CD4 T-cell Proliferation Research Articles

Antigen-specific modulation of chronic experimental autoimmune encephalomyelitis in humanized mice by TCR-like antibody targeting autoreactive T-cell epitope.

The development and application of human TCR-like (TCRL) antibodies recognizing disease-specific MHC-peptide complexes mayprove asan important tool for basic research and therapeutic applications. Multiple sclerosis is characterized by aberrant CD4 T-cell response to self-antigens presented by MHC class II molecules. This led us to select a panel of TCRL Abs targeting the immunodominant autoantigenic epitope MOG35-55 derived from myelin oligodendrocyte glycoprotein (MOG) presented on HLA-DR2, which is associated with multiple sclerosis (MS). We demonstrate that these TCRL Abs bind with high specificity to human HLA-DR2/MOG35-55-derived MHC class II molecules and can detect APCs that naturally present the MS-associated autoantigen in the humanized EAE transgenic mouse model. The TCRL Abs can block ex vivo and in vivo CD4 T-cell proliferation in response to MOG35-55 stimulation in an antigen-specific manner. Most significantly, administration of TCRL Abs to MOG35-55-induced EAE model in HLA-DR2 transgenic mice both prevents and regresses established EAE. TCRL function was associated with a reduction in autoreactive pathogenic T-cell infiltration into the CNS, along with modulation of activated CD11b+ macrophages/microglial APCs. Collectively, these findings demonstrate the combined action of TCRL Abs in blocking TCR-MHC interactions and modulating APC presentation and activation, leading to a profound antigen-specific inhibitory effect on the neuroinflammatory process, resulting in regression of EAE. Our study constitutes an in vivo proof of concept for the utility of TCR-like antibodies as antigen-specific immunomodulators for CD4-mediated autoimmune diseases such as MS, validating the importance of the TCR-MHC axis as a therapeutic target for various autoimmune and inflammatory diseases.

VISTA Emerges as a Promising Target against Immune Evasion Mechanisms in Medulloblastoma.

Relapsed medulloblastoma (MB) poses a significant therapeutic challenge due to its highly immunosuppressive tumor microenvironment. Immune checkpoint inhibitors (ICIs) have struggled to mitigate this challenge, largely due to low T-cell infiltration and minimal PD-L1 expression. Identifying the mechanisms driving low T-cell infiltration is crucial for developing more effective immunotherapies. We utilize a syngeneic mouse model to investigate the tumor immune microenvironment of MB and compare our findings to transcriptomic and proteomic data from human MB. Flow cytometry reveals a notable presence of CD45hi/CD11bhi macrophage-like and CD45int/CD11bint microglia-like tumor-associated macrophages (TAMs), alongside regulatory T-cells (Tregs), expressing high levels of the inhibitory checkpoint molecule VISTA. Compared to sham control mice, the CD45hi/CD11bhi compartment significantly expands in tumor-bearing mice and exhibits a myeloid-specific signature composed of VISTA, CD80, PD-L1, CTLA-4, MHCII, CD40, and CD68. These findings are corroborated by proteomic and transcriptomic analyses of human MB samples. Immunohistochemistry highlights an abundance of VISTA-expressing myeloid cells clustering at the tumor-cerebellar border, while T-cells are scarce and express FOXP3. Additionally, tumor cells exhibit immunosuppressive properties, inhibiting CD4 T-cell proliferation in vitro. Identification of VISTA's binding partner, VSIG8, on tumor cells, and its correlation with increased VISTA expression in human transcriptomic analyses suggests a potential therapeutic target. This study underscores the multifaceted mechanisms of immune evasion in MB and highlights the therapeutic potential of targeting the VISTA-VSIG axis to enhance anti-tumor responses.

