Sex Differences in Immunity.

Strong epidemiological evidence now exists that sex is an important biologic variable in immunity. Recent studies, for example, have revealed that sex differences are associated with the severity of symptoms and mortality due to coronavirus disease 2019 (COVID-19). Despite this evidence, much remains to be learned about the mechanisms underlying associations between sex differences and immune-mediated conditions. A growing body of experimental data has made significant inroads into understanding sex-influenced immune responses. As physicians seek to provide more targeted patient care, it is critical to understand how sex-defining factors (e.g., chromosomes, gonadal hormones) alter immune responses in health and disease. In this review, we highlight recent insights into sex differences in autoimmunity; virus infection, specifically severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; and cancer immunotherapy. A deeper understanding of underlying mechanisms will allow the development of a sex-based approach to disease screening and treatment.

The X-linked epigenetic regulator UTX controls NK cell-intrinsic sex differences.

Viral infection outcomes are sex biased, with males generally more susceptible than females. Paradoxically, the numbers of antiviral natural killer (NK) cells are increased in males. We demonstrate that while numbers of NK cells are increased in male mice, they display decreased effector function compared to females in mice and humans. These differences were not solely dependent on gonadal hormones, because they persisted in gonadectomized mice. Kdm6a (which encodes the protein UTX), an epigenetic regulator that escapes X inactivation, was lower in male NK cells, while NK cell-intrinsic UTX deficiency in female mice increased NK cell numbers and reduced effector responses. Furthermore, mice with NK cell-intrinsic UTX deficiency showed increased lethality to mouse cytomegalovirus. Integrative multi-omics analysis revealed a critical role for UTX in regulating chromatin accessibility and gene expression critical for NK cell homeostasis and effector function. Collectively, these data implicate UTX as a critical molecular determinant of sex differences in NK cells.

AIM2 in regulatory T cells restrains autoimmune diseases.

The inflammasome initiates innate defence and inflammatory responses by activating caspase-1 and pyroptotic cell death in myeloid cells1,2. It consists of an innate immune receptor/sensor, pro-caspase-1, and a common adaptor molecule, ASC. Consistent with their pro-inflammatory function, caspase-1, ASC and the inflammasome component NLRP3 exacerbate autoimmunity during experimental autoimmune encephalomyelitis by enhancing the secretion of IL-1ß and IL-18 in myeloid cells3-6. Here we show that the DNA-binding inflammasome receptor AIM27-10 has a T cell-intrinsic and inflammasome-independent role in the function of T regulatory (Treg) cells. AIM2 is highly expressed by both human and mouse Treg cells, is induced by TGFß, and its promoter is occupied by transcription factors that are associated with Treg cells such as RUNX1, ETS1, BCL11B and CREB. RNA sequencing, biochemical and metabolic analyses demonstrated that AIM2 attenuates AKT phosphorylation, mTOR and MYC signalling, and glycolysis, but promotes oxidative phosphorylation of lipids in Treg cells. Mechanistically, AIM2 interacts with the RACK1-PP2A phosphatase complex to restrain AKT phosphorylation. Lineage-tracing analysis demonstrates that AIM2 promotes the stability of Treg cells during inflammation. Although AIM2 is generally accepted as an inflammasome effector in myeloid cells, our results demonstrate a T cell-intrinsic role of AIM2 in restraining autoimmunity by reducing AKT-mTOR signalling and altering immune metabolism to enhance the stability of Treg cells.

The transcriptional regulator Aire coopts the repressive ATF7ip-MBD1 complex for the induction of immunotolerance.

The maintenance of immunological tolerance requires the deletion of self-reactive T cells in the thymus. The expression of genes encoding tissue-specific antigens (TSAs) by thymic epithelial cells is critical for this process and depends on activity of the transcriptional regulator Aire; however, the molecular mechanisms Aire uses to target loci encoding TSAs are unknown. Here we identified two Aire-interacting proteins known to be involved in gene repression, ATF7ip and MBD1, that were required for Aire's targeting of loci encoding TSAs. Moreover, Mbd1(-/-) mice developed pathological autoimmunity and had a defect in Aire-dependent thymic expression of genes encoding TSAs, which underscores the importance of Aire's interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.

Cutting Edge: Hypoxia Sensing by the Histone Demethylase UTX (KDM6A) Limits Colitogenic CD4+ T Cells in Mucosal Inflammation.

