CD4+ T cells reactive to a hybrid peptide from insulin-chromogranin A adopt a distinct effector fate and are pathogenic in autoimmune diabetes.

T cell-mediated islet destruction is a hallmark of autoimmune diabetes. Here, we examined the dynamics and pathogenicity of CD4+ T cell responses to four different insulin-derived epitopes during diabetes initiation in non-obese diabetic (NOD) mice. Single-cell RNA sequencing of tetramer-sorted CD4+ T cells from the pancreas revealed that islet-antigen-specific T cells adopted a wide variety of fates and required XCR1+ dendritic cells for their activation. Hybrid-insulin C-chromogranin A (InsC-ChgA)-specific CD4+ T cells skewed toward a distinct T helper type 1 (Th1) effector phenotype, whereas the majority of insulin B chain and hybrid-insulin C-islet amyloid polypeptide-specific CD4+ T cells exhibited a regulatory phenotype and early or weak Th1 phenotype, respectively. InsC-ChgA-specific CD4+ T cells were uniquely pathogenic upon transfer, and an anti-InsC-ChgA:IAg7 antibody prevented spontaneous diabetes. Our findings highlight the heterogeneity of T cell responses to insulin-derived epitopes in diabetes and argue for the feasibility of antigen-specific therapies that blunts the response of pathogenic CD4+ T cells causing autoimmunity.

Limited proliferation capacity of aortic intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression.

Early atherosclerosis depends upon responses by immune cells resident in the intimal aortic wall. Specifically, the healthy intima is thought to be populated by vascular dendritic cells (DCs) that, during hypercholesterolemia, initiate atherosclerosis by being the first to accumulate cholesterol. Whether these cells remain key players in later stages of disease is unknown. Using murine lineage-tracing models and gene expression profiling, we reveal that myeloid cells present in the intima of the aortic arch are not DCs but instead specialized aortic intima resident macrophages (MacAIR) that depend upon colony-stimulating factor 1 and are sustained by local proliferation. Although MacAIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaques is overtaken by recruited monocytes, which induce MacAIR-defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

Adoptive T Cell Therapy with IL-12-Preconditioned Low-Avidity T Cells Prevents Exhaustion and Results in Enhanced T Cell Activation, Enhanced Tumor Clearance, and Decreased Risk for Autoimmunity.

Optimal ex vivo expansion protocols of tumor-specific T cells followed by adoptive cell therapy must yield T cells able to home to tumors and effectively kill them. Our previous study demonstrated ex vivo activation in the presence of IL-12-induced optimal CD8+ T cell expansion and melanoma regression; however, adverse side effects, including autoimmunity, can occur. This may be due to transfer of high-avidity self-specific T cells. In this study, we compared mouse low- and high-avidity T cells targeting the tumor Ag tyrosinase-related protein 2 (TRP2). Not surprisingly, high-avidity T cells provide superior tumor control, yet low-avidity T cells can promote tumor regression. The addition of IL-12 during in vitro expansion boosts low-avidity T cell responsiveness, tumor regression, and prevents T cell exhaustion. In this study, we demonstrate that IL-12-primed T cells are resistant to PD-1/PD-L1-mediated suppression and retain effector function. Importantly, IL-12 preconditioning prevented exhaustion as LAG-3, PD-1, and TOX were decreased while simultaneously increasing KLRG1. Using intravital imaging, we also determined that high-avidity T cells have sustained contacts with intratumoral dendritic cells and tumor targets compared with low-avidity T cells. However, with Ag overexpression, this defect is overcome, and low-avidity T cells control tumor growth. Taken together, these data illustrate that low-avidity T cells can be therapeutically beneficial if cocultured with IL-12 cytokine during in vitro expansion and highly effective in vivo if Ag is not limiting. Clinically, low-avidity T cells provide a safer alternative to high-avidity, TCR-engineered T cells, as IL-12-primed, low-avidity T cells cause less autoimmune vitiligo.

The Role of Programmed Death-1 in Type 1 Diabetes.