Identification of pediatric activated T-cell hepatitis using clinical immune studies

Background and aimsThe majority of indeterminate pediatric acute liver failure (PALF) cases are secondary to immune dysregulation, labeled activated T-cell hepatitis (TCHep). We aimed to describe a cohort of children with acute severe hepatitis and PALF and define how clinical immune labs may help identify the TCHep group. MethodsRetrospective review of children with acute hepatitis and PALF between March 2020 and August 2022. Patients were classified as known diagnosis, indeterminate hepatitis (IND-Hep), or TCHep (defined by liver biopsy with predominant CD8 T-cell inflammation or development of aplastic anemia). Results124 patients were identified: 83 with known diagnoses, 16 with TCHep, and 25 with IND-Hep. Patients with TCHep had significantly increased median total bilirubin levels (7.5 mg/dL (IQR 6.8–8.9) vs 1.5 mg/dL (IQR 1.0–3.6), p < 0.0001), soluble interleukin-2 receptor levels (4512 IU/mL (IQR 4073–5771) vs 2997 IU/mL (IQR 1957–3237), p = 0.02), and percent of CD8+ T-cells expressing perforin (14.5 % (IQR 8.0–20.0) vs 1.0 % (IQR 0.8–1.0), p = 0.004) and granzyme (37.5 % (IQR 15.8–54.8) vs 4.0 % (IQR 2.5–5.5), p = 0.004) compared to IND-Hep patients. Clinical flow cytometry showed that TCHep patients had significantly increased percent CD8+ T cells (29.0 % (IQR 24.5–33.5) vs 23.6 % (IQR 19.8–25.8), p = 0.04) and HLA-DR+ (16.0 % (IQR 14.5–24.5) vs 2.7 (1.8–5.3), p < 0.001) compared to IND-Hep patients indicative of increase in CD8+ T cells that are activated. ConclusionsPeripheral blood clinical immune studies demonstrate increased markers of CD8 T-cell activation, proliferation, and cytotoxic function for TCHep patients. These readily available immune function labs can be used to help distinguish patients with TCHep from those with other causes. This provides a non-invasive tool for early detection of potential TCHep before progression to liver failure.

IMT030122, A novel engineered EpCAM/CD3/4-1BB tri-specific antibody, enhances T-cell recruitment and demonstrates anti-tumor activity in mouse models of colorectal cancer

Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.

A novel strategy to elicit enduring anti-morphine immunity and relief from addiction by targeting Acr1 protein nano vaccine through TLR-2 to dendritic cells

Morphine addiction poses a significant challenge to global healthcare. Current opioid substitution therapies, such as buprenorphine, naloxone and methadone are effective but often lead to dependence. Thus, exploring alternative treatments for opioid addiction is crucial. We have developed a novel vaccine that presents morphine and Pam3Cys (a TLR-2 agonist) on the surface of Acr1 nanoparticles. This vaccine has self-adjuvant properties and targets TLR-2 receptors on antigen-presenting cells, particularly dendritic cells. Our vaccination strategy promotes the proliferation and differentiation of morphine-specific B-cells and Acr1-reactive CD4 T-cells. Additionally, the vaccine elicited the production of high-affinity anti-morphine antibodies, effectively eliminating morphine from the bloodstream and brain in mice. It also reduced the expression of addiction-associated μ-opioid receptor and dopamine genes. The significant increase in memory CD4 T-cells and B-cells indicates the vaccine's ability to induce long-lasting immunity against morphine. This vaccine holds promise as a prophylactic measure against morphine addiction.

Progressive multifocal leukoencephalopathy (PML) treated with pembrolizumab in a patient with mosaic STAT3 gain-of-function mutation

