Metabolic modeling of single Th17 cells reveals regulators of autoimmunity.

Metabolism is a major regulator of immune cell function, but it remains difficult to study the metabolic status of individual cells. Here, we present Compass, an algorithm to characterize cellular metabolic states based on single-cell RNA sequencing and flux balance analysis. We applied Compass to associate metabolic states with T helper 17 (Th17) functional variability (pathogenic potential) and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known Th17/regulatory T cell (Treg) differences, which we validated by metabolic assays. Compass also predicted that Th17 pathogenicity was associated with arginine and downstream polyamine metabolism. Indeed, polyamine-related enzyme expression was enhanced in pathogenic Th17 and suppressed in Treg cells. Chemical and genetic perturbation of polyamine metabolism inhibited Th17 cytokines, promoted Foxp3 expression, and remodeled the transcriptome and epigenome of Th17 cells toward a Treg-like state. In vivo perturbations of the polyamine pathway altered the phenotype of encephalitogenic T cells and attenuated tissue inflammation in CNS autoimmunity.

Regulatory T Cells in Tumor-Associated Tertiary Lymphoid Structures Suppress Anti-tumor T Cell Responses.

Infiltration of regulatory T (Treg) cells into many tumor types correlates with poor patient prognoses. However, mechanisms of intratumoral Treg cell function remain to be elucidated. We investigated Treg cell function in a genetically engineered mouse model of lung adenocarcinoma and found that Treg cells suppressed anti-tumor responses in tumor-associated tertiary lymphoid structures (TA-TLSs). TA-TLSs have been described in human lung cancers, but their function remains to be determined. TLSs in this model were spatially associated with >90% of tumors and facilitated interactions between T cells and tumor-antigen-presenting dendritic cells (DCs). Costimulatory ligand expression by DCs and T cell proliferation rates increased in TA-TLSs upon Treg cell depletion, leading to tumor destruction. Thus, we propose that Treg cells in TA-TLSs can inhibit endogenous immune responses against tumors, and targeting these cells might provide therapeutic benefit for cancer patients.

Lupus Susceptibility Loci Predispose Mice to Clonal Lymphocytic Responses and Myeloid Expansion.

Lupus susceptibility results from the combined effects of numerous genetic loci, but the contribution of these loci to disease pathogenesis has been difficult to study due to the large cellular heterogeneity of the autoimmune immune response. We performed single-cell RNA, BCR, and TCR sequencing of splenocytes from mice with multiple polymorphic lupus susceptibility loci. We not only observed lymphocyte and myeloid expansion, but we also characterized changes in subset frequencies and gene expression, such as decreased CD8 and marginal zone B cells and increased Fcrl5- and Cd5l-expressing macrophages. Clonotypic analyses revealed expansion of B and CD4 clones, and TCR repertoires from lupus-prone mice were distinguishable by algorithmic specificity prediction and unsupervised machine learning classification. Myeloid differential gene expression, metabolism, and altered ligand-receptor interaction were associated with decreased Ag presentation. This dataset provides novel mechanistic insight into the pathophysiology of a spontaneous model of lupus, highlighting potential therapeutic targets for autoantibody-mediated disease.

CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.

T cell help in the autoreactive germinal center.

The germinal center serves as a site of B cell selection and affinity maturation, critical processes for productive adaptive immunity. In autoimmune disease tolerance is broken in the germinal center reaction, leading to production of autoreactive B cells that may propagate disease. Follicular T cells are crucial regulators of this process, providing signals necessary for B cell survival in the germinal center. Here, we review the emerging roles of follicular T cells in the autoreactive germinal center. Recent advances in immunological techniques have allowed study of the gene expression profiles and repertoire of follicular T cells at unprecedented resolution. These studies provide insight into the potential role follicular T cells play in preventing or facilitating germinal center loss of tolerance. Improved understanding of the mechanisms of T cell help in autoreactive germinal centers provides novel therapeutic targets for diseases of germinal center dysfunction.

Prior immunosuppressive therapy is associated with mortality in COVID-19 patients: A retrospective study of 835 patients.

