RESUMO
MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8(+) T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database.
Assuntos
Biologia Computacional , Epitopos , Antígenos HLA-C/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Bases de Dados Factuais , Expressão Gênica , Antígenos HLA-A/química , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Antígenos HLA-B/química , Antígenos HLA-B/imunologia , Antígenos HLA-B/metabolismo , Antígenos HLA-C/química , Antígenos HLA-C/imunologia , Humanos , Radioisótopos do Iodo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Família Multigênica , Biblioteca de Peptídeos , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human ß2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8(+) T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ-producing CD8(+) T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8(+) T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell-based vaccines.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Genes MHC Classe I , Animais , Epitopos de Linfócito T/imunologia , Feminino , Antígeno HLA-A1/biossíntese , Antígeno HLA-A1/genética , Antígeno HLA-A24/biossíntese , Antígeno HLA-A24/genética , Antígeno HLA-B27/biossíntese , Antígeno HLA-B27/genética , Antígeno HLA-B35/biossíntese , Antígeno HLA-B35/genética , Antígeno HLA-B44/biossíntese , Antígeno HLA-B44/genética , Antígeno HLA-B8/biossíntese , Antígeno HLA-B8/genética , Antígenos HLA-C/biossíntese , Antígenos HLA-C/genética , Humanos , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos AnimaisRESUMO
HFE, an MHC class Ib molecule that controls iron metabolism, can be directly targeted by cytotoxic TCR αß T lymphocytes. Transgenic DBA/2 mice expressing, in a Rag 2 KO context, an αß TCR that directly recognizes mouse HFE (mHFE) were created to further explore the interface of HFE with the immune system. TCR-transgenic mHfe WT mice deleted mHFE-reactive T cells in the thymus, but a fraction of reprogrammed cells were able to escape deletion. In contrast, TCR-transgenic mice deprived of mHFE molecules (mHfe KO mice) or expressing a C282âY mutated mHFE molecule - the most frequent mutation associated with human hereditary hemochromatosis - positively selected mHFE-reactive CD8(+) T lymphocytes and were not tolerant toward mHFE. By engrafting these mice with DBA/2 WT (mHFE(+)) skin, it was established, as suspected on the basis of similar engraftments performed on DBA/2 mHfe KO mice, that mHFE behaves as an autonomous skin-associated histocompatibility antigen, even for mHFE-C282âY mutated mice. By contrast, infusion of DBA/2 mHFE(+) mice with naïve mHFE-reactive transgenic CD8(+) T lymphocytes did not induce GVHD. Thus, tolerance toward HFE in mHfe WT mice can be acquired at either thymic or peripheral levels but is disrupted in mice reproducing human familial hemochromatosis.