Progressive multifocal leukoencephalopathy (PML) is a rare and often fatal disease of the central nervous system caused by JC virus. It occurs exclusively in immunocompromised hosts and is strongly associated with HIV infection and iatrogenic immunosuppression. Monogenic defects underlying the pathogenesis and reactivation of JC virus are not well understood, although some reports of primary immunodeficiencies (including four cases of STAT1 gain-of-function) have been previously described.A 77 year old male with history of prostate cancer (in remission; treated with radiation and hormonal therapy >10 years ago) presented with dysarthria, ataxia, left-sided weakness, diagnosed with CSF and biopsy-confirmed PML. He underwent an extensive workup upon which no other chronic infectious or oncologic processes were identified. He has never received any form of immunomodulation (including chemotherapy) in the past. Workup revealed low CD4 (347/mm3) and CD8 (282/mm3) T cell counts with normal proportions of naive vs. memory populations. Mitogen stimulation demonstrated poor CD8T cell proliferation. Targeted sequencing revealed a heterozygous mosaic mutation in the DNA-binding domain of STAT3 (c.1229 A > G p.H410R, 14% in 96 reads). This variant had been previously identified in two individuals with large granular lymphocyte (LGL) leukemia and validated as a gain-of-function via downstream signaling assays.Our patient demonstrated a favorable response to PD-1 blockade with pembrolizumab (2 mg/kg) and was discharged to a rehabilitation facility. His MRI brain showed no further progression of cerebellar white matter lesions following three doses of pembrolizumab.To our knowledge, this is the first case to implicate that STAT3 gain-of-function may confer a predisposition to JC virus reactivation and/or control in the pathogenesis of PML. Furthermore, we propose that this patient’s response to pembrolizumab may be partially attributed to STAT3-mediated regulation of the PD-1/PD-L1 axis. Further studies are currently underway to characterize this patient’s mosaic variant in flow cytometry-sorted subsets (including CD8T cells). Additional studies may include measurement of PD-1/PD-L1 expression and basal vs. induced STAT3 phosphorylation in sorted cell populations.

Immunomodulatory Functions of Adipose Mesenchymal Stromal/Stem Cell Derived From Donors With Type 2 Diabetes and Obesity on CD4 T Cells.

For adipose stromal/stem cell (ASCs)-based immunomodulatory therapies, it is important to study how donor characteristics, such as obesity and type 2 diabetes (T2D), influence ASCs efficacy. Here, ASCs were obtained from 2 groups, donors with T2D and obesity (dASCs) or nondiabetic donors with normal-weight (ndASCs), and then cultured with anti-CD3/CD28-stimulated allogeneic CD4 T cells. ASCs were studied for the expression of the immunomodulators CD54, CD274, and indoleamine 2, 3 dioxygenase 1 (IDO) in inflammatory conditions. CD4 T cells cultured alone or in cocultures were assessed to evaluate proliferation, activation marker surface expression, apoptosis, the regulatory T cells (Tregs; CD4+ CD25high FOXP3+) frequency, and intracellular cytokine expression using flow cytometry. Modulation of T-cell subset cytokines was explored via ELISA. In inflammatory conditions, the expression of CD54, CD274, and IDO was significantly upregulated in ASCs, with no significant differences between ndASCs and dASCs. dASCs retained the potential to significantly suppress CD4 T-cell proliferation, with a slightly weaker inhibitory effect than ndASCs, which was associated with significantly reduced abilities to decrease IL-2 production and increase IL-8 levels in cocultures. Such attenuated potentials were significantly correlated with increasing body mass index. dASCs and ndASCs comparably reduced CD4 T-cell viability, HLA-DR expression, and interferon-gamma production and conversely increased CD69 expression, the Tregs percentage, and IL-17A production. Considerable amounts of the immunomodulators prostaglandin E2 (PGE2) and IL-6 were detected in the conditioned medium of cocultures. These findings suggest that ASCs obtained from donors with T2D and obesity are receptive to the inflammatory environment and able to modulate CD4 T cells accordingly.

Drastic transformation of visceral adipose tissue and peripheral CD4 T cells in obesity.

