Vδ1 Effector and Vδ2 γδ T-Cell Subsets Shift in Frequency and Are Linked to Plasma Inflammatory Markers During Antiretroviral Therapy-Suppressed HIV Infection.

BACKGROUND: Chronic inflammation is prevalent with antiretroviral therapy (ART)-suppressed human immunodeficiency virus (HIV) infection and one immune cell subset putatively driving this phenomenon is TIGIT+ γδ T cells. METHODS: To elucidate γδ T-cell phenotypic diversity, spectral flow cytometry was performed on blood lymphocytes from individuals of a HIV and aging cohort and data were analyzed using bioinformatic platforms. Plasma inflammatory markers were measured and correlated with γδ T-cell subset frequencies. RESULTS: Thirty-nine distinct γδ T-cell subsets were identified (22 Vδ1+, 14 Vδ2+, and 3 Vδ1-Vδ2-Vγ9+) and TIGIT was nearly exclusively found on the Vδ1+CD45RA+CD27- effector populations. People with ART-suppressed HIV infection (PWH) exhibited high frequencies of distinct clusters of Vδ1+ effectors distinguished via CD8, CD16, and CD38 expression. Among Vδ2+ cells, most Vγ9+ (innate-like) clusters were lower in PWH; however, CD27+ subsets were similar in frequency between participants with and without HIV. Comparisons by age revealed lower 'naive' Vδ1+CD45RA+CD27+ cells in older individuals, regardless of HIV status. Plasma inflammatory markers were selectively linked to subsets of Vδ1+ and Vδ2+ cells. CONCLUSIONS: These results further elucidate γδ T-cell subset complexity and reveal distinct alterations and connections with inflammatory pathways of Vδ1+ effector and Vδ2+ innate-like subsets during ART-suppressed HIV infection.

Comparison of Anti-SARS-CoV-2-Specific Antibody Signatures in Maternal and Infant Blood after COVID-19 Infection versus COVID-19 Vaccination during Pregnancy.

OBJECTIVE: The Advisory Committee on Immunization Practices and The American College of Obstetricians and Gynecologists recommend coronavirus disease 2019 (COVID-19) vaccine for pregnant persons to prevent severe illness and death. The objective was to examine levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG, IgM, and IgA against spike protein receptor binding domain (RBD) and nucleocapsid protein (NCP) in maternal and infant/cord blood at delivery after COVID 19 vaccination compared with SARS-CoV-2 infection at in mother-infant dyads at specified time points. STUDY DESIGN: Mothers with SARS-CoV-2 infection (n = 31) or COVID-19 vaccination (n = 25) during pregnancy were enrolled between July 2020 and November 2021. Samples were collected at delivery and IgG, IgM, and IgA to RBD of spike and NCPs compared in the infected and vaccinated groups. Timing of infection/vaccination prior to delivery and correlation with antibody levels was performed. RESULTS: The majority of participants received vaccination within 90 days of delivery and over half received the Pfizer BioNTech vaccine. There were no significant correlations between antibody levels and timing of infection or vaccination. Infant IgG levels to the RBD domain of spike protein were higher in the vaccinated group (n = 25) as compared with the infants born to mothers with infection (n = 31). Vaccination against COVID-19 during pregnancy was associated with detectable maternal and infant anti-RBD IgG levels at delivery irrespective of the timing of vaccination. CONCLUSION: Timing of vaccination had no correlation to the antibody levels suggesting that the timing of maternal vaccination in the cohort did not matter. There was no IgM detected in infants from vaccinated mothers. Infants from vaccinated mothers had robust IgG titers to RBD, which have a lasting protective effect in infants. KEY POINTS: · COVID-19 vaccination during pregnancy had detectable antibody.. · No correlation between antibody levels and timing of vaccination.. · Infants from vaccinated mothers had robust IgG titers to RBD..

HIV-1 Transcription but Not Intact Provirus Levels are Associated With Systemic Inflammation.