Hypoxia is a hallmark of inflammatory conditions (e.g., inflammatory bowel disease [IBD]), and adaptive responses have consequently evolved to protect against hypoxia-associated tissue injury. Because augmenting hypoxia-induced protective responses is a promising therapeutic approach for IBD, a more complete understanding of these pathways is needed. Recent work has demonstrated that the histone demethylase UTX is oxygen-sensitive, but its role in IBD is unclear. In this study, we show that hypoxia-induced deactivation of UTX downregulates T cell responses in mucosal inflammation. Hypoxia results in decreased T cell proinflammatory cytokine production and increased immunosuppressive regulatory T cells, and these findings are recapitulated by UTX deficiency. Hypoxia leads to T cell accumulation of H3K27me3 histone modifications, suggesting that hypoxia impairs UTX's histone demethylase activity to dampen T cell colitogenic activity. Finally, T cell-specific UTX deletion ameliorates colonic inflammation in an IBD mouse model, implicating UTX's oxygen-sensitive demethylase activity in counteracting hypoxic inflammation.

IL-11 induces NLRP3 inflammasome activation in monocytes and inflammatory cell migration to the central nervous system.

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes, and IL-11R+ neutrophils in comparison to matched healthy controls. IL-11+ and granulocyte-macrophage colony-stimulating factor (GM-CSF)+ monocytes, CD4+ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B. All CD4+ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R+-sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65+, NLRP3+, and IL-1ß+ monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

Pathogenic TNF-α drives peripheral nerve inflammation in an Aire-deficient model of autoimmunity.

Immune cells infiltrate the peripheral nervous system (PNS) after injury and with autoimmunity, but their net effect is divergent. After injury, immune cells are reparative, while in inflammatory neuropathies (e.g., Guillain Barré Syndrome and chronic inflammatory demyelinating polyneuropathy), immune cells are proinflammatory and promote autoimmune demyelination. An understanding of immune cell phenotypes that distinguish these conditions may, therefore, reveal new therapeutic targets for switching immune cells from an inflammatory role to a reparative state. In an autoimmune regulator (Aire)-deficient mouse model of inflammatory neuropathy, we used single-cell RNA sequencing of sciatic nerves to discover a transcriptionally heterogeneous cellular landscape, including multiple myeloid, innate lymphoid, and lymphoid cell types. Analysis of cell-cell ligand-receptor interactions uncovered a macrophage-mediated tumor necrosis factor-α (TNF-α) signaling axis that is induced by interferon-γ and required for initiation of autoimmune demyelination. Developmental trajectory visualization suggested that TNF-α signaling is associated with metabolic reprogramming of macrophages and polarization of macrophages from a reparative state in injury to a pathogenic, inflammatory state in autoimmunity. Autocrine TNF-α signaling induced macrophage expression of multiple genes (Clec4e, Marcksl1, Cxcl1, and Cxcl10) important in immune cell activation and recruitment. Genetic and antibody-based blockade of TNF-α/TNF-α signaling ameliorated clinical neuropathy, peripheral nerve infiltration, and demyelination, which provides preclinical evidence that the TNF-α axis may be effectively targeted to resolve inflammatory neuropathies.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

Inhibition of IL-17A Protects against Thyroid Immune-Related Adverse Events while Preserving Checkpoint Inhibitor Antitumor Efficacy.

Immune checkpoint inhibitor (ICI) immunotherapy leverages the body's own immune system to attack cancer cells but leads to unwanted autoimmune side effects in up to 60% of patients. Such immune-related adverse events (IrAEs) may lead to treatment interruption, permanent organ dysfunction, hospitalization, and premature death. Thyroiditis is one of the most common IrAEs, but the cause of thyroid IrAEs remains unknown. In this study, we use a new, physiologically relevant mouse model of ICI-associated autoimmunity to identify a key role for type 3 immune cells in the development of thyroid IrAEs. Multiple lineages of IL-17A-producing T cells expand in thyroid tissue with ICI treatment. Intrathyroidal IL-17A-producing innate-like γδT17 cells were increased in tumor-free mice, whereas adaptive Th17 cells were also prominent in tumor-bearing mice, following ICI treatment. Furthermore, Ab-based inhibition of IL-17A, a clinically available therapy, significantly reduced thyroid IrAE development in ICI-treated mice with and without tumor challenge. Finally, combination of IL-17A neutralization with ICI treatment in multiple tumor models did not reduce ICI antitumor efficacy. These studies suggest that targeting Th17 and γδT17 cell function via the IL-17A axis may reduce IrAEs without impairing ICI antitumor efficacy and may be a generalizable strategy to address type 3 immune-mediated IrAEs.

An Inconvenient Variable: Sex Hormones and Their Impact on T Cell Responses.

Epidemiologic data demonstrate sex differences in autoimmune diseases, immune responses against infection, and antitumor immunity, and accumulating evidence suggests a major role for sex hormones in mediating these differences. In this study, we review recent advances in understanding how sex hormones regulate T cell responses to alter susceptibility to autoimmunity. Although sex hormones can directly alter gene transcriptional programs of T cells, we focus in this study on how sex hormones alter T cell development and function through their effects on thymic stromal cells and innate cell types. In addition to contributing to our understanding of sex differences, these findings also have implications for the therapeutic use of sex hormones and sex hormone modulators, which are now being prescribed to increasing numbers of patients for a wide variety of indications.

