mice exhibit a constitutively activated phenotype, and depletion of these cells abolished the autoimmune disorders. In addition, adoptive transfer of CD8+ ILLs from Kb-/-Db-/-CIITA-/- mice to Rag-1-/-pfn-/- mice also resulted in IBD and insulitis. These findings provide direct evidence that CD8+ ILLs are sufficient to initiate and mediate autoimmune diseases.
Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present several self- and foreign antigens to Kb-, Db-, or Qa-1b-restricted CD8+ cytotoxic T cells, presentation of some antigens was unaffected or significantly enhanced. Consistent with these findings, mice generated defective CD8+ T cell responses against class I-presented antigens. These findings reveal an important in vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.
Hepatic expression levels of class I MHC Ags are generally regarded as very low. Because the status of these Ags and their ability to present peptides are important for the understanding of pathogen clearance and tolerogenic properties of the liver, we set out to identify the factors contributing to the reported phenotype. Unexpectedly, we found that the surface densities of K(b) and D(b) on C57BL/6 mouse hepatocytes are nearly as high as on splenocytes, as are the lysate concentrations of mRNA encoding H chain and beta(2)-microglobulin (beta(2)m). In contrast, the components of the peptide-loading pathway are reduced in hepatocytes. Despite the difference in the stoichiometric ratios of H chain/beta(2)m/peptide-loading machineries, both cell types express predominantly thermostable class I and are critically dependent on TAP and tapasin for display of surface Ags. Minor differences in the expression patterns in tapasin(-/-) background suggest cell specificity in class I assembly. Under immunostimulatory conditions, such as exposure to IFN-gamma or Listeria monocytogenes, hepatocytes respond with a vigorous mRNA synthesis of the components of the Ag presentation pathway (up to 10-fold enhancement) but up-regulate H chain and beta(2)m to a lesser degree (<2-fold). This type of response should promote rapid influx of newly generated peptides into the endoplasmic reticulum and preferential presentation of foreign/induced Ag by hepatic class I.
Cytotoxic T lymphocytes (CTL) play a significant role in the clearance of respiratory syncytial virus (RSV) infection in humans and mice. Identification of class I MHC-restricted CTL epitopes is critical in elucidating mechanisms of CTL responses against viral infections. However, only four H-2d-restricted epitopes have been reported in mice. Because of the diversity of transgenic and knockout mice available to study immune responses, new epitopes in additional strains of mice must be identified. We therefore attempted to discover novel CTL epitopes in C57Bl/6 mice. Our efforts revealed a new H-2D(b)-restricted CTL epitope from the RSV M protein, corresponding to aa 187-195 (NAITNAKII). Also, M187-195-specific CTLs were activated with kinetics similar to the immunodominant BALB/c epitope, M2 82-90. This is the first RSV-specific CTL epitope described in a strain of mice other than BALB/c. Furthermore, identification of this H-2b-restricted CTL epitope provides access to genetically modified H-2b mice for more detailed studies of CTL mechanisms in RSV infection.
The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.
Copyright 2004 The American Association of Immunologists, Inc.
Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin(-/-) cells and experiments with tapasin(-/-) and TAP1(-/-) macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin(-/-) cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin(-/-) cells might be peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both tapasin(-/-) and TAP1(-/-) macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin(-/-) and TAP1(-/-) macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin(-/-) or TAP1(-/-) cells at 26 degrees C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments.
Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.
Our previous results showed that TAP1 mutant mice rejected heart and skin grafts from donors with no H-2 disparity that express normal density of MHC class I molecules at the cell surface. During rejection, CD4 cells were predominant and essentially, no CD8 cells were found infiltrating the grafts. We hypothesized that TAP1 mutant mice, which developed and matured in an MHC class I-deficient environment, may have selected a repertoire of T cells with distinct reactivity to self class I molecules. The rejection of grafts with no H-2 disparity could be mediated by CD4+ T cells reactive to wild type H-2b class I molecules, or derived peptides, in the context of self-APC. Accordingly, we observed that transplanted TAP1 mutant mice presented a significant amplification of the proliferative T cell response to H-2Kb peptides, indicating that the stimulus with the graft was sufficient to induce peripheral expansion of these T cell repertoires. Therefore, the response to H-2Kb molecules could be a relevant pathway of activating T cells and triggering rejection of grafts expressing normal levels of these class I molecules. To test our hypothesis, we investigate the effect of pre-transplantation H-2Kb peptide-immunization on TAP1 mutant, which were then transplanted with C57BL/6 skin grafts (H-2b). Mice were immunized with a pool of five peptides derived from the polymorphic region of Kb alpha chain, before tail skin grafting. To study the role of CD4+ T cells in the rejection of C57BL/6 skin grafts, mice were in vivo depleted with an anti-CD4 monoclonal antibody GK1.5, and transplant evolution was observed. Sensitization of TAP1 mutant mice with H-2Kb peptides accelerated the rejection of skin grafts. Immunized mice rejected grafts with a MST of 13 days, compared to 16 days for the non-immunized mice (P=0.0089). The significant acceleration of graft rejection, induced by immunization with H-2Kb peptides, indicates that these peptides are capable of mobilizing effector T-cells that participate in rejection. These results support our hypothesis that class I molecules may be a target in the rejection of grafts with no MHC disparity. Depletion of CD4 T-cells resulted in a significant delay in rejection compared with the untreated control group. The MST of skin grafts in the controls was 16 days, whereas CD4-depleted recipients rejected skin grafts with a MST of 41 days (P=0.025). Moreover, some animals did not show macroscopic signs of rejection up to > 100 days posttransplantation. The contribution of CD4+ T cells to skin graft rejection, in our model, may reflect the occurrence of the presentation of H-2b peptides during graft rejection, in the context of self-APC. In conclusion, our results demonstrate an important role for H-2b molecules and CD4 T cells in the rejection of C57BL/6 grafts by TAP1 mutant mice. The low expression of MHC-I molecules on TAP1-/- mice may be determinant in the selection of a T cell repertoire strongly reactive to self MHC class I molecules which probably escapes the control of peripheral regulatory mechanisms.