Individuals infected with human immunodeficiency virus (HIV) 1 have increased inflammation, which has been associated with age-associated diseases. Plasma markers, cell-associated virus levels, and ability to stimulate RNA transcription in latently infected cell lines was examined in younger and older HIV-1-infected individuals with suppressed virus. Cell-associated RNA, but not intact provirus level, had positive correlation with plasma D-dimer levels. Compared with the younger group, the older group had higher D-dimer levels and a trend toward more cell-associated RNA but similar levels of intact proviruses. Even though all measured inflammatory markers were relatively higher in the older group, this greater inflammation did not induce more HIV-1 transcription in latently infected cell lines. Inflammation and HIV-1 RNA expression increase with age despite similar levels of intact infectious HIV DNA. While plasma inflammation is correlated with HIV-1 RNA expression in peripheral blood mononuclear cells, it does not induce HIV-1 transcription in latently infected cell lines.

Inhibition of Ubc13-mediated Ubiquitination by GPS2 Regulates Multiple Stages of B Cell Development.

Non-proteolytic ubiquitin signaling mediated by Lys63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells.

B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile.

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell-null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell-null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

Invariant natural killer T (iNKT) cell exhaustion in sarcoidosis.

Invariant natural killer T (iNKT) cells are integral components of immune responses during many chronic diseases, yet their surface phenotypes, subset distribution, and polyfunctional capacity in this environment are largely unknown. Therefore, using flow cytometry, we determined iNKT cell phenotypic and functional characteristics in subjects with chronic inflammatory disease sarcoidosis and matched controls. We found that sarcoidosis subjects displayed lower iNKT-cell frequencies, which correlated with lung fibrosis, C-reactive protein levels, and other measures of clinical disease. The CD4(-) CD8(-) (double negative, DN) iNKT-cell population was selectively lower in diseased individuals and the remaining DN iNKT cells exhibited higher frequencies of the activation markers CD69 and CD56. Functionally, both total IFN-γ(+) and the dual-functional IFN-γ(+) TNF-α(+) iNKT cells were decreased in sarcoidosis subjects and these functional defects correlated with total iNKT-cell circulating frequencies. As the loss of polyfunctionality can reflect functional exhaustion, we measured the surface antigens programmed death-1 receptor and CD57 and found that levels inversely correlated with dual-functional iNKT-cell percentages. These findings reveal that, similar to traditional T cells, iNKT cells may also undergo functional exhaustion, and that circulating iNKT-cell frequencies reflect these defects. Programmed death-1 receptor antagonists may therefore be attractive therapeutic candidates for sarcoidosis and other iNKT-cell-mediated chronic diseases.

Cytokine levels in maternal and infant blood after COVID-19 vaccination during pregnancy in comparison with unvaccinated controls.

The objective of this study was to compare maternal and infant cytokine profiles at delivery among those vaccinated against SARS-CoV-2 during pregnancy to unvaccinated controls. Mother-infant dyads were enrolled in this prospective cohort study, and maternal blood and infant and/or cord blood collected. Samples were analyzed utilizing a LEGENDplex 13-plex human anti-viral response cytokine panel. Maternal IP-10 and IFN-λ2/3 were lower in the vaccinated cohort. In the infants, levels were lower for IL-1ß, IFN-λ2/3, and GM-CSF, and higher for IFN-λ1 in the vaccinated cohort. Vaccination against SARS-CoV-2 during pregnancy did not lead to elevations in cytokines in mothers or infants.

Maternal, Infant, and Breast Milk Antibody Response Following COVID-19 Infection in Early Versus Late Gestation.