PURPOSE OF REVIEW: Programmed death-1 (PD-1) is an inhibitory receptor that controls T and B cell proliferation and function through interacting with its ligand PD-L1 or PD-L2. PD-1/PD-L1 blockade reboots anti-tumor immunity and is currently used to treat > 15 different types of cancer. However, the response rate is not at 100% and some patients relapse. Importantly, up to 37% of patients treated with PD-1/PD-L1 blocking antibodies develop immune-related adverse events, including overt autoimmunity, such as type 1 diabetes (T1D). Herein, we discuss the role of PD-1, PD-L1, and PD-L2 signaling in pre-clinical models of T1D, including recent work from our laboratory. RECENT FINDINGS: We highlight ongoing efforts to harness PD-1/PD-L1 signaling and treat autoimmunity. We also evaluate studies aimed at defining biomarkers that could reliably predict the development of immune-related adverse events after clinical PD-1/PD-L1 blockade. With increasing use of PD-1 blockade in the clinic, onset of autoimmunity is a growing health concern. In this review, we discuss what is known about the role of PD-1 pathway signaling in T1D and comment on ongoing efforts to identify patients at risk of T1D development after PD-1 pathway blockade.

Programmed Death-1 Restrains the Germinal Center in Type 1 Diabetes.

Programmed death-1 (PD-1) inhibits T and B cell function upon ligand binding. PD-1 blockade revolutionized cancer treatment, and although numerous patients respond, some develop autoimmune-like symptoms or overt autoimmunity characterized by autoantibody production. PD-1 inhibition accelerates autoimmunity in mice, but its role in regulating germinal centers (GC) is controversial. To address the role of PD-1 in the GC reaction in type 1 diabetes, we used tetramers to phenotype insulin-specific CD4+ T and B cells in NOD mice. PD-1 or PD-L1 deficiency, and PD-1 but not PD-L2 blockade, unleashed insulin-specific T follicular helper CD4+ T cells and enhanced their survival. This was concomitant with an increase in GC B cells and augmented insulin autoantibody production. The effect of PD-1 blockade on the GC was reduced when mice were treated with a mAb targeting the insulin peptide:MHC class II complex. This work provides an explanation for autoimmune side effects following PD-1 pathway inhibition and suggests that targeting the self-peptide:MHC class II complex might limit autoimmunity arising from checkpoint blockade.

Enhanced CD4+ and CD8+ T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses.

The notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4+ and CD8+ T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate immune cell infiltration into pancreatic islets of Langerhans for the study of disease progression or therapeutic efficacy in murine T1D. However, the accuracy of this approach is often limited by subjective and potentially biased qualitative assessment of immune cell subsets. In addition, attempts at quantitative measurements require significant time for manual analysis and often involve sophisticated and expensive imaging software. In this study, we developed and illustrate here a streamlined analytical strategy for the rapid, automated and unbiased investigation of islet area and immune cell infiltration within (insulitis) and around (peri-insulitis) pancreatic islets. To this end, we demonstrate swift and accurate detection of islet borders by modeling cross-sectional islet areas with convex polygons (convex hulls) surrounding islet-associated insulin-producing ß cell and glucagon-producing α cell fluorescent signals. To accomplish this, we used a macro produced with the freeware software ImageJ equipped with the Fiji Is Just ImageJ (FIJI) image processing package. Our image analysis procedure allows for direct quantification and statistical determination of islet area and infiltration in a reproducible manner, with location-specific data that more accurately reflect islet areas as insulitis proceeds throughout T1D. Using this approach, we quantified the islet area infiltrated with CD4+ and CD8+ T cells allowing statistical comparison between different age groups of non-obese diabetic (NOD) mice progressing towards T1D. We found significantly more CD4+ and CD8+ T cells infiltrating the convex hull-defined islet mass of 13-week-old non-diabetic and 17-week-old diabetic NOD mice compared to 4-week-old NOD mice. We also determined a significant and measurable loss of islet mass in mice that developed T1D. This approach will be helpful for the location-dependent quantitative calculation of islet mass and cellular infiltration during T1D pathogenesis and can be combined with other markers of inflammation or activation in future studies.

Interferon-gamma drives programmed death-ligand 1 expression on islet ß cells to limit T cell function during autoimmune diabetes.

