Project description:HLA DRB1*15:01 is overrepresentated in Parkinson's disease patients and binds with high affinity to the ⍺-synuclein peptide, 32-46. Immunization of humanized mice expressing HLA DRB1*15:01 with ⍺-syn32-46 induces enteric phenotypes similar to those of prodromal Parkinson's disease. We collected the ileum from HLA mice immunized with either Complete Freund's Adjuvant (CFA) with ⍺-syn32-46 or CFA alone, and sequenced the tissue with bulk RNA sequencing at 21 days post immunization to determine changes in gene expression.
Project description:HLA DRB1*15:01 is overrepresentated in Parkinson's disease patients and binds with high affinity to the ⍺-synuclein peptide, 32-46. Immunization of humanized mice expressing HLA DRB1*15:01 with ⍺-syn32-46 induces enteric phenotypes similar to those of prodromal Parkinson's disease. We collected the small intestines from HLA mice immunized with either Complete Freund's Adjuvant (CFA) with ⍺-syn32-46 or CFA alone, sorted the CD45+ immune cells, then performed single-cell RNA sequencing to evaluate gene expression at single cell resolution.
Project description:Few non-classical HLA-E restricted HIV-specific epitopes have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope (KAFSPEVIPMF or KF11) based on their restriction by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from 8 people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8s were analyzed through single-cell (sc) RNA and TCR sequencing. Additionally, supernatants were analyzed for soluble proteomics using a Luminex assay. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFNγ, while E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27confirmed through scRNAseq. Despite distinct cytokine profiles, TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03. In vitro T cell reporter assays clearly demonstrated this cross-restriction. A TRAV5-containing metaclonotype cluster was seen in PWH with lower viral loads. These findings demonstrate that HIV-specific CD8s in PWH exhibit cross HLA-B*57 and HLA-E*01/03 restriction, resulting in functionally distinct immune responses that may contribute to HIV control.
Project description:Few non-classical HLA-E restricted HIV-specific epitopes have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope (KAFSPEVIPMF or KF11) based on their restriction by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from 8 people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8s were analyzed through single-cell (sc) RNA and TCR sequencing. Additionally, supernatants were analyzed for soluble proteomics using a Luminex assay. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFNγ, while E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27confirmed through scRNAseq. Despite distinct cytokine profiles, TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03. In vitro T cell reporter assays clearly demonstrated this cross-restriction. A TRAV5-containing metaclonotype cluster was seen in PWH with lower viral loads. These findings demonstrate that HIV-specific CD8s in PWH exhibit cross HLA-B*57 and HLA-E*01/03 restriction, resulting in functionally distinct immune responses that may contribute to HIV control.
Project description:HLA-DR15 is a haplotype associated with multiple sclerosis. It contains the two DRB* genes DRB1*1501 (DR2b) and DRB5*0101 (DR2a). The reported anchor motif of the corresponding HLA-DR molecules was determined years ago based on a small number of peptide ligands and binding assays. DR2a could display a set of peptides complementary to that presented by DR2b or, alternatively, a similar peptide repertoire but recognized in a different manner by T cells. It is known that DR2a and DR2b share some peptide ligands, although the degree of similarity of their associated peptidomes remains unclear. In addition, the contribution of each molecule to the global peptide repertoire presented by the HLA-DR15 haplotype has not been evaluated. We used mass spectrometry to analyze the peptide pools bound to DR2a and DR2b, identifying 169 and 555 unique peptide ligands of DR2a and DR2b, respectively. The analysis of these sets of peptides allowed the refinement of the corresponding binding motifs revealing novel anchor residues that had been overlooked in previous analyses. Moreover, the number of shared ligands between both molecules was low, indicating that DR2a and DR2b present complementary peptide repertoires to T cells. Finally, our analysis suggests that, quantitatively, both molecules contribute to the peptide repertoire presented by cells expressing the HLA-DR15 haplotype.