BACKGROUND: Coronavirus disease 2019 [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] infection at varying time points during the pregnancy can influence antibody levels after delivery. We aimed to examine SARS-CoV-2 IgG, IgM and IgA receptor binding domain of the spike protein and nucleocapsid protein (N-protein) reactive antibody concentrations in maternal blood, infant blood and breastmilk at birth and 6 weeks after SARS-CoV-2 infection in early versus late gestation. METHODS: Mothers with SARS-CoV-2 infection during pregnancy were enrolled between July 2020 and May 2021. Maternal blood, infant blood and breast milk samples were collected at delivery and 6 weeks postpartum. Samples were analyzed for SARS-CoV-2 spike and N-protein reactive IgG, IgM and IgA antibodies. Antibody concentrations were compared at the 2 time points and based on trimester of infection ("early" 1st/2nd vs. "late" 3rd). RESULTS: Dyads from 20 early and 11 late trimester infections were analyzed. For the entire cohort, there were no significant differences in antibody levels at delivery versus 6 weeks with the exception of breast milk levels which declined over time. Early gestation infections were associated with higher levels of breastmilk IgA to spike protein ( P = 0.04). Infant IgG levels to spike protein were higher at 6 weeks after late infections ( P = 0.04). There were strong correlations between maternal and infant IgG levels at delivery ( P < 0.01), and between breastmilk and infant IgG levels. CONCLUSIONS: SARS-CoV-2 infection in early versus late gestation leads to a persistent antibody response in maternal blood, infant blood and breast milk over the first 6 weeks after delivery.

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients.

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M. leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.007-0.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.032-0.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.030-0.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-γ after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M. leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.

The impact of maternal SARS-CoV-2 vaccination and first trimester infection on feto-maternal immune responses.

PROBLEM: COVID-19 infection during pregnancy increases maternal and fetal morbidity and mortality. Infection in the second or third trimester leads to changes in the decidual leukocyte populations. However, it is not known whether COVID-19 infection in the first trimester or COVID-19 vaccination during pregnancy alters the decidual immune environment. METHOD OF STUDY: We examined decidual biopsies obtained at delivery from women who had COVID-19 in the first trimester (n = 8), were fully vaccinated against COVID-19 during pregnancy (n = 17), or were neither infected nor vaccinated during pregnancy (n = 9). Decidual macrophages, NK cells, and T cells were quantified by immunofluorescence. Decidual IL-6, IL-10, and IP-10 were quantified by ELISA. RESULTS: There were no differences in decidual macrophages, NK cells, T cells, or cytokines between the first trimester COVID-19 group and the control group. The vaccinated cohort had lower levels of macrophages and NK cells compared to the control group. There were no differences in cytokines between the vaccinated and control groups. CONCLUSIONS: COVID-19 infection in the first trimester did not cause significant decidual leukocyte or cytokine changes at the maternal-fetal interface. Additionally, vaccination was not associated with decidual inflammation, supporting the safety of SARS-CoV-2 vaccination during pregnancy.

Evaluation of maternal-infant dyad inflammatory cytokines in pregnancies affected by maternal SARS-CoV-2 infection in early and late gestation.

OBJECTIVE: SARS-CoV-2 infection induces significant inflammatory cytokine production in adults, but infant cytokine signatures in pregnancies affected by maternal SARS-CoV-2 are less well characterized. We aimed to evaluate cytokine profiles of mothers and their infants following COVID-19 in pregnancy. STUDY DESIGN: Serum samples at delivery from 31 mother-infant dyads with maternal SARS-CoV-2 infection in pregnancy (COVID) were examined in comparison to 29 control dyads (Control). Samples were evaluated using a 13-plex cytokine assay. RESULTS: In comparison with controls, interleukin (IL)-6 and interferon gamma-induced protein 10 (IP-10) were higher in COVID maternal and infant samples (p < 0.05) and IL-8 uniquely elevated in COVID infant samples (p < 0.05). Significant elevations in IL-6, IP-10, and IL-8 were found among both early (1st/2nd Trimester) and late (3rd Trimester) maternal SARS-CoV-2 infections. CONCLUSIONS: Maternal SARS-CoV-2 infections throughout gestation are associated with increased maternal and infant inflammatory cytokines at birth with potential to impact long-term infant health.

Functionally distinct subsets of human NK cells and monocyte/DC-like cells identified by coexpression of CD56, CD7, and CD4.

