T-cell Proliferative Capacity Research Articles

Diverse BCR usage and T cell activation induced by different COVID-19 sequential vaccinations.

Using the same laboratory test to avoid unnecessary interference due to cohort ethnicity, and experimental and statistical errors, we have compared the T/B cell immune response in the same cohort sequential vaccinated by different types of COVID-19 vaccine. We found that different sequential vaccinations can induce different dominant BCR usage with no significant neutralizing titers and RBD+ B-cell phenotype. Recombinant protein vaccine can induce higher numbers of regulatory T cells, circulating TFH (CTFH)1, CTFH17, and CTFH-CM, and lower SMNE+ T-cell proliferative capacity than the other two groups, whereas I-I-A showed higher proportion and number of virus-specific CD4+ T cells than I-I-R. Overall, our study provides a deep insight about the source of differences in immune protection of different types of COVID-19 vaccines, which further improves our understanding of the mechanisms underlying the immune response to SARS-CoV-2.

T Cell Dysfunction and Exhaustion in Patients with CLL: The Impact of Long Term Ibrutinib Treatment

Introduction: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with defects in the immune system and is dependent on the microenvironmental niche for survival. Ibrutinib, the first irreversible inhibitor of Bruton's tyrosine kinase targets B-cell receptor signaling to kill tumor cells. In addition, ibrutinib also appears to regulate the tumor microenvironment and T-cell immunity, but this mechanism unknown. Increased expression of inhibitory receptors, and functional defects such as reduced proliferative capacity and cytotoxicity are common in T cells from patients with CLL. The effect of ibrutinib on T-cell immunity has not been extensively studied in the setting of long term ibrutinib treatment. Therefore, we investigated the phenotype and function of CD4+ and CD8+ T-cells from patients with CLL treated with ibrutinib, treatment naïve CLL and age-matched healthy controls. Methods: CLL patient samples were obtained from treatment naïve patients at diagnosis, and at the 3- and 5-year timepoints. Samples from patients on continuous ibrutinib who had not relapsed at the 3- and 5-year timepoints; and pre-ibrutinib were obtained as well. Age matched healthy donor-derived peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll. PBMCs from CLL samples and healthy donors were labeled with CellTrace Violet and treated with anti-CD3 and anti-CD28 (ThermoFisher) for 7 days. T-cells were stimulated to identify proliferation capacity and to identify immune profiles. Results: Efficient T-cell activation and proliferation requires multiple signals such as T-cell receptor (TCR) stimulation, a costimulatory signal, and cytokine production. We analyzed the proliferation of T cells in-vitro upon stimulation with anti-CD3/CD28 beads using CellTrace Violet on PBMCs and found that this stimulation resulted in increased expansion of CD4+ T cells and CD8+ T cells in all the samples. We also found significant difference in the proliferation between untreated and treated CLL samples. Interestingly, proliferating T cells had different expression pattern of the exhaustion markers and inhibitory molecules such as TIM3, TIGIT, LAG3, CD160, KLRG1, CD244 and PD-1 at different phases of the disease. Increased expression of CD160, CD244, and PD1 are the key features of T-cell exhaustion. We found significant increase in the percentage of T cells from CLL patients expressing CD244, CD160 and PD1 compared to healthy donor controls but not between naïve and ibrutinib treated samples. Long term ibrutinib treatment in 3yr and 5yr samples does not change the exhaustion state in patients. We also found T-cells from ibrutinib treated samples express higher levels of CD57 which is known as a marker of senescence (Figure 1A). The phenotypes of untreated CLL samples are CCR7+CD45RA- central memory T-cells (35%) and CCR7+CD45RA+ naïve T cells (25%) (p<0.05) after stimulation in-vitro on day 3. In the same setting, ibrutinib treated samples had higher levels of effector memory T-cells (p<0.05) (80%) compared to untreated CLL samples (Figure 1B). These results suggest that the phenotype has changed to central memory phenotype in untreated samples. Functional T-cell exhaustion in CLL also leads to failure in cytokine production. Therefore, we measured different cytokine levels after stimulation in all the patient samples and in healthy controls. We found significant increase in the levels of inflammatory cytokines such as IFN-γ (p<0.05) and TNF-α (p<0.05) in long term ibrutinib treated samples compared to naïve CLL samples and healthy controls. Conclusion: Several studies have showed that T-cell counts are increased after treatment with ibrutinib in patients with CLL but not in extended therapy. Our findings suggest that long term ibrutinib treatment does increase T-cell number and proliferative capacity in CLL. The proliferating T cells express higher levels of exhaustion markers and inhibitory molecules in-vitro making them dysfunctional. However, these T-cells still maintained good proliferative potential, on par to healthy donors and better than untreated patients which can be applied in expanding improved T cell grafts for various types of adoptive T cell therapy, such as TCR gene therapy, and CAR therapy. Also better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells.

