STAT3 regulates cytotoxicity of human CD57+ CD4+ T cells in blood and lymphoid follicles

A subset of human follicular helper T cells (TFH) cells expresses CD57 for which no distinct function has been identified. We show that CD57+ TFH cells are universally PD-1hi, but compared to their CD57− PD-1hi counterparts, express little IL-21 or IL-10 among others. Instead, CD57 expression on TFH cells marks cytotoxicity transcriptional signatures that translate into only a weak cytotoxic phenotype. Similarly, circulating PD-1+ CD57+ CD4+ T cells make less cytokine than their CD57− PD-1+ counterparts, but have a prominent cytotoxic phenotype. By analysis of responses to STAT3-dependent cytokines and cells from patients with gain- or loss-of-function STAT3 mutations, we show that CD4+ T cell cytotoxicity is STAT3-dependent. TFH formation also requires STAT3, but paradoxically, once formed, PD-1hi cells become unresponsive to STAT3. These findings suggest that changes in blood and germinal center cytotoxicity might be affected by changes in STAT3 signaling, or modulation of PD-1 by therapy.This work was supported by National Health and Medical Research Council (Australia) program grants APP1016953 and APP427620 (CGV, CCG, SGT, MCC)

Impact of a delayed second dose of mRNA vaccine (BNT162b2) and inactivated SARS-CoV-2 vaccine (CoronaVac) on risks of all-cause mortality, emergency department visit, and unscheduled hospitalization

BACKGROUND: Safety after the second dose of the SARS-CoV-2 vaccine remains to be elucidated, especially among individuals reporting adverse events after their first dose. This study aims to evaluate the impact of a delayed second dose on all-cause mortality and emergency services. METHODS: A territory-wide, retrospective cohort of people who had completed two doses of mRNA (BNT162b2) or inactivated SARS-CoV-2 (CoronaVac) vaccine between February 23 and July 3, 2021, in Hong Kong was analyzed, with linkage to electronic health records retrieved from the Hong Kong Hospital Authority. Vaccine recipients were classified as receiving a second dose within recommended intervals (21-28 days for BNT162b2; 14-28 days for CoronaVac) or delayed. Study outcomes were all-cause mortality, emergency department (ED) visits, and unscheduled hospitalizations within 28 days after the second dose of vaccination. RESULTS: Among 417,497 BNT162b2 and 354,283 CoronaVac second dose recipients, 3.8% and 28.5% received the second dose beyond the recommended intervals (mean 34.4 and 31.8 days), respectively. During the study period, there were < 5 daily new cases of COVID-19 infections in the community. Delaying the second dose was not associated with all-cause mortality (hazard ratio [HR] = 1.185, 95% CI 0.478-2.937, P = 0.714), risk of ED visit (HR = 0.966, 95% CI 0.926-1.008, P = 0.113), and risk of unscheduled hospitalization (HR = 0.956, 95% CI 0.878-1.040, P = 0.294) compared to that within the recommended interval for CoronaVac recipients. No statistically significant differences in all-cause mortality (HR = 4.438, 95% CI 0.951-20.701, P = 0.058), ED visit (HR = 1.037, 95% CI 0.951-1.130, P = 0.411), and unscheduled hospitalization (HR = 1.054, 95% CI 0.867-1.281, P = 0.597) were identified between people who received a second dose of BNT162b2 within and beyond the recommended intervals. CONCLUSIONS: No significant association between delayed second dose of BNT162b2 or CoronaVac and all-cause mortality, ED visit, and unscheduled hospitalization was observed in the present cohort. Regardless of the recommended or delayed schedule for SARS-CoV-2 vaccination, a second dose of both vaccines should be administered to obtain better protection against infection and serious disease. The second dose should be administered within the recommended interval following the manufacturer's product information, until further studies support the benefits of delaying vaccination outweighing the risks

Enteropathy-associated T cell lymphoma subtypes are characterized by loss of function of SETD2