The lack of natural killer (NK) cell-specific markers, as well as the overlap among several common surface antigens and functional properties, has obscured the delineation between NK cells and dendritic cells. Here, novel subsets of peripheral blood CD3/14/19(neg) NK cells and monocyte/dendritic cell (DC)-like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56+ monocyte/DC-like cells, which lack CD7. In contrast to CD7+CD56+ NK cells, CD7(neg)CD56+ cells lack expression of NK cell-associated markers, but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7, we observed approximately 60% of CD4+CD56+ cells were CD7(neg) cells, indicating the actual frequency of activated CD4+ NK cells is much lower in the blood than previously recognized. Functionally, only CD7+ NK cells secrete gamma interferon (IFNgamma) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore, using CD7 to separate CD56+ NK cells and CD56+ myeloid cells, we demonstrate that unlike resting CD7+CD56+ NK cells, the CD7(neg)CD56+ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7(neg)CD56+ monocyte/DC-like cells, thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo.

Novel ELISA Protocol Links Pre-Existing SARS-CoV-2 Reactive Antibodies With Endemic Coronavirus Immunity and Age and Reveals Improved Serologic Identification of Acute COVID-19 via Multi-Parameter Detection.

The COVID-19 pandemic has drastically impacted work, economy, and way of life. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into pre-existing immunity, virus transmission dynamics, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient reliable detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. Very low optical densities from samples added to buffer coated wells at as low as a 1:5 dilution are reported using this 'BU ELISA' method. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA) in 44 and 100 percent of pre-pandemic subjects, respectively, and the magnitude of these antibodies tracked with antibody levels of analogous viral proteins from endemic coronavirus (eCoV) strains. The disease status (HIV, SLE) of unexposed subjects was not linked with SARS-CoV-2 reactive antibody levels; however, quantities were significantly lower in subjects over 70 years of age compared with younger counterparts. Also, we measured SARS-CoV-2 RBD- and N- specific IgM, IgG, and IgA antibodies from 29 SARS-CoV-2 infected individuals at varying disease states, including 10 acute COVID-19 hospitalized subjects with negative serology results by the EUA approved Abbott IgG chemiluminescent microparticle immunoassay. Measurements of SARS-CoV-2 RBD- and N- specific IgM, IgG, IgA levels measured by the BU ELISA revealed higher signal from 9 of the 10 Abbott test negative COVID-19 subjects than all pre-pandemic samples for at least one antibody specificity/isotype, implicating improved serologic identification of SARS-CoV-2 infection via multi-parameter, high sensitive antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and immunity and has promising implications for improved detection of all analytes measurable by this platform.

Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation.

Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes.

Automated optimized parameters for T-distributed stochastic neighbor embedding improve visualization and analysis of large datasets.

Accurate and comprehensive extraction of information from high-dimensional single cell datasets necessitates faithful visualizations to assess biological populations. A state-of-the-art algorithm for non-linear dimension reduction, t-SNE, requires multiple heuristics and fails to produce clear representations of datasets when millions of cells are projected. We develop opt-SNE, an automated toolkit for t-SNE parameter selection that utilizes Kullback-Leibler divergence evaluation in real time to tailor the early exaggeration and overall number of gradient descent iterations in a dataset-specific manner. The precise calibration of early exaggeration together with opt-SNE adjustment of gradient descent learning rate dramatically improves computation time and enables high-quality visualization of large cytometry and transcriptomics datasets, overcoming limitations of analysis tools with hard-coded parameters that often produce poorly resolved or misleading maps of fluorescent and mass cytometry data. In summary, opt-SNE enables superior data resolution in t-SNE space and thereby more accurate data interpretation.

Automated analysis of two- and three-color fluorescent Elispot (Fluorospot) assays for cytokine secretion.

The Elispot effectively measures the frequencies of cells secreting particular molecules, especially low-frequency cells such as antigen-specific T cells. The Fluorospot assay adapted this analysis to two products per cell, and this has now been extended to three-color measurement of both mouse and human cytokine-secreting cells. Due to the increased data complexity, and particularly the need to define single-, double- and triple-producing cells, it is critical to objectively quantify spot number, size, intensity, and coincidence with other spots. An automated counting program, Exploraspot, was therefore developed to detect and quantify Fluorospots in automated fluorescence microscope images. Morphological parameters, including size, intensity, location, circularity and others are calculated for each spot, exported in FCS format, and further analyzed by gating and graphical display in popular flow cytometry analysis programs. The utility of Exploraspot is demonstrated by identification of single-, double- and triple-secreting T cells; tolerance of variable background fluorescence; and estimation of the numbers of genuine versus random multiple events.