Obesity has a pronounced effect on the immune response in systemic organs that results in not only insulin resistance but also altered immune responses to infectious diseases and malignant tumors. Obesity-associated microenvironmental changes alter transcriptional expression and metabolism in T cells, leading to alterations in T-cell differentiation, proliferation, function, and survival. Adipokines, cytokines, and lipids derived from obese visceral adipose tissue (VAT) may also contribute to the systemic T-cell phenotype, resulting in obesity-specific pathogenesis. VAT T cells, which have multiple roles in regulating homeostasis and energy utilization and defending against pathogens, are most susceptible to obesity. In particular, many studies have shown that CD4 T cells are deeply involved in the homeostasis of VAT endocrine and metabolic functions and in obesity-related chronic inflammation. In obesity, macrophages and adipocytes in VAT function as antigen-presenting cells and contribute to the obesity-specific CD4 T-cell response by inducing CD4 T-cell proliferation and differentiation into inflammatory effectors via interactions between major histocompatibility complex class II and T-cell receptors. When obesity persists, prolonged stimulation by leptin and circulating free fatty acids, repetitive antigen stimulation, activating stress responses, and hypoxia induce exhaustion of CD4 T cells in VAT. T-cell exhaustion is characterized by restricted effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector and memory T cells. Moreover, obesity causes thymic regression, which may result in homeostatic proliferation of obesity-specific T-cell subsets due to changes in T-cell metabolism and gene expression in VAT. In addition to causing T-cell exhaustion, obesity also accelerates cellular senescence of CD4 T cells. Senescent CD4 T cells secrete osteopontin, which causes further VAT inflammation. The obesity-associated transformation of CD4 T cells remains a negative legacy even after weight loss, causing treatment resistance of obesity-related conditions. This review discusses the marked transformation of CD4 T cells in VAT and systemic organs as a consequence of obesity-related microenvironmental changes.

A pilot phase Ib study to evaluate tadalafil to overcome immunosuppression during chemoradiotherapy for IDH-wild-type glioblastoma.

Myeloid-derived suppressor cells (MDSCs) are critical regulators of immunosuppression and radioresistance in glioblastoma (GBM). The primary objective of this pilot phase Ib study was to validate the on-target effect of tadalafil on inhibiting MDSCs in peripheral blood and its safety when combined with chemoradiotherapy in GBM patients. Patients with newly diagnosed IDH-wild-type GBM received radiation therapy (RT) and temozolomide (TMZ) combined with oral tadalafil for 2 months. A historical cohort of 12 GBM patients treated with RT and TMZ was used as the comparison group. The ratio of MDSCs, T cells, and cytokines at week 6 of RT compared to baseline were analyzed using flow cytometry. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan-Meier method. Tadalafil was well tolerated with no dose-limiting toxicity among 16 evaluable patients. The tadalafil cohort had a significantly lower ratio of circulating MDSCs than the control: granulocytic-MDSCs (mean 0.78 versus 3.21, respectively, P = 0.01) and monocytic-MDSCs (1.02 versus 1.96, respectively, P = 0.006). Tadalafil increased the CD8 ratio compared to the control (1.99 versus 0.70, respectively, P < 0.001), especially the PD-1-CD8 T cells expressing Ki-67, CD38, HLA-DR, CD28, and granzyme B. Proinflammatory cytokine IL-1β was also significantly increased after tadalafil compared to the control. The tadalafil cohort did not have significantly different PFS and OS than the historical control. Concurrent tadalafil is well tolerated during chemoradiotherapy for GBM. Tadalafil is associated with a reduction of peripheral MDSCs after chemoradiotherapy and increased CD8 T-cell proliferation and activation.

Unbalanced Glutamine Partitioning between CD8T Cells and Cancer Cells Accompanied by Immune Cell Dysfunction in Hepatocellular Carcinoma.

Glutamine metabolism is critical both for the proliferation of cancer cells and the activation of CD8T cells to kill cancer cells. We aim to explore the relationship between the glutamine metabolism of CD8T cells and cancer cells and tumor immunity in the tumor microenvironment. In a TCGA cohort, we found that patients with high scores of glutamine-metabolism-related genes showed poor prognoses, and that a high score of glutamine-metabolism-related genes was an independent risk factor for HCC patients. In single-cell RNA-seq data, we found that, in some patients, the glutamine metabolism gene scores of tumor cells were significantly higher than those of CD8T cells, while decreased ratios of CD8-Tef-GZMA and suppressed tumor-killing activity of CD8-Tef-APOC2 were observed. A further genetic dynamics pseudotime analysis suggested that immune remodeling of these two subpopulations was accompanied by metabolic reprogramming. CD8-Tef-APOC2 in the dominant group tended to metabolize exogenous lipids, while the metabolic program of CD8-Tef-GZMA in the nondominant group was characterized by amino acid and endogenous lipid synthesis. In addition, we found that the glutamine metabolism inhibitor JHU083 promoted the proliferation of CD8T cells and improved the efficacy of PD-1 blockers. We proposed a new tool to quantify the glutamine partitioning between tumor cells and CD8T cells, through which the unique immune microenvironment could be identified at the transcriptome level. Furthermore, the simultaneous destruction of the glutamine metabolism in tumor cells and CD8T cells facilitated the enrichment of tumor-infiltrating CD8T cells and enhanced the efficacy of immunotherapy.

