Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling

BACKGROUND T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain RESULTS Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data, we identify differentially expressed candidate genes, including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. CONCLUSION The data provide a molecular map of the negative selection response in vivo and, by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death.This work was supported by grants from the NHMRC and the Juvenile Diabetes Research Foundation

Response to COVID-19 vaccination in patients on cancer therapy:Analysis in a SARS-CoV-2-naïve population

Background: Cancer patients have increased morbidity and mortality from COVID-19, but may respond poorly to vaccination. The Evaluation of COVID-19 Vaccination Efficacy and Rare Events in Solid Tumors (EVEREST) study, comparing seropositivity between cancer patients and healthy controls in a low SARS-CoV-2 community-transmission setting, allows determination of vaccine response with minimal interference from infection. Methods: Solid tumor patients from The Canberra Hospital, Canberra, Australia, and healthy controls who received COVID-19 vaccination between March 2021 and January 2022 were included. Blood samples were collected at baseline, pre-second vaccine dose and at 1, 3 (primary endpoint), and 6 months post-second dose. SARS-CoV-2 anti-spike-RBD (S-RBD) and anti-nucleocapsid IgG antibodies were measured. Results: Ninety-six solid tumor patients and 20 healthy controls were enrolled, with median age 62 years, and 60% were female. Participants received either AZD1222 (65%) or BNT162b2 (35%) COVID-19 vaccines. Seropositivity 3 months post vaccination was 87% (76/87) in patients and 100% (20/20) in controls (p =.12). Seropositivity was observed in 84% of patients on chemotherapy, 80% on immunotherapy, and 96% on targeted therapy (differences not satistically significant). Seropositivity in cancer patients increased from 40% (6/15) after first dose, to 95% (35/37) 1 month after second dose, then dropped to 87% (76/87) 3 months after second dose. Conclusion: Most patients and all controls became seropositive after two vaccine doses. Antibody concentrations and seropositivity showed a decrease between 1 and 3 months post vaccination, highlighting need for booster vaccinations. SARS-CoV-2 infection amplifies S-RBD antibody responses; however, cannot be adequately identified using nucleocapsid serology. This underlines the value of our COVID-naïve population in studying vaccine immunogenicity.</p

Early T Cell Infiltration Correlates with Anti-CTLA4 Treatment Response in Murine Cancer Models

Immune checkpoint inhibitor (ICI) Abs are a revolutionary class of cancer treatment, but only ~30% of patients receive a lasting benefit from therapy. Preclinical studies using animals from the same genetic backgrounds, challenged with the same cancer models, also show nonuniform responses. Most mouse studies that have evaluated tumor-infiltrating leukocytes after ICI therapy cannot directly correlate their findings with treatment outcomes, because terminal methods were used to acquire immune infiltrate data. In the present study, we used fine-needle aspiration (a nonterminal sampling method) to collect multiple aspirates over several days from s.c. implanted P815, CT26, and 4T1 mouse cancer models treated with ICI Abs. These aspirates were then analyzed with flow cytometry to directly correlate tumor-infiltrating leukocyte populations with treatment success. We found that the P815 and CT26 models respond well to anti-CTLA4 therapies. Among P815-challenged animals, mice that regressed following anti-CTLA4 treatment showed significant increases in CD8+ T cells on days 3, 5, and 7 and in CD4+ T cells on days 5 and 7 and a decrease in macrophages and monocytes on days 3, 5, and 7 after treatment. Similar results were obtained in the CT26 model on day 11 posttreatment. Our study is the first, to our knowledge, to directly correlate early tumor infiltration of T cells with anti-CTLA4 treatment success, thus providing a mechanistic clue toward understanding why alloidentical mice challenged with identical tumors do not respond uniformly to ICI therapies.</p

Analysis of A47, an Immunoprevalent Protein of Vaccinia Virus, Leads to a Reevaluation of the Total Antiviral CD8+ T Cell Response▿

Vaccinia virus (VACV) is the prototypic orthopoxvirus and was the live vaccine used to eradicate smallpox. In addition, VACV is a possible vector for recombinant vaccines. Despite these reasons for study, the roles of many VACV genes are unknown, and some fundamental aspects, such as the total size of immune responses, remain poorly characterized. VACV gene A47L is of interest because it is highly transcribed, has no sequence similarity to any nonpoxvirus gene, and contains a larger-than-expected number of CD8+ T cell epitopes. Here it is shown that A47L is not required for growth in vitro and does not contribute to virulence in mice. However, we confirmed that this one protein primes CD8+ T cells to three different epitopes in C57BL/6 mice. In the process, one of these epitopes was redefined and shown to be the most dominant in A47 and one of the more highly ranked in VACV as a whole. The relatively high immunogenicity of this epitope led to a reevaluation of the total CD8+ T cell response to VACV. By the use of two methods, the true size of the response was found to be around double previous estimates and at its peak is on the order of 60% of all CD8+ T cells. We speculate that more CD8+ T cell epitopes remain to be mapped for VACV and that underestimation of responses is unlikely to be unique to VACV, so there would be merit in revisiting this issue for other viruses

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling

Background: T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain.status: publishe