Enteropathy-associated T cell lymphoma (EATL) is a lethal, and the most common, neoplastic complication of celiac disease. Here, we defined the genetic landscape of EATL through whole-exome sequencing of 69 EATL tumors. SETD2 was the most frequently silenced gene in EATL (32% of cases). The JAK-STAT pathway was the most frequently mutated pathway, with frequent mutations in STAT5B as well as JAK1 , JAK3 , STAT3 , and SOCS1 . We also identified mutations in KRAS , TP53 , and TERT . Type I EATL and type II EATL (monomorphic epitheliotropic intestinal T cell lymphoma) had highly overlapping genetic alterations indicating shared mechanisms underlying their pathogenesis. We modeled the effects of SETD2 loss in vivo by developing a T cell–specific knockout mouse. These mice manifested an expansion of γδ T cells, indicating novel roles for SETD2 in T cell development and lymphomagenesis. Our data render the most comprehensive genetic portrait yet of this uncommon but lethal disease and may inform future classification schemes

Characterisation of a Novel Human IL-21R Mutation

Inborn errors of immunity (IEI) encompass a group of inherited disorders that manifest in various forms, including increased susceptibility to infection, allergy, autoinflammatory or autoimmune disease, and malignancy. Rapid advances in genomics have enabled the identification of more causative mutations that can confer either loss- or gain-of-function upon the affected translated protein. Identification of new inborn errors of immunity can provide explanations for patients without a clear diagnosis. Functional characterisation of variants that confer altered function can provide important biological insights. Inborn errors of immunity affecting different genes can present with similar clinical and cellular phenotypes, and this can provide insight into interactions within biochemical pathways that regulate those phenotypes. IL-21 is a pleiotropic cytokine important for immune regulation and T and B cell effector functions. Several reports describing germline loss-of-function mutations in either IL-21 or IL21R revealed the significance of IL-21 signaling in T cell-dependent B cell activation, germinal centre reactions, and humoral immunity. However, there are currently no reports on hypermorphic mutations affecting IL-21 signaling. In this thesis, I describe the immunological consequences of a novel IL21R mutation identified in a kindred with common variable immunodeficiency (CVID) characterised by hypogammaglobulinemia and susceptibility to fungal and mycobacterial infections. The novel variant is a missense mutation substituting a serine 492 for arginine in the translated protein. Using a combination of ex vivo phenotyping and in vitro functional assays, I identified a distinct expansion in the circulating follicular helper T cells of the patients and a robust response to B cell proliferative cues antithetically accompanied by a functional impairment in plasmablast formation. I employed a bespoke mouse model bearing the orthologous Il21r mutation to demonstrate and test the effect of this variant in primary and secondary lymphoid organs. Extensive phenotyping of cellular subsets from the various lymphoid organs revealed an expansion in the germinal centre B cells and follicular helper T cell subsets. Additionally, in vitro assays of naive CD4+ T cells from IL21R mutant mice responded more robustly to Th1 and Th17 polarising stimuli. However, introducing recombinant IL-21 to these conditions resulted in reduced differentiation of Th1 cells but not Th17. Overall, these phenotypic features recapitulate and extend the immunological phenotype identified in the patients. Using biochemical analysis of splenocytes from the IL21R mouse model, I demonstrated a delay in STAT1 and STAT3 dephosphorylation in cells carrying the novel IL21R variant, consistent with a gain-of-function mutation. Having established that PBMCs from STAT1 and STAT3 GoF patients have impaired plasmablast formation similar to our IL21R mutant patients, I induced plasmablasts from PBMCs from healthy donors in the presence of cytokines predominantly signalling through either STAT1 or STAT3. Surprisingly, our findings indicate that additional STAT1 and STAT3 signals diminish plasmablast formation in response to IL-21. In summary, my thesis describes and demonstrates the effect of a missense mutation in IL21R on T and B cell subsets in both humans and mice. The characterisation of this IL21RS492R mutation will be the first description of an IEI caused by a hypermorphic IL21R variant. Furthermore, my thesis highlights the importance of maintaining the fragile balance between STAT1 and STAT3 signaling for IL-21-induced B cell differentiation, which has broad implications for immunity and immune modulating therapy

IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model.