COVID-19 Outcomes of Patients With Differentiated Thyroid Cancer: A Multicenter Los Angeles Cohort Study.

OBJECTIVE: Cancer may be a risk factor for worse outcomes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infections. However, there is a significant variability across cancer types in the extent of disease burden and modalities of cancer treatment that may impact morbidity and mortality from coronavirus disease-19 (COVID-19). Therefore, we evaluated COVID-19 outcomes in patients with a differentiated thyroid cancer (DTC) history. METHODS: This is a retrospective cohort study of patients with a history of DTC and SARS-CoV2 infection from 2 academic Los Angeles healthcare systems. Demographic, thyroid cancer, and treatment data were analyzed for associations with COVID-19 outcomes. RESULTS: Of 21 patients with DTC and COVID-19, 8 (38.1%) were hospitalized and 2 (9.5%) died from COVID-19. Thyroid cancer initial disease burden and extent, treatment, or current response to therapy (eg, excellent vs incomplete) were not associated with COVID-19 severity in DTC patients. However, older age and the presence of a comorbidity other than DTC were significantly associated with COVID-19 hospitalization (P = .047 and P = .024, respectively). COVID-19-attributed hospitalization and mortality in DTC patients was lower than that previously reported in cancer patients, although similar to patients with nonthyroid malignancies in these centers. CONCLUSION: These data suggest that among patients with DTC, advanced age and comorbid conditions are significant contributors to the risk of hospitalization from SARS-CoV2 infection, rather than factors associated with thyroid cancer diagnosis, treatment, or disease burden. This multicenter report of clinical outcomes provides additional data to providers to inform DTC patients regarding their risk of COVID-19.

BP180 dysfunction triggers spontaneous skin inflammation in mice.

BP180, also known as collagen XVII, is a hemidesmosomal component and plays a key role in maintaining skin dermal/epidermal adhesion. Dysfunction of BP180, either through genetic mutations in junctional epidermolysis bullosa (JEB) or autoantibody insult in bullous pemphigoid (BP), leads to subepidermal blistering accompanied by skin inflammation. However, whether BP180 is involved in skin inflammation remains unknown. To address this question, we generated a BP180-dysfunctional mouse strain and found that mice lacking functional BP180 (termed ΔNC16A) developed spontaneous skin inflammatory disease, characterized by severe itch, defective skin barrier, infiltrating immune cells, elevated serum IgE levels, and increased expression of thymic stromal lymphopoietin (TSLP). Severe itch is independent of adaptive immunity and histamine, but dependent on increased expression of TSLP by keratinocytes. In addition, a high TSLP expression is detected in BP patients. Our data provide direct evidence showing that BP180 regulates skin inflammation independently of adaptive immunity, and BP180 dysfunction leads to a TSLP-mediated itch. The newly developed mouse strain could be a model for elucidation of disease mechanisms and development of novel therapeutic strategies for skin inflammation and BP180-related skin conditions.

An essential role of the transcription factor GATA-3 for the function of regulatory T cells.

Forkhead Box P3 (Foxp3)-expressing regulatory T (Treg) cells are central to maintaining self-tolerance and immune homeostasis. How Treg cell function and Foxp3 expression are regulated is an important question under intensive investigation. Here, we have demonstrated an essential role for the transcription factor GATA-3, a previously recognized Th2 cell master regulator, in controlling Treg cell function. Treg cell-specific GATA-3 deletion led to a spontaneous inflammatory disorder in mice. GATA-3-null Treg cells were defective in peripheral homeostasis and suppressive function, gained Th17 cell phenotypes, and expressed reduced amounts of Foxp3. In addition, GATA-3 controlled Foxp3 expression by binding to and promoting the activity of cis-acting elements of Foxp3. Furthermore, the combined function of GATA-3 and Foxp3 was essential for Foxp3 expression. These findings provide insights into immune regulatory mechanisms and uncover a critical function of GATA-3 in Treg cells and immune tolerance.

IL-10 Paradoxically Promotes Autoimmune Neuropathy through S1PR1-Dependent CD4+ T Cell Migration.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is a debilitating condition caused by autoimmune demyelination of peripheral nerves. CIDP is associated with increased IL-10, a cytokine with well-described anti-inflammatory effects. However, the role of IL-10 in CIDP is unclear. In this study, we demonstrate that IL-10 paradoxically exacerbates autoimmunity against peripheral nerves. In IL-10-deficient mice, protection from neuropathy was associated with an accrual of highly activated CD4+ T cells in draining lymph nodes and absence of infiltrating immune cells in peripheral nerves. Accumulated CD4+ T cells in draining lymph nodes of IL-10-deficient mice expressed lower sphingosine-1-phosphate receptor 1 (S1pr1), a protein important in lymphocyte egress. Additionally, IL-10 stimulation in vitro induced S1pr1 expression in lymph node cells in a STAT3-dependent manner. Together, these results delineate a novel mechanism in which IL-10-induced STAT3 increases S1pr1 expression and CD4+ T cell migration to accelerate T cell-mediated destruction of peripheral nerves.