Type 1 diabetes is caused by autoreactive T cell-mediated ß cell destruction. Even though co-inhibitory receptor programmed death-1 (PD-1) restrains autoimmunity, the expression and regulation of its cognate ligands on ß cell remains unknown. Here, we interrogated ß cell-intrinsic programmed death ligand-1 (PD-L1) expression in mouse and human islets. We measured a significant increase in the level of PD-L1 surface expression and the frequency of PD-L1+ ß cells as non-obese diabetic (NOD) mice aged and developed diabetes. Increased ß cell PD-L1 expression was dependent on T cell infiltration, as ß cells from Rag1-deficient mice lacked PD-L1. Using Rag1-deficient NOD mouse islets, we determined that IFN-γ promotes ß cell PD-L1 expression. We performed analogous experiments using human samples, and found a significant increase in ß cell PD-L1 expression in type 1 diabetic samples compared to type 2 diabetic, autoantibody positive, and non-diabetic samples. Among type 1 diabetic samples, ß cell PD-L1 expression correlated with insulitis. In vitro experiments with human islets from non-diabetic individuals showed that IFN-γ promoted ß cell PD-L1 expression. These results suggest that insulin-producing ß cells respond to pancreatic inflammation and IFN-γ production by upregulating PD-L1 expression to limit self-reactive T cells.

STAT3 Signaling Is Required for Optimal Regression of Large Established Tumors in Mice Treated with Anti-OX40 and TGFß Receptor Blockade.

In preclinical tumor models, αOX40 therapy is often successful at treating small tumors, but is less effective once the tumors become large. For a tumor immunotherapy to be successful to cure large tumors, it will most likely require not only an agonist to boost effector T-cell function but also inhibitors of T-cell suppression. In this study, we show that combining αOX40 antibodies with an inhibitor of the TGFß receptor (SM16) synergizes to elicit complete regression of large established MCA205 and CT26 tumors. Evaluation of tumor-infiltrating T cells showed that SM16/αOX40 dual therapy resulted in an increase in proliferating granzyme B(+) CD8 T cells, which produced higher levels of IFNγ, compared with treatment with either agent alone. We also found that the dual treatment increased pSTAT3 expression in both CD4 and CD8 T cells isolated from tumors. Because others have published that STAT3 signaling is detrimental to T-cell function within the tumor microenvironment, we explored whether deletion of STAT3 in OX40-expressing cells would affect this potent combination therapy. Surprisingly, we found that deletion of STAT3 in OX40-expressing cells decreased the efficacy of this combination therapy, showing that the full therapeutic potential of this treatment depends on STAT3 signaling, most likely in the T cells of tumor-bearing mice.

Detailed characterization of tumor infiltrating lymphocytes in two distinct human solid malignancies show phenotypic similarities.

BACKGROUND: We examined the phenotype and function of lymphocytes collected from the peripheral blood (PBL) and tumor (TIL) of patients with two different solid malignancies: colorectal cancer liver metastases (CRLM) and ovarian cancer (OVC). METHODS: Tumor and corresponding peripheral blood were collected from 16 CRLM and 22 OVC patients; immediately following resection they were processed and analyzed using a multi-color flow cytometry panel. Cytokine mRNA from purified PBL and TIL CD4(+) T cells were also analyzed by qPCR. RESULTS: Overall, we found similar changes in the phenotypic and cytokine profiles when the TIL were compared to PBL from patients with two different malignancies. The percentage of Treg (CD4(+)/CD25(+)/FoxP3(+)) in PBL and TIL was similar: 8.1% versus 10.2%, respectively in CRLM patients. However, the frequency of Treg in primary OVC TIL was higher than PBL: 19.2% versus 4.5% (p <0.0001). A subpopulation of Treg expressing HLA-DR was markedly increased in TIL compared to PBL in both tumor types, CRLM: 69.0% versus 31.7% (p = 0.0002) and OVC 74.6% versus 37.0% (p <0.0001), which suggested preferential Treg activation within the tumor. The cytokine mRNA profile showed that IL-6, a cytokine known for its immunosuppressive properties through STAT3 upregulation, was increased in TIL samples in patients with OVC and CRLM. Both TIL populations also contained a significantly higher proportion of activated CD8(+) T cells (HLA-DR(+)/CD38(+)) compared to PBL (CRLM: 30.2% vs 7.7%, (p = 0.0012), OVC: 57.1% vs 12.0%, (p <0.0001)). CONCLUSION: This study demonstrates that multi-color flow cytometry of freshly digested tumor samples reveals phenotypic differences in TIL vs PBL T cell sub-populations. The TIL composition in primary and metastatic tumors from two distinct histologies were remarkably similar, showing a greater proportion of activated/suppressive Treg (HLA-DR(+), CD39(+), CTLA-4(+) and Helios(+)) and activated cytotoxic T cells (CD8(+)/HLA-DR(+)/CD38(+)) when compared to PBL and an increase in IL-6 mRNA from CD4 TIL.