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Exhausted CD8 T cells (T EX ) are a distinct state of T cell differentiation associated with failure to clear chronic viruses and cancer. Immunotherapies such as PD-1 blockade can reinvigorate T EX cells, but reinvigoration is not durable. A major unanswered question is whether T EX cells differentiate into functional durable memory T cells (T MEM ) upon antigen clearance. Here, using a mouse model, we found that upon eliminating chronic antigenic stimulation, T EX cells partially (re)acquire phenotypic and transcriptional features of T MEM cells. These ‘recovering’ T EX cells originated from the T cell factor (TCF-1 + ) T EX progenitor subset. Nevertheless, the recall capacity of these recovering T EX cells remained compromised as compared to T MEM cells. Chromatin-accessibility profiling revealed a failure to recover core memory epigenetic circuits and maintenance of a largely exhausted open chromatin landscape. Thus, despite some phenotypic and transcriptional recovery upon antigen clearance, exhaustion leaves durable epigenetic scars constraining future immune responses. These results support epigenetic remodeling interventions for T EX cell–targeted immunotherapies. Wherry and colleagues examine whether exhausted T cells (T EX ) can differentiate into functional memory T cells (T MEM ) when chronic antigen is withdrawn. Using the chronic LCMV infection mouse model, they show that ‘recovering’ T EX cells (REC-T EX ) only partially recover immunophenotypic and functional characteristics of T MEM cells. The epigenomic status of REC-T EX cells more closely resembles that of T EX cells, and, upon rechallenge, the REC-T EX cells were still compromised in their ability to respond to virus.

SARS-CoV-2 messenger RNA vaccination in healthy individuals generates immune protection against COVID-19. However, little is known about SARS-CoV-2 mRNA vaccine-induced responses in immunosuppressed patients. We investigated induction of antigen-specific antibody, B cell and T cell responses longitudinally in patients with multiple sclerosis (MS) on anti-CD20 antibody monotherapy ( n  = 20) compared with healthy controls ( n  = 10) after BNT162b2 or mRNA-1273 mRNA vaccination. Treatment with anti-CD20 monoclonal antibody (aCD20) significantly reduced spike-specific and receptor-binding domain (RBD)-specific antibody and memory B cell responses in most patients, an effect ameliorated with longer duration from last aCD20 treatment and extent of B cell reconstitution. By contrast, all patients with MS treated with aCD20 generated antigen-specific CD4 and CD8 T cell responses after vaccination. Treatment with aCD20 skewed responses, compromising circulating follicular helper T (T FH ) cell responses and augmenting CD8 T cell induction, while preserving type 1 helper T (T H 1) cell priming. Patients with MS treated with aCD20 lacking anti-RBD IgG had the most severe defect in circulating T FH responses and more robust CD8 T cell responses. These data define the nature of the SARS-CoV-2 vaccine-induced immune landscape in aCD20-treated patients and provide insights into coordinated mRNA vaccine-induced immune responses in humans. Our findings have implications for clinical decision-making and public health policy for immunosuppressed patients including those treated with aCD20. SARS-CoV-2-specific antibodies and memory B cells are significantly reduced, but CD4 + and CD8 + T cells are robustly activated, in patients with multiple sclerosis on anti-CD20 monotherapy versus healthy controls after BNT162b2 or mRNA-1273 mRNA vaccination.

Immunological tolerance has evolved to curtail immune responses against self-antigens and prevent autoimmunity. One mechanism that contributes to immunological tolerance is the expression of inhibitory receptors by lymphocytes that signal to dampen immune responses during the course of an infection and to prevent immune-mediated collateral damage to the host. The understanding that tumors exploit these physiological mechanisms to avoid elimination has led to remarkable, but limited, success in the treatment of cancer through the use of biologics that interfere with the ability of cancers to suppress immune function. This therapy, based on the understanding of how T lymphocytes are normally activated and suppressed, has led to the development of therapeutic blocking antibodies, referred to as immune checkpoint blockade, which either directly or indirectly promote the activation of CD8 T cells to eradicate cancer. Here, we highlight the distinct signaling mechanisms, timing and location of inhibition used by the CTLA-4 and PD-1 inhibitory receptors compared to a novel inhibitory signaling axis comprised of the bioactive lipid, lysophosphatidic acid (LPA), signaling via the LPA5 receptor expressed by CD8 T cells. Importantly, abundant evidence indicates that an LPA-LPA5 signaling axis is also exploited by diverse cancers to suppress T cell activation and function. Clearly, a thorough molecular and biochemical understanding of how diverse T cell inhibitory receptors signal to suppress T cell antigen receptor signaling and function will be important to inform the choice of which complimentary checkpoint blockade modalities might be used for a given cancer.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Recent advances in cytometry have enabled high-throughput data collection with multiple single-cell protein expression measurements. The significant biological and technical variance in cytometry has posed a formidable challenge during the gating process, especially for the initial pre-gates which deal with unpredictable events, such as debris and technical artifacts. To mitigate the labor-intensive manual gating process, we propose UNITO, a framework to rigorously identify the hierarchical cytometric subpopulations. UNITO transforms a cell-level classification task into an image-based segmentation problem. The framework is validated on three independent cohorts (two mass cytometry and one flow cytometry datasets). We compare its results with previous automated methods using the consensus of at least four experienced immunologists. UNITO outperforms existing methods and deviates from human consensus by no more than any individual does. UNITO can reproduce a similar contour compared to manual gating for post-hoc inspection, and it also allows parallelization of samples for faster processing. High-throughput cytometry generates complex single-cell data with challenging manual gating process. Here, authors introduce UNITO, a framework that transforms cell classification into image segmentation, outperforming existing methods in identifying cytometric subpopulations across diverse datasets.

