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The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4+ memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of “private” TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4+ T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4+ memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.

Type 1 Diabetes (T1D) is caused by beta cell destruction, eventually resulting in loss of glycemic control. Following onset, subjects with T1D exhibit diverse amounts of residual c-peptide, indicating varying levels of beta cell function. Persistence of c-peptide is correlated with reduced risk of complications and its preservation has been used as an endpoint in clinical trials. However, the immunologic factors that differentiate subjects who retain measurable c-peptide from those who do not are not well characterized. We investigated the number and phenotype of beta cell specific CD8+ T-cells in peripheral blood samples from a cohort of T1D subjects, stratified into slow progressors (c-peptide > 0.1ng/ml) and rapid progressors (undetectable c-peptide), including longitudinal samples. Using tetramers corresponding to HLA-A2 epitopes, we enumerated epitope specific T-cells and sorted single cells for RNA-Seq. Differential gene expression between slow and rapid progressors indicated differences in effector, exhaustion, and regulatory pathways which were consistent with a recently published high dimensional CyTOF data set. Assembly of autoantigen specific TCRs revealed differences in clonal expansion for specific cell clusters. Pseudotime trajectory analysis of the scRNAseq data revealed that CD8+ T cells expressing the same TCR resided in different phenotypic end states. Furthermore, flow cytometric analyses demonstrated differential expression of lineage-specific cell surface markers, which correlated well with the transcriptional profiles within each cluster. Cumulatively, we discovered specific aspects of T cell function and repertoire that differ between slow- and rapid-progressing T1D subjects.

CD8 T Cell exhaustion is associated with poor clinical outcome in cancer and chronic infection, but positive outcome in autoimmunity. Recently, phenotypic and functional heterogeneity within exhausted CD8 T cells suggests lineage subsets. We observed similar heterogeneity in the phenotype of exhausted cells in the context of T1D. Here we further explore the phenotypic and functional features of exhausted subsets, and ask about correlation with features of T1D. Using a CyTOF panel, we identified 12 major phenotypes (clusters) among total CD8 T cells in T1D subjects. Four of these clusters were characterized by features of exhaustion, namely PD-1, EOMES, KLRG-1, and TIGIT expression. Of these four, a CD127 high, but Granzyme B and TBET low cluster was enriched for islet-specific autoreactive CD8 T cells, and was associated with slower disease progression. As compared with other exhausted clusters, CD127-high exhausted cells proliferated more and produced increased IFNγ and TNFα upon in vitro anti-CD3 stimulation, measured by flow cytometry. This phenotype and functionality is consistent with a stem-like exhausted population. In comparison, the other three other clusters expressed higher TBET and Granzyme B but lower CD127, two of which were enriched for insulin-specific cells. This is consistent with a transitory exhausted phenotype driven by repeated antigen exposure as occurs with insulin treatment of T1D. Thus, varying stages of exhaustion are present in T1D and the stem-like exhausted phenotype may be beneficial in slowing disease progression in T1D. Understanding exhausted CD8 T cell heterogeneity and the role these subsets play in T1D will lead to improved therapies.

Studies conducted in mice and humans have shown that IFN-γ+ CD8 T cell are the key effectors against liver-stage (LS) malaria; however, owing to complexities associated with the development of Plasmodia parasites, specificities of CD8 T cell effectors have been difficult to establish. Direct detection of Ag-specific CD8 T cells in high-throughput manner became possible with the development of caged MHC-tetramer technology, based on conditional photo-cleavable MHC class I ligands that can be displaced during UV induced exchange with a peptide of interest. On the basis of previously identified P. falciparum (Pf) genes expressed exclusively during LS infection, we identified P. berghei (Pb) orthologues of Pf genes and validated their expression by qRT-PCR in Pb infected C57Bl/6 mice. Several Pb LS antigens, administered as DNA vaccines, significantly reduced LS parasite burden in mice infected with Pb sporozoites. Using sequences of the 28 novel Pb LS genes we screened ~400 peptides by the caged MHC class I tetramer approach. Our preliminary results demonstrate that 2 H-2Kb- and 1 H-2Db-restricted Pb peptides that correspond to protective Pb LS Ags form MHC class I tetramers that specifically bound to CD8 T cells from mice immunized with radiation-attenuated Pb sporozoites. Identification of these epitopes is crucial towards resolving the role of antigen-specific CD8 T cell responses and establishing these responses as correlates of protection to Plasmodia infection.

A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by deletion of 2 pre-erythrocytic genes, was administered to 6 subjects by bites from Anopheles mosquitoes. Previous studies of immune responses elicited by recombinant malaria vaccines show that protection is linked to Ag-specific T cells producing IFN-γ, TNF, and/or IL-2. In this study we asked if Pf GAP vaccine induced Ag-specific T cell responses characterized by inflammatory cytokines. We analyzed pre- and post vaccination PBMC for Ag-specific T cell responses to well characterized pre-erythrocytic and erythrocytic P. falciparum protein and peptide Ags. Additionally, we examined responses to several novel Pf liver-stage Ag (Pf LSA) because Pf LSA-dependent immunity might contribute to GAP-induced protection. IFN-γ and TNF responses to multiple antigens were significantly enhanced post vaccination; IFN-γ responses were primarily attributed to CD8+ T cells while TNF was secreted primarily by CD4+ T cells. Notably, a subject with a breakthrough peripheral parasitemia did show positive IFN-γ responses to erythrocytic-stage Ags, but no responses were recalled with the novel Pf LSA. In summary, two exposures to a Pf GAP vaccine induced persisting T cell responses specific for Pf LSA in humans. Future trials that assess the efficacy of Pf GAP vaccines should examine the role of LSA-specific responses in protection.