Marginal Zone B Cells Facilitate CD4 T Cell Proliferation in Response to Factor VIII in Mice with Hemophilia Α

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies represents a significant barrier to FVIII replacement therapy in patients with hemophilia A. Despite this, the immune factors that regulate anti-FVIII antibody formation remain incompletely understood. We have previously shown that shortly after injection, FVIII co-localizes with marginal zone (MZ) B cells in the marginal sinus of the spleen in mice with hemophilia A. Moreover, mice depleted of MZ B cells did not produce detectable anti-FVIII IgG antibodies. Given the ability of MZ B cells to trap and rapidly respond to blood-borne antigens, produce both IgM and IgG antibodies and activate CD4 T cells, while also trafficking antigen to B cell follicles, these results suggest that MZ B cells likely play a critical role in the initiation and subsequent orchestration of an immune response to FVIII. In contrast to other model antigens, anti-FVIII antibody formation occurs only following multiple FVIII exposures in both human and mouse models. As CD4 T cells facilitate long-lasting antibody formation, and have previously been shown to play an important role in anti-FVIII antibody development (Bray GL, et al. Am J Hematol 1993; Reding MT, et al. Thromb Haemost 2000), it is possible that each FVIII exposure event eventually propagates a CD4 T cell response to a threshold necessary to generate an optimal antibody response. Consistent with this, we have previously shown that FVIII-specific CD4 T cell proliferation requires more than 1 prior exposure to FVIII. An improved understanding of the overall timing and factors influencing CD4 T cell activation in the immune response to FVIII is necessary for the development of strategies to prevent antibody formation. OBJECTIVE: Define the key factors that regulate CD4T cell activation following FVIII exposure and the number of exposure events required to observe FVIII-specific CD4 T cell proliferation. METHODS: Hemophilia A mice +/- CD4T cell depletion or MZ B cell depletion or S1PR1-TSS mice were challenged with 1-4 weekly exposures to FVIII. Anti-FVIII IgM and IgG was examined in plasma by enzyme-linked immunoassay (ELISA). To assess FVIII-specific CD4T cell proliferation, we engineered FVIII-OVA, which contains the ovalbumin peptide recognized by OTII T cell receptor transgenic T cells. CFSE-labeled OTII splenocytes were adoptively transferred into mice with hemophilia A, mice lacking B cells and MZ B cell-depleted mice, followed by administration of FVIII-OVA. RESULTS: Anti-FVIII IgG formation is CD4T cell-dependent, however IgM is readily produced in the absence of CD4T cells. In contrast, MZ B cell depletion prevented the formation of both IgM and IgG antibodies against FVIII. FVIII is unable to cause proliferation of CD4T cells or produce IgG antibodies following initial exposure, but instead significantly increases following the 3rd exposure to FVIII. Mapping the kinetics of antibody formation has uncovered that anti-FVIII IgM formation peaks and anti-FVIII IgG formation begins after the 3rd FVIII exposure, at the time of CD4 T cell proliferation. We next examined the influence of B cells on CD4 T cell proliferation, and observed that FVIII-specific CD4T cell proliferation is significantly impaired in B cell-deficient recipients. Moreover, in mice who have undergone MZ B cell depletion, there is likewise an absence of anti-FVIII IgM and IgG and a lack of measurable CD4 T cell proliferation. Finally, in S1PR1-TSS mice with MZ B cells that are unable to traffic antigen to the B cell follicle, anti-FVIII IgM and IgG formation occurs at the same rate as control mice. CONCLUSION: In summary, our findings suggest that MZ B cells are required for anti-FVIII IgM and IgG antibody formation as well as FVIII-specific CD4 T cell proliferation, which requires 3 antigen exposures. The finding that MZ B cells are not required to traffic antigen to the B cell follicle contributes to our hypothesis that MZ B cell production of IgM, with or without direct activation of CD4 T cells contributes to FVIII-specific CD4 T cell activation. These results suggest that targeted approaches designed to prevent anti-FVIII antibody formation may be most effectively achieved by defining key factors that regulate MZ B cell activation following initial FVIII exposure. In doing so, the present findings and future work will likely uncover important pathways to effectively target to reduce, or event prevent anti-FVIII antibody production.