It is common practice for researchers to use antibodies to remove a specific cell type to infer its function. However, it is difficult to completely eliminate a cell type and there is often limited or no information as to how the cells which survive depletion are affected. This is particularly important for CD8+ T cells for two reasons. First, they are more resistant to mAb-mediated depletion than other lymphocytes. Second, targeting either the CD8α or CD8β chain could induce differential effects. We show here that two commonly used mAbs, against either the CD8α or CD8β subunit, can differentially affect cellular metabolism. Further, in vivo treatment leaves behind a population of CD8+ T cells with different phenotypic and functional attributes relative to each other or control CD8+ T cells. The impact of anti-CD8 antibodies on CD8+ T cell phenotype and function indicates the need to carefully consider the use of these, and possibly other \"depleting\" antibodies, as they could significantly complicate the interpretation of results or change the outcome of an experiment. These observations could impact how immunotherapy and modulation of CD8+ T cell activation is pursued.

Background Lipids may influence cellular penetrance by viral pathogens and the immune response that they evoke. We deeply phenotyped the lipidomic response to SARs‐CoV‐2 and compared that with infection with other pathogens in patients admitted with acute respiratory distress syndrome to an intensive care unit (ICU). Methods Mass spectrometry was used to characterise lipids and relate them to proteins, peripheral cell immunotypes and disease severity. Results Circulating phospholipases (sPLA2, cPLA2 (PLA2G4A) and PLA2G2D) were elevated on admission in all ICU groups. Cyclooxygenase, lipoxygenase and epoxygenase products of arachidonic acid (AA) were elevated in all ICU groups compared with controls. sPLA2 predicted severity in COVID‐19 and correlated with TxA2, LTE4 and the isoprostane, iPF2α‐III, while PLA2G2D correlated with LTE4. The elevation in PGD2, like PGI2 and 12‐HETE, exhibited relative specificity for COVID‐19 and correlated with sPLA2 and the interleukin‐13 receptor to drive lymphopenia, a marker of disease severity. Pro‐inflammatory eicosanoids remained correlated with severity in COVID‐19 28 days after admission. Amongst non‐COVID ICU patients, elevations in 5‐ and 15‐HETE and 9‐ and 13‐HODE reflected viral rather than bacterial disease. Linoleic acid (LA) binds directly to SARS‐CoV‐2 and both LA and its di‐HOME products reflected disease severity in COVID‐19. In healthy marines, these lipids rose with seroconversion. Eicosanoids linked variably to the peripheral cellular immune response. PGE2, TxA2 and LTE4 correlated with T cell activation, as did PGD2 with non‐B non‐T cell activation. In COVID‐19, LPS stimulated peripheral blood mononuclear cell PGF2α correlated with memory T cells, dendritic and NK cells while LA and DiHOMEs correlated with exhausted T cells. Three high abundance lipids – ChoE 18:3, LPC‐O‐16:0 and PC‐O‐30:0 – were altered specifically in COVID. LPC‐O‐16:0 was strongly correlated with T helper follicular cell activation and all three negatively correlated with multi‐omic inflammatory pathways and disease severity. Conclusions A broad based lipidomic storm is a predictor of poor prognosis in ARDS. Alterations in sPLA2, PGD2 and 12‐HETE and the high abundance lipids, ChoE 18:3, LPC‐O‐16:0 and PC‐O‐30:0 exhibit relative specificity for COVID‐19 amongst such patients and correlate with the inflammatory response to link to disease severity. Sepsis is accompanied with eicosanoid storm. Secretory phospholipase A2 (sPLA2) serves as an immunolipidomic hub regulating COVID‐19 induced sepsis. sPLA2‐driven selective PGD2 elevation was observed in COVID‐19 subjects. sPLA2‐driven depletion of ether lipids may function as a restraint on the inflammatory response.