Though it is widely believed that chronic immunosuppressive medications increase the severity of coronavirus disease 2019 (COVID-19) illness, there is little data to support this. We performed a retrospective study of COVID-19 positive patients diagnosed at a single academic medical center between March 10, 2020 and October 13, 2020. A total of 835 patients diagnosed with COVID-19 by polymerase chain reaction were included (median age 64 years; 52% female). Of these, 46 (5.5%) had a prescription for an immunosuppressive therapy before diagnosis, most commonly oral steroids (20, 43%), mycophenolate (12, 26%), or tacrolimus (11, 24%). Patients on immunosuppressive therapy with COVID-19 had increased mortality (30% vs. 17%, p = 0.036; odds ratio 2.1, 95% confidence interval 1.11-4.04), which remained significant (p = 0.040) after performing multivariate logistic regression controlling for gender, age, race, and comorbidity status. Laboratory markers of inflammation were uniformly elevated in both patients on or not on immunosuppressive therapies who died, but lymphocytes and neutrophils were decreased in both COVID-19 patients on immunosuppressive therapies who died and who remained alive. These findings demonstrate that COVID-19 disease is more severe in patients taking prior immunosuppressive medications. This finding emphasizes the need for aggressive monitoring and supportive care for immunosuppressed patients who are diagnosed with COVID-19.

Weighing the value of memory loss in the surgical evaluation of left temporal lobe epilepsy: a decision analysis.

OBJECTIVES: Anterior temporal lobectomy is curative for many patients with disabling medically refractory temporal lobe epilepsy, but carries an inherent risk of disabling verbal memory loss. Although accurate prediction of iatrogenic memory loss is becoming increasingly possible, it remains unclear how much weight such predictions should have in surgical decision making. Here we aim to create a framework that facilitates a systematic and integrated assessment of the relative risks and benefits of surgery versus medical management for patients with left temporal lobe epilepsy. METHODS: We constructed a Markov decision model to evaluate the probabilistic outcomes and associated health utilities associated with choosing to undergo a left anterior temporal lobectomy versus continuing with medical management for patients with medically refractory left temporal lobe epilepsy. Three base-cases were considered, representing a spectrum of surgical candidates encountered in practice, with varying degrees of epilepsy-related disability and potential for decreased quality of life in response to post-surgical verbal memory deficits. RESULTS: For patients with moderately severe seizures and moderate risk of verbal memory loss, medical management was the preferred decision, with increased quality-adjusted life expectancy. However, the preferred choice was sensitive to clinically meaningful changes in several parameters, including quality of life impact of verbal memory decline, quality of life with seizures, mortality rate with medical management, probability of remission following surgery, and probability of remission with medical management. SIGNIFICANCE: Our decision model suggests that for patients with left temporal lobe epilepsy, quantitative assessment of risk and benefit should guide recommendation of therapy. In particular, risk for and potential impact of verbal memory decline should be carefully weighed against the degree of disability conferred by continued seizures on a patient-by-patient basis.

Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.

T cell help shapes B cell tolerance.

T cell help is a crucial component of the normal humoral immune response, yet whether it promotes or restrains autoreactive B cell responses remains unclear. Here, we observe that autoreactive germinal centers require T cell help for their formation and persistence. Using retrogenic chimeras transduced with candidate TCRs, we demonstrate that a follicular T cell repertoire restricted to a single autoreactive TCR, but not a foreign antigen-specific TCR, is sufficient to initiate autoreactive germinal centers. Follicular T cell specificity influences the breadth of epitope spreading by regulating wild-type B cell entry into autoreactive germinal centers. These results demonstrate that TCR-dependent T cell help can promote loss of B cell tolerance and that epitope spreading is determined by TCR specificity.

Family history as the strongest predictor of aortic and peripheral aneurysms in patients with intracranial aneurysms.