Deconvolution of Adult T-Cell Leukemia/Lymphoma With Single-Cell RNA-Seq Using Frozen Archived Skin Tissue Reveals New Subset of Cancer-Associated Fibroblast.

Adult T-cell Leukemia/Lymphoma (ATLL) is a rare aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. However, little is known about the underlying activated molecular pathways at the single cell level. Moreover, the intercellular communications between the tumor microenvironment (TME) and tumor cells in this malignancy are currently unknown. Difficulties in harvesting fresh tissue in a clinical setting have hampered our deeper understanding of this malignancy. Herein, we examined ATLL using archived fresh frozen tissue after biopsy using single-cell RNA sequencing (scRNA-seq) with T-cell receptor (TCR) clonal analysis. Highly clonal tumor cells showed multiple activating pathways, suggesting dynamic evolution of the malignancy. By dissecting diverse cell types comprising the TME, we identified a novel subset of cancer-associated fibroblast, which showed enriched epidermal growth factor receptor (EGFR)-related transcripts including early growth response 1 and 2 (EGR1 and EGR2). Cancer associated fibroblasts (CAFs) of ATLL play an important role for CD4 T-cell proliferation via FGF7-FGF1 and PDGFA-PDGFRA/B signaling, and CAFs, particularly EGR-enriched, are also associated with CD8 and NKT expansion by EGFR. These findings suggest a potential targeted therapeutic pathway to better treat this neoplasm.

Metabolic Reprogramming of Alloreactive T Cells Through TCR/MYC/mTORC1/E2F6 Signaling in aGvHD Patients.

Acute graft-versus-host disease (aGvHD) is the most common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and significantly linked with morbidity and mortality. Although much work has been engaged to investigate aGvHD pathogenesis, the understanding of alloreactive T-cell activation remains incomplete. To address this, we studied transcriptional activation of carbohydrate, nucleotide, tricarboxylic acid (TCA) cycle, and amino acid metabolism of T cells before aGvHD onset by mining the Gene Expression Omnibus (GEO) datasets. Glycolysis had the most extensive correlation with other activated metabolic sub-pathways. Through Pearson correlation analyses, we found that glycolytic activation was positively correlated with activated CD4 memory T-cell subset and T-cell proliferation and migration. T-cell receptor (TCR), mechanistic target of rapamycin complex 1 (mTORC1), myelocytomatosis oncogene (MYC) signaling pathways and E2F6 might be “master regulators” of glycolytic activity. aGvHD predictive model constructed by glycolytic genes (PFKP, ENO3, and GAPDH) through logistic regression showed high predictive and discriminative value. Furthermore, higher expressions of PFKP, ENO3, and GAPDH in alloreactive T cells were confirmed in our pre-aGvHD patient cohort. And the predictive value of the aGvHD risk model was also validated. In summary, our study demonstrated that glycolytic activation might play a pivotal function in alloreactive T-cell activation before aGvHD onset and would be the potential target for aGvHD therapy.

The β 1 -Adrenergic Receptor Contributes to Sepsis-Induced Immunosuppression Through Modulation of Regulatory T-Cell Inhibitory Function.