Disparate COVID-19 outcomes have been observed between Hispanic, non-Hispanic Black, and White patients. The underlying causes for these disparities are not fully understood. This was a retrospective study utilizing electronic medical record data from five hospitals within a single academic health system based in New York City. Multivariable logistic regression models were used to identify demographic, clinical, and lab values associated with in-hospital mortality. A total of 3,086 adult patients with self-reported race/ethnicity information presenting to the emergency department and hospitalized with COVID-19 up to April 13, 2020, were included in this study. While older age (multivariable odds ratio (OR) 1.06, 95% CI 1.05-1.07) and baseline hypoxia (multivariable OR 2.71, 95% CI 2.17-3.36) were associated with increased mortality overall and across all races/ethnicities, non-Hispanic Black (median age 67, interquartile range (IQR) 58-76) and Hispanic (median age 63, IQR 50-74) patients were younger and had different comorbidity profiles as compared to non-Hispanic White patients (median age 73, IQR 62-84; p < 0.05 for both comparisons). Among inflammatory markers associated with COVID-19 mortality, there was a significant interaction between the non-Hispanic Black population and interleukin-1-beta (interaction p-value 0.04). This analysis of a multiethnic cohort highlights the need for inclusion and consideration of diverse populations in ongoing COVID-19 trials targeting inflammatory cytokines.

BackgroundFms-like tyrosine kinase 3 ligand (FLT3L) is a potent hematopoietic growth factor that mobilizes stem cells and increases the number of circulating dendritic cells (DCs) in blood and organs. In a murine lung adenocarcinoma model, FLT3L had modest activity as monotherapy but demonstrated synergy when combined with high-dose radiotherapy.1 Clinical trials have demonstrated that FLT3L is safe but has minimal activity in cancer patients. We performed a prospective trial for patients with advanced non-small cell lung cancer (NSCLC) to evaluate the immune correlates of combining FLT3L with stereotactic body radiotherapy (SBRT).MethodsTwenty-nine subjects were enrolled between October 2016 and January 2020 in a Phase II clinical, and all subjects failed at least one previous round of treatment (median 3 lines, range 1 to 5). Subcutaneous CDX-301 (75 µg/kg) was administered daily for five days with concomitant treatment of SBRT (figure 1). To identify immune cell signatures of response, we developed two high dimensional flow cytometry panels measuring 31 unique subsets found in peripheral blood mononuclear cells (PBMCs) and performed multiplex proteomic analysis measuring 92 different proteins at baseline, 2 weeks, 4 weeks, and 8 weeks post treatment.ResultsIn our trial, 55% of patients achieved progression free survival four months (PSF4) after treatment with 31% of the patients achieving abscopal responses. SBRT and CDX-301 induced significant increases in the myeloid compartment, including subsets of monocytes, myeloid-derived suppressor cells (MDSCs) and DCs (figure 2). Within the DC compartment, we found heterogeneity, with FceR1-expressing DC2 and DC3 being the most responsive to treatment. Despite the strong responses from DCs and MDSCs to SBRT and CDX-301, cellular responses returned to baseline 8 weeks post treatment. Notably, the treatment induced greater co-expression of HLADR and Ki67 on CD4 T cells than CD8 T cells. In abscopal responders, CD4 T cell phenotype continued to evolve over a longer time period with concomitant increases of Th1-like CD4 T cells, IL21, and DC1 over 4 weeks (figure 3).ConclusionsCDX-301 and SBRT emerge as a compelling approach to reactivate the immune system that has facilitated the progression of NSCLC. Patients with abscopal responses exhibit distinct cellular responses to FLT3L and SBRT that align with the emerging significance of DC1 and CD4 T cells. This sustained Type 1 response over 4 weeks in abscopal responders independent of high grade adverse events, indicating a unique therapeutic axis to harness systemic immunity against metastatic lesions in NSCLC.AcknowledgementsThe authors would like to thank patients and families who enrolled into the study. Furthermore, the authors thank the Wherry and Guha labs for valuable scientific discussions.ReferenceChakravarty PC. Flt3-ligand administration after radiation therapy prolongs survival in a murine model of metastatic lung cancer. Cancer Res.1999;59;6028–32.Ethics ApprovalThis study (2015–5267) was approved by the Albert Einstein College of Medicine IRB on 9/30/2015. All participants provided informed consentAbstract 1522 Figure 1Trial schemeAbstract 1522 Figure 2Patient level PCA using aggregated flow cytometry data representing 31 unique immune populations. Each point is colored by timepoint with the green arrow representing the mean of PC1 and PC2 at each timepoint. Variable contributions for the PC1 axis (right) and PC2 axis (bottom) are ranked by magnitude and direction of contribution and colored by parent populationAbstract 1522 Figure 3Spearman rank correlations of the fold change from T4 to T0 (baseline) in DC1, preDC, pDC, CD14-DC3, CD14+DC3, CD5+DC2, CDS- DC2, Th1-like CD4 T cells (CXCR5+), exhausted CD8 T cells (PD1+CD39+), activated CD4 T cells (HLADR+Ki67+), activated CD8 T cells (HLADR+Ki67+), and the top 10 cytokines from that are changing the most with time. Nodes are colored by associated group (cytokine, T cells, and DCs). Green edges represent positive correlations and red edges represent negative correlations. Correlations >0.7 for inclusion in correlation network