Acknowledgements: The authors thank the Australian Phenomics Facility staff for husbandry and genotyping, and the Flow Cytometry facility, Biomolecular Resource Facility, the Phenomics Translation Initiative team, and the ANU Bioinformatics Consultancy at the John Curtin School of Medical Research for their services. This work was funded by the National Health and Medical Research Council Program Grant APP1113577 (CGV, MCC), CRE APP1079648 (CGV, MCC) and Project Grant APP1107464 (MCC); the Alan Harvey CVID Research Endowment (CC); Royal Society Wolfson Fellowship RSWF\R2\222004 (MCC). This study utilized the Australian Phenomics Network Histopathology and Organ Pathology Service of the University of Melbourne. The Phenomics Translation Initiative is supported by the Medical Research Future Fund (EPCD000035).Funder: Alan Harvey CVID Research EndowmentLoss-of-function mutations have provided crucial insights into the immunoregulatory actions of Foxp3+ regulatory T cells (Tregs). By contrast, we know very little about the consequences of defects that amplify aspects of Treg function or differentiation. Here we show that mice heterozygous for an Ikbkb gain-of-function mutation develop psoriasis. Doubling the gene dose (IkbkbGoF/GoF) results in dactylitis, spondylitis, and characteristic nail changes, which are features of psoriatic arthritis. IkbkbGoF mice exhibit a selective expansion of Foxp3 + CD25+ Tregs of which a subset express IL-17. These modified Tregs are enriched in both inflamed tissues, blood and spleen, and their transfer is sufficient to induce disease without conventional T cells. Single-cell transcriptional and phenotyping analyses of isolated Tregs reveal expansion of non-lymphoid tissue (tissue-resident) Tregs expressing Th17-related genes, Helios, tissue-resident markers including CD103 and CD69, and a prominent NF-κB transcriptome. Thus, IKK2 regulates tissue-resident Treg differentiation, and overactivity drives dose-dependent skin and systemic inflammation

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4 + T cells

Acknowledgements: We thank Harpreet Vohra and Michael Devoy at the Flow Cytometry Facility and Maxim Nekrasov at the Australian Cancer Research Foundation (ACRF) Biomolecular Resource Facility of the John Curtin School of Medical Research for technical support and Ann-Maree Hatch and Anastasia Wilson for assistance with obtaining blood and tonsil samples. We thank Dominik Spensberger and Gaetan Burgio at John Curtin School of Medical Research for their help with mouse model construction. The study was supported by NHMRC grants APP1113577 (MCC, CGV) and APP1079648 (MCC, CGV), and grant APP1130330 awarded through the Priority-drive Collaborative Cancer Research Scheme and funded by Cancer Australia (MCC, DY, SY).As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells.

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection

Enteropathy-associated T cell lymphoma subtypes are characterized by loss of function of SETD2.

Enteropathy-associated T cell lymphoma (EATL) is a lethal, and the most common, neoplastic complication of celiac disease. Here, we defined the genetic landscape of EATL through whole-exome sequencing of 69 EATL tumors. SETD2 was the most frequently silenced gene in EATL (32% of cases). The JAK-STAT pathway was the most frequently mutated pathway, with frequent mutations in STAT5B as well as JAK1, JAK3, STAT3, and SOCS1 We also identified mutations in KRAS, TP53, and TERT Type I EATL and type II EATL (monomorphic epitheliotropic intestinal T cell lymphoma) had highly overlapping genetic alterations indicating shared mechanisms underlying their pathogenesis. We modeled the effects of SETD2 loss in vivo by developing a T cell-specific knockout mouse. These mice manifested an expansion of γδ T cells, indicating novel roles for SETD2 in T cell development and lymphomagenesis. Our data render the most comprehensive genetic portrait yet of this uncommon but lethal disease and may inform future classification schemes