OBJECTIVE: Intracranial aneurysms (IA) and aortic aneurysms (AA) are both abnormal dilations of arteries with familial predisposition and have been proposed to share co-prevalence and pathophysiology. Associations of IA and non-aortic peripheral aneurysms are less well-studied. The goal of the study was to understand the patterns of aortic and peripheral (extracranial) aneurysms in patients with IA, and risk factors associated with the development of these aneurysms. METHODS: 4701 patients were included in our retrospective analysis of all patients with intracranial aneurysms at our institution over the past 26 years. Patient demographics, comorbidities, and aneurysmal locations were analyzed. Univariate and multivariate analyses were performed to study associations with and without extracranial aneurysms. RESULTS: A total of 3.4% of patients (161 of 4701) with IA had at least one extracranial aneurysm. 2.8% had thoracic or abdominal aortic aneurysms. Age, male sex, hypertension, coronary artery disease, history of ischemic cerebral infarction, connective tissues disease, and family history of extracranial aneurysms in a 1st degree relative were associated with the presence of extracranial aneurysms and a higher number of extracranial aneurysms. In addition, family history of extracranial aneurysms in a second degree relative is associated with the presence of extracranial aneurysms and atrial fibrillation is associated with a higher number of extracranial aneurysms. CONCLUSION: Significant comorbidities are associated with extracranial aneurysms in patients with IA. Family history of extracranial aneurysms has the strongest association and suggests that IA patients with a family history of extracranial aneurysms may benefit from screening.

Complex subsets but redundant clonality after B cells egress from spontaneous germinal centers.

Affinity matured self-reactive antibodies are found in autoimmune diseases like systemic lupus erythematous. Here, we used fate-mapping reporter mice and single-cell transcriptomics coupled to antibody repertoire analysis to characterize the post-germinal center (GC) B cell compartment in a new mouse model of autoimmunity. Antibody-secreting cells (ASCs) and memory B cells (MemBs) from spontaneous GCs grouped into multiple subclusters. ASCs matured into two terminal clusters, with distinct secretion, antibody repertoire and metabolic profiles. MemBs contained FCRL5+ and CD23+ subsets, with different in vivo localization in the spleen. GC-derived FCRL5+ MemBs share transcriptomic and repertoire properties with atypical B cells found in aging and infection and localize to the marginal zone, suggesting a similar contribution to recall responses. While transcriptomically diverse, ASC and MemB subsets maintained an underlying clonal redundancy. Therefore, self-reactive clones could escape subset-targeting therapy by perpetuation of self-reactivity in distinct subsets.

Regulation of immunological tolerance by the p53-inhibitor iASPP.

Maintenance of immunological homeostasis between tolerance and autoimmunity is essential for the prevention of human diseases ranging from autoimmune disease to cancer. Accumulating evidence suggests that p53 can mitigate phagocytosis-induced adjuvanticity thereby promoting immunological tolerance following programmed cell death. Here we identify Inhibitor of Apoptosis Stimulating p53 Protein (iASPP), a negative regulator of p53 transcriptional activity, as a regulator of immunological tolerance. iASPP-deficiency promoted lung adenocarcinoma and pancreatic cancer tumorigenesis, while iASPP-deficient mice were less susceptible to autoimmune disease. Immune responses to iASPP-deficient tumors exhibited hallmarks of immunosuppression, including activated regulatory T cells and exhausted CD8+ T cells. Interestingly, iASPP-deficient tumor cells and tumor-infiltrating myeloid cells, CD4+, and γδ T cells expressed elevated levels of PD-1H, a recently identified transcriptional target of p53 that promotes tolerogenic phagocytosis. Identification of an iASPP/p53 axis of immune homeostasis provides a therapeutic opportunity for both autoimmune disease and cancer.

p53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation-driven pancreatic cancer.