Although cardiovascular benefits of β 1 -adrenergic receptor blockade have been described in sepsis, little is known about its impact on the adaptive immune response, specifically CD4 T cells. Herein, we study the effects of β 1 -adrenergic receptor modulation on CD4 T-cell function in a murine model of sepsis. Experimental study. University laboratory. C57BL/6 mice. High-grade sepsis was induced by cecal ligation and puncture in wild-type mice (β 1+/+ ) with or without esmolol (a selective β 1 -adrenergic receptor blocker) or in β 1 -adrenergic receptor knockout mice (β 1-/- ). At 18 hours after surgery, echocardiography was performed with blood and spleen collected to analyze lymphocyte function. At 18 hours, β 1+/+ cecal ligation and puncture mice exhibited characteristics of high-grade sepsis and three surrogate markers of immunosuppression, namely decreased splenic CD4 T cells, reduced CD4 T-cell proliferation, and increased regulatory T lymphocyte cell proportions. Pharmacologic and genetic β 1 -adrenergic receptor blockade reversed the impact of sepsis on CD4 T and regulatory T lymphocyte proportions and maintained CD4 T-cell proliferative capacity. β 1 -adrenergic receptor blocked cecal ligation and puncture mice also exhibited a global decrease in both pro- and anti-inflammatory mediators and improved in vivo cardiovascular efficiency with maintained cardiac power index despite the expected decrease in heart rate. β 1 -adrenergic receptor activation enhances regulatory T lymphocyte inhibitory function and thus contributes to sepsis-induced immunosuppression. This can be attenuated by β 1 -adrenergic receptor blockade, suggesting a potential immunoregulatory role for this therapy in the management of sepsis.

SUMOylation of PDPK1 Is required to maintain glycolysis-dependent CD4 T-cell homeostasis

The immune system is finely tuned to fight against infections, eradicate neoplasms, and prevent autoimmunity. Protein posttranslational modification (PTM) constitutes a molecular layer of regulation to guarantee the proper intensity of immune response. Herein, we report that UBC9-mediated protein SUMOylation plays an essential role in peripheral CD4 T-cell proliferation, but without a perceptible impact on T-cell polarization. Both conventional T-cell (Tcon) and regulatory T-cell (Treg) maintenance are differentially affected, which was likely caused by a shared deficit in cell glycolytic metabolism. Mechanistically, PDPK1 (3-phosphoinositide-dependent protein-kinase 1) was identified as a novel SUMOylation substrate, which occurred predominantly at lysine 299 (K299) located within the protein-kinase domain. Loss of PDPK1 SUMOylation impeded its autophosphorylation at serine 241 (S241), thereby leading to hypoactivation of downstream mTORC1 signaling coupled with incompetence of cell proliferation. Altogether, our results revealed a novel regulatory mechanism in peripheral CD4 T-cell homeostatic proliferation, which involves SUMOylation regulation of PDPK1–mTORC1 signaling-mediated glycolytic process.

Repression of the stress granule protein G3BP2 inhibits immune checkpoint molecule PD-L1.

Mounting evidence suggests that cancer stemness and immunosuppression are related, but the underlying mechanisms behind these are not clear. We previously reported that the stress granule-associated protein G3BP2 is involved in the regulation of tumor-initiating (stem) cells. In this study, we show that this protein also upregulates the immune checkpoint molecule PD-L1 under conditions of stress in breast and glioblastoma cancer cells, revealing a previously unknown connection between stemness programs, stress responses, and immune checkpoint control. We also identified a significant correlation between G3BP2 and PD-L1 co-expression in tumor tissues from cancer patients. To assess the targetability of G3BP2, we employed a small molecule (C108) that binds G3BP2 and interferes with the stress response. Tumors treated with C108 had increased CD8 T-cell proliferation and infiltration. Moreover, treatment of breast tumor-bearing mice with C108 resulted in a significant survival benefit and long-term cures. Cancer cells treated with C108 or cancer cells with genetically repressed G3BP2 had decreased PD-L1 expression due to enhanced mRNA degradation. Our study provides a compelling mechanism linking stress granule formation and immune checkpoint program of cancer, suggesting this link may provide new opportunities for improving anticancer immunotherapy.

Mitochondrial Exhaustion of Memory CD4 T-Cells in Treated HIV-1 Infection.

People living with HIV (PLWH) who are immune non-responders (INR) to therapy are unable to restore their CD4 T-cell count and remain at great risk of morbidity and mortality. Here the mitochondrial defects that characterize memory CD4 T-cells in INR and causes of this mitochondrial exhaustion are reviewed. This review also describes the various reagents used to induce the expression of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), the master regulator of mitochondrial biogenesis, which can restore mitochondria fitness and CD4 T-cell proliferation in INR. Due to sustained heightened inflammation in INR, the mitochondrial network is unable to be rejuvenated and requires attenuation of mediators of inflammation to rescue mitochondria and CD4 T-cell counts in INR.