Dynamic metrics-based biomarkers to predict responders to anti-PD-1 immunotherapy.

BACKGROUND: Anti-PD-1 immunotherapies have shown clinical benefit in multiple cancers, but response was only observed in a subset of patients. Predicting which patients will respond is an urgent clinical need, but current companion diagnosis based on PD-L1 IHC staining shows limited predictability. METHODS: A dynamic, metrics-based biomarker was developed to discriminate responders from non-responders for anti-PD-1 immunotherapy in B16F10 melanoma-bearing mice. RESULTS: Similar to patients, there was considerable heterogeneity in response to anti-PD-1 immunotherapy in mice. Compared with the control group, 45% of anti-PD-1 antibody-treated mice displayed improved survival (defined as responders) and the remainder only gained little, if any, survival benefit from PD-1 blockade (non-responders). Interestingly, the dynamics of IFN-γ secretion by peripheral lymphocytes was associated with faster secretion onset (shorter lag time), stronger exponential phase, shorter time to half magnitude, and higher magnitude of secretion in responders at day 10 after tumour inoculation. To sufficiently predict responders from non-responders, IFN-γ secretion descriptors as well as phenotypic markers were subjected to multivariate analysis using orthogonal partial least-squares discriminant analysis (OPLS-DA). CONCLUSIONS: By integrating phenotypic markers, IFN-γ secretion descriptors sufficiently predict response to anti-PD-1 immunotherapy. Such a dynamic, metrics-based biomarker holds high diagnostic potential for anti-PD-1 checkpoint immunotherapy.

Correction: Dynamic metrics-based biomarkers to predict responders to anti-PD-1 immunotherapy.

Since the publication of this paper, the authors noticed that the funding information for Maureen A. Su was not included. Therefore the authors would like to add the following information to the Acknowledgements section.

Epigenetic Dysfunction in Turner Syndrome Immune Cells.

Turner syndrome (TS) is a chromosomal condition associated with partial or complete absence of the X chromosome that involves characteristic findings in multiple organ systems. In addition to well-known clinical characteristics such as short stature and gonadal failure, TS is also associated with T cell immune alterations and chronic otitis media, suggestive of a possible immune deficiency. Recently, ubiquitously transcribed tetratricopeptide repeat on the X chromosome (UTX), a histone H3 lysine 27 (H3K27) demethylase, has been identified as a downregulated gene in TS immune cells. Importantly, UTX is an X-linked gene that escapes X-chromosome inactivation and thus is haploinsufficient in TS. Mice with T cell-specific UTX deficiency have impaired clearance of chronic viral infection due to decreased frequencies of T follicular helper (Tfh) cells, which are critical for B cell antibody generation. In parallel, TS patients have decreased Tfh frequencies in peripheral blood. Together, these findings suggest that haploinsufficiency of the X-linked UTX gene in TS T cells underlies an immune deficit, which may manifest as increased predisposition to chronic otitis media.

Differential T cell responses to residual viral antigen prolong CD4+ T cell contraction following the resolution of infection.

The contraction phase of the T cell response is a poorly understood period after the resolution of infection when virus-specific effector cells decline in number and memory cells emerge with increased frequencies. CD8(+) T cells plummet in number and quickly reach stable levels of memory following acute lymphocytic choriomeningitis virus infection in mice. In contrast, virus-specific CD4(+) T cells gradually decrease in number and reach homeostatic levels only after many weeks. In this study, we provide evidence that MHCII-restricted viral Ag persists during the contraction phase following this prototypical acute virus infection. We evaluated whether the residual Ag affected the cell division and number of virus-specific naive and memory CD4(+) T cells and CD8(+) T cells. We found that naive CD4(+) T cells underwent cell division and accumulated in response to residual viral Ag for >2 mo after the eradication of infectious virus. Surprisingly, memory CD4(+) T cells did not undergo cell division in response to the lingering Ag, despite their heightened capacity to recognize Ag and make cytokine. In contrast to CD4(+) T cells, CD8(+) T cells did not undergo cell division in response to the residual Ag. Thus, CD8(+) T cells ceased division within days after the infection was resolved, indicating that CD8(+) T cell responses are tightly linked to endogenous processing of de novo synthesized virus protein. Our data suggest that residual viral Ag delays the contraction of CD4(+) T cell responses by recruiting new populations of CD4(+) T cells.