Oncogenic KRAS activation, inflammation and p53 mutation are key drivers of pancreatic cancer (PC) development. Here we report iASPP, an inhibitor of p53, as a paradoxical suppressor of inflammation and oncogenic KRASG12D-driven PC tumorigenesis. iASPP suppresses PC onset driven by KRASG12D alone or KRASG12D in combination with mutant p53R172H. iASPP deletion limits acinar-to-ductal metaplasia (ADM) in vitro but accelerates inflammation and KRASG12D-induced ADM, pancreatitis and PC tumorigenesis in vivo. KRASG12D/iASPPΔ8/Δ8 tumours are well-differentiated classical PCs and their derivative cell lines form subcutaneous tumours in syngeneic and nude mice. Transcriptomically, either iASPP deletion or p53 mutation in the KRASG12D background altered the expression of an extensively overlapping gene set, comprised primarily of NF-κB and AP1-regulated inflammatory genes. All these identify iASPP as a suppressor of inflammation and a p53-independent oncosuppressor of PC tumorigenesis.

Unbiased identification of clinical characteristics predictive of COVID-19 severity.

There is currently limited clinical ability to identify COVID-19 patients at risk for severe outcomes. To unbiasedly identify metrics associated with severe outcomes in COVID-19 patients, we conducted a retrospective study of 835 COVID-19 positive patients at a single academic medical center between March 10, 2020 and October 13, 2020. As of December 1, 2020, 656 (79%) patients required hospitalization and 149 (18%) died. Unbiased comparisons of all clinical characteristics and mortality revealed that abnormal pH (OR 8.54, 95% CI 5.34-13.6), abnormal creatinine (OR 6.94, 95% CI 4.22-11.4), and abnormal PTT (OR 4.78, 95% CI 3.11-7.33) were most significantly associated with mortality. Correlation with ordinal severity scores confirmed these associations, in addition to associations between respiratory rate (Spearman's rho  = -0.56), absolute neutrophil count (Spearman's rho  = -0.5), and C-reactive protein (Spearman's rho  =  0.59) with disease severity. Unsupervised principal component analysis and machine learning model classification of patient demographics, laboratory results, medications, comorbidities, signs and symptoms, and vitals are capable of separating patients on the basis of COVID-19 mortality (AUC 0.82). This retrospective analysis identifies laboratory and clinical metrics most relevant to predict COVID-19 severity.

Dosimetric Modeling of Lymphopenia in Patients With Metastatic Cancer Receiving Palliative Radiation and PD-1 Immune Checkpoint Inhibitors.

PURPOSE: Radiation therapy (RT)-associated lymphopenia may adversely affect treatment outcomes, particularly in the era of immunotherapy. We sought to determine dosimetric factors correlated with lymphopenia after palliative RT in a cohort of patients with advanced cancer treated with anti-PD-1 immune checkpoint inhibitors. METHODS AND MATERIALS: We included patients with metastatic lung cancer, melanoma, or renal cell carcinoma who were treated with either pembrolizumab or nivolumab and received palliative RT to an extracranial site. Baseline and nadir absolute lymphocyte counts (ALCs) within 6 weeks of RT were recorded. Dosimetric factors were extracted from the corresponding dose-volume histograms and also used to model the dose to circulating lymphocytes via a whole-body blood flow model that simulates the spatiotemporal distribution of blood particles in major organs during RT. RESULTS: We analyzed 55 patients who underwent 80 total courses of palliative RT; most (94%) were treated with 3-dimensional conformal RT. Doses to the whole body, bone, and large blood vessels (LBVs) were negatively correlated with the ALC nadir, with the strongest correlations seen at V15 (rs, -0.38, -0.43, and -0.37, and P = .0004, .0001, and .0008, respectively). Doses to other organs were not significantly correlated with the ALC nadir. The modeled dose to circulating lymphocytes was also negatively correlated with the ALC nadir and percent ALC change (for D2%, rs, -0.31 and -0.44, and P = .005 and .0001, respectively). Grade ≥3 lymphopenia was associated with LBV V15 (odds ratio [OR], 1.16; 95% CI, 1.07-1.26; P < .001), bone V15 (OR, 1.04; 95% CI, 1.01-1.08; P = .03), body V15 (OR, 1.003; 95% CI, 1.001-1.006; P = .008), and modeled lymphocyte dose (OR, 1.45; 95% CI, 1.16-1.82; P < .001). CONCLUSIONS: The RT dose to the whole body, bone, and LBVs and the modeled dose to circulating lymphocytes were correlated with lymphopenia in patients treated with palliative RT and anti-PD-1 immune checkpoint inhibitors. These findings may inform future radiation planning in this setting.