Aging, immune senescence, and immunotherapy: A comprehensive review.

The advent of immune checkpoint inhibitors (ICIs) has changed the landscape of cancer treatment. Older adults represent the majority of cancer patients; however, direct data evaluating ICIs in this patient population is lacking. Aging is associated with changes in the immune system known as "immunosenescence" that could impact the efficacy and safety profile of ICIs. In this paper, we review aging-associated changes in the immune system as they may relate to cancer and immunotherapy, with mention of the effect of chronic viral infections and frailty. Furthermore, we summarize the current clinical evidence of ICI effectiveness and toxicity among older adults with cancer.

HIV-1 intron-containing RNA expression induces innate immune activation and T cell dysfunction.

Low levels of type I interferon (IFN-I) are thought to be a driving force for immune activation and T-cell exhaustion in HIV-1 infected individuals on combination antiretroviral therapy (cART), though the causative mechanisms for persistent IFN-I signaling have remained unclear. Here, we show Rev-CRM1-dependent nuclear export and peripheral membrane association of intron-containing HIV-1 RNA, independent of primary viral sequence or viral protein expression, is subject to sensing and signaling via MAVS, resulting in IFN-I-dependent pro-inflammatory responses in macrophages. Additionally, HIV-1 intron-containing-RNA-induced innate immune activation of macrophages leads to upregulation of inhibitory receptor expression and functional immune exhaustion of co-cultured T cells. Our findings suggest that persistent expression of HIV-1 intron-containing RNA in macrophages contributes to chronic immune activation and T-cell dysfunction and that use of HIV RNA expression inhibitors as adjunct therapy might abrogate aberrant inflammation and restore immune function in HIV-infected individuals on cART.

Increased Expression and Modulated Regulatory Activity of Coinhibitory Receptors PD-1, TIGIT, and TIM-3 in Lymphocytes From Patients With Systemic Sclerosis.

OBJECTIVE: Immune dysfunction is an important component of the disease process underlying systemic sclerosis (SSc), but the mechanisms contributing to altered immune cell function in SSc remain poorly defined. This study was undertaken to measure the expression and function of the coinhibitory receptors (co-IRs) programmed cell death 1 (PD-1), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin domain 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in lymphocyte subsets from the peripheral blood of patients with SSc. METHODS: Co-IR expression levels on subsets of immune cells were analyzed using a 16-color flow cytometry panel. The functional role of co-IRs was determined by measuring cytokine production after in vitro stimulation of SSc and healthy control peripheral blood mononuclear cells (PBMCs) in the presence of co-IR-blocking antibodies. Supernatants from cultures of stimulated PBMCs were added to SSc fibroblasts, and their impact on fibroblast gene expression was measured. Mathematical modeling was used to reveal differences between co-IR functions in SSc patients and healthy controls. RESULTS: Levels of the co-IRs PD-1 and TIGIT were increased, and each was coexpressed, in distinct T cell subsets from SSc patients compared to healthy controls. Levels of TIM-3 were increased in SSc natural killer cells. PD-1, TIGIT, and TIM-3 antibody blockade revealed patient-specific roles of each of these co-IRs in modulating activation-induced T cell cytokine production. In contrast to healthy subjects, blockade of TIGIT and TIM-3, but not PD-1, failed to reverse inhibited cytokine production in SSc patients, indicating that enhanced T cell exhaustion is present in SSc. Finally, cytokines secreted in anti-TIM-3-treated PBMC cultures distinctly changed the gene expression profile in SSc fibroblasts. CONCLUSION: The altered expression and regulatory capacity of co-IRs in SSc lymphocytes may contribute to disease pathophysiology by modulating the cytokine-mediated cross-talk of immune cells and fibroblasts at sites of inflammation and/or fibrosis.