Hydroxychloroquine inhibits the mitochondrial antioxidant system in activated T cells

Although hydroxychloroquine (HCQ) has long been used to treat autoimmune diseases, its mechanism of action remains poorly understood. In CD4 T-cells, we found that a clinically relevant concentration of HCQ inhibited the mitochondrial antioxidant system triggered by TCR crosslinking, leading to increased mitochondrial superoxide, impaired activation-induced autophagic flux, and reduced proliferation of CD4 T-cells. In antigen-presenting cells, HCQ also reduced constitutive activation of the endo-lysosomal protease legumain and toll-like receptor 9, thereby reducing cytokine production, but it had little apparent impact on constitutive antigen processing and peptide presentation. HCQ's effects did not require endo-lysosomal pH change, nor impaired autophagosome-lysosome fusion. We explored the clinical relevance of these findings in patients with celiac disease-a prototypic CD4 T-cell-mediated disease-and found that HCQ limits exvivo antigen-specific Tcell responses. We report a T-cell-intrinsic immunomodulatory effect from HCQ and suggest potential re-purposing of HCQ for celiac disease.

Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.

Chronic Lymphocytic Leukemia Actively Induces T-Cell Dysfunction By Contact-Dependent Signaling Via CD24 and CD52

IntroductionSuccess rates of autologous T cell-based therapies, such as CAR-T cell therapy, in chronic lymphocytic leukemia (CLL) have been suboptimal and correlate with failure of activation and proliferation of T cells in vitro and in vivo. Previous data showing that impaired CD8 T-cell activation, proliferation and metabolic reprogramming could be restored by purifying CLL T cells via cell-sorting (van Bruggen et al., Blood, 2019) indicating that an as yet unknown, CLL-derived factor is responsible for acquired T-cell dysfunction. In this study we aim to elucidate the mechanistic basis of CLL-mediated T-cell dysfunction.ResultsDynamic analysis of αCD3/CD28 stimulated autologous T cells in presence of CLL cells over a period of 9 days revealed that T-cell activation (CD25, CD71, CD95 and PD-1) in CLL is in fact not impaired but occurs in a delayed fashion. CLL T cells reached peak activation after 5-6 days in contrast to 2-3 days for age-matched healthy donors. (Fig. 1A). This delayed T cell receptor-induced T cell activation was largely normalized with tumor cell depletion by flow-sorting prior to activation. Accordingly, in absence versus presence of autologous CLL cells, CAR-T cells derived from CLL patients showed enhanced proliferation, cytokine production and cytotoxicity, indicating potential clinical relevance. These findings show that T cells in CLL are not (terminally) exhausted but that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We attempted to identify the mechanism of action in which CLL cells induce T cell dysfunction and whether these suppressive effects are mediated through a soluble factor secreted by CLL cells or by contact-dependent mechanisms.Previous studies have shown that CD40 activation of CLL cells results in increased expression of key surface-expressed adhesion and costimulatory molecules, but also in alterations of immune-modulatory cytokines secretion. This model was therefore used to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation in a contact-dependent manner (based on trans-well experiments; Fig. 1B ).Several clinically approved kinase inhibitors were tested to identify signaling cascades involved in CD40-mediated alleviation of T-cell dysfunction. Only pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce expression of co-stimulatory markers on CD40-activated CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. RNA sequencing of CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL, which we confirmed at the protein level. We also found increased expression of Siglec-10 on CLL T cells, suggesting a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, inhibition of Siglec-10 ligation by blocking CD24, and CD52 antibodies subsequently improved T-cell activation despite presence of CLL cells (Fig. 1C).ConclusionThese results demonstrate that T cells derived from CLL patients are not terminally dysfunctional and can be revived. Our observations indicate that CLL cells actively suppress (CAR) T-cell function in a contact-dependent fashion through CD24- and CD52-mediated Siglec-10 ligation. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell function in CLL. [Display omitted] DisclosuresKater: Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.