Prospective Clinical Investigation of the Efficacy of Combination Radiation Therapy With Immune Checkpoint Inhibition.

Immune checkpoint inhibitors (ICIs) lead to durable responses in a subset of patients with cancer, but most patients do not respond to ICI, prompting interest in combining immunotherapy with other therapeutic regimens. Preclinical evidence supports the potential for therapeutic synergy between immunotherapy and radiation therapy through modulation of the tumor microenvironment and antitumor immune responses. Local therapy also has the potential to overcome localized sites of relative immune suppression and resistance. Prospective clinical trials have been initiated to test these hypotheses in the clinic as well as to investigate the toxicities and adverse events associated with combination immunotherapy and radiation therapy. In this review, we discuss the emerging results from prospective clinical trials of combination immunotherapy and radiation therapy, the safety and efficacy of their combination, concordance with preclinical and retrospective data, and some of the remaining open questions to be addressed by future clinical trials.

Follicular T cells are clonally and transcriptionally distinct in B cell-driven mouse autoimmune disease.

Pathogenic autoantibodies contribute to tissue damage and clinical decline in autoimmune disease. Follicular T cells are central regulators of germinal centers, although their contribution to autoantibody-mediated disease remains unclear. Here we perform single cell RNA and T cell receptor (TCR) sequencing of follicular T cells in a mouse model of autoantibody-mediated disease, allowing for analyses of paired transcriptomes and unbiased TCRαß repertoires at single cell resolution. A minority of clonotypes are preferentially shared amongst autoimmune follicular T cells and clonotypic expansion is associated with differential gene signatures in autoimmune disease. Antigen prediction using algorithmic and machine learning approaches indicates convergence towards shared specificities between non-autoimmune and autoimmune follicular T cells. However, differential autoimmune transcriptional signatures are preserved even amongst follicular T cells with shared predicted specificities. These results demonstrate that follicular T cells are phenotypically distinct in B cell-driven autoimmune disease, providing potential therapeutic targets to modulate autoantibody development.

Outcomes of a Student-Led Telemedicine Clinic in Response to COVID-19.

In response to the coronavirus disease-2019 (COVID-19) pandemic, we developed and launched a student-led telemedicine program in Chelsea. From April to November 2020, over 200 student volunteers contacted over 1000 patients to assess COVID-19 symptoms, provide counseling, and triage patients. Through a retrospective cohort study, we determined that student triage decision was associated with patient outcomes, including hospitalization status, COVID-19 test administration, and COVID-19 test result. These results quantify the outcomes of a student-led telemedicine clinic to combat the ongoing pandemic and may serve as a model for implementation of similar clinics to alleviate mounting health care system burden.

Inducible de novo expression of neoantigens in tumor cells and mice.

Inducible expression of neoantigens in mice would enable the study of endogenous antigen-specific naïve T cell responses in disease and infection, but has been difficult to generate because leaky antigen expression in the thymus results in central T cell tolerance. Here we develop inversion-induced joined neoantigen (NINJA), using RNA splicing, DNA recombination and three levels of regulation to prevent leakiness and allow tight control over neoantigen expression. We apply NINJA to create tumor cell lines with inducible neoantigen expression, which could be used to study antitumor immunity. We also show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cell responses on neoantigen induction in peripheral tissues. NINJA will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, transplantation, autoimmune diseases and cancer.

Rlf-Mycl Gene Fusion Drives Tumorigenesis and Metastasis in a Mouse Model of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf-Mycl-driven mouse model of SCLC. RLF-MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF-MYCL genetically engineered mouse model displayed gene expression similarities with human RLF-MYCL SCLC. Together, our studies support RLF-MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. SIGNIFICANCE: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF-MYCL gene fusion by developing a Rlf-Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945.