Exploring BiTE-Integrated CAR T-Cell Therapy to Overcome Tumor Antigen Escape and Reinforce CAR-T Therapy in Mantle Cell Lymphoma

Background T cell engagers (BiTEs) are bispecific T-cell engaging antibodies that redirect T cells to target antigen-expressing tumor cells. Combining a second antigen-targeted bispecific T cell engager (BiTE) with CAR T cells or incorporating bispecific engaging antibody-secreting T cells (engager-T cells) is expected to augment T-cell activation and proliferative capacity, and antitumor cytotoxicity, even towards tumor cells expressing tumor antigens heterogeneously. Here, we report a BiTE-integrated T-cell therapeutic strategy to overcome tumor antigen escape and reinforce the efficacy of CAR-T therapy in mantle cell lymphoma (MCL). Methods CD20-directed bispecific T-cell engager (CD20-BiTE) was constructed with a publicly available VHH fragment from an anti-CD20 single-domain antibody, and anti-CD3 scFv from OKT3 monoclonal antibody. CD19-directed CAR construct is comprised of anti-CD19 scFv from publicly available FMC63 monoclonal antibody, CD8 transmembrane-, 4-1BB costimulatory-, and CD3ζ signaling-domains. Human primary pan-T cells were isolated from PBMC derived from normal human donors (MD Anderson Blood Bank) or patients with MCL. Activated T cells were used for effector functional assay or transduced with lentivirus to produce CAR T or engager-T cells. Effector function of bispecific antibodies and T cells was determined via T-cell activation, cytokine release, and T-cell-dependent cytotoxicity assay in a co-culture model. Results Treatment with CD20-BiTE enhances the effector function of naïve T cells: the BiTE-Fc fusion protein was ectopically expressed in Expi293 and purified by immobilized Protein-A. The binding specificity of scFv(s) of CD20-BiTE was confirmed to bind strongly and specifically to CD20-expressing cells. In vitro functional analyses demonstrated that co-culture of naïve T cells with CD20-exressing cells in the presence of CD20-BiTE induced antigen-specific activation of the T cells (increase in the expression of CD25 and CD69). In the presence of naïve T cells and antigen-positive MCL cells, CD20-BiTE enhanced the secretion of cytokines (IFNγ and TNFα, > 2-fold increase) and CD8 T-cell cytotoxic molecule granzyme B by T cells, in a BiTE-dependent and CD20-specific manner. The enhanced effector function translated into improved antitumor cytotoxicity, as activated T cells treated with CD20-BiTE exerted 43- 72% increase in the killing efficacy of JeKo-1 BTK-KD cells. CD20-BiTE-combined CAR T cells endowed with enhanced antitumor activity overcome tumor antigen escape and resistance to current CD19-CAR T cell therapy: when primary T cells and CD19-CAR T cells were co-cultured with MCL cells, addition of CD20-BiTE potentiated T cell-dependent cytotoxicity ( p < 0.001). Since both CD19-dependent and -independent CAR T cell-resistance often develop during adoptive cell therapy in MCL, we sought to overcome the resistance in MCL by combining a second antigen-targeted CD20-BiTE with CAR T cells or incorporating CD20-BiTE secreting T cells (engager-T cells). Remarkably, when combined with CD19-CAR T cells, CD20-BiTE mediates significant increase in the antitumor cytotoxicity, compared to T cells alone ( p ≤ 0,001 and p < 0.001 for 2nM and 5nM of the BiTE, respectively), in a CD20-specific fashion, in both MCL cells with diminished CD19 expression, and CD19-CAR T-resistant primary MCL cells. Importantly, in a transwell assay, CD19-CAR T cells demonstrated restored potent killing (>3-fold increase over CAR T cells alone) of the CD19-low or CAR T therapy resistant tumor cells, but only when the engager-T cells were present in the insert, suggesting the engaging molecules were able to redirect CD19-CAR T cells to CD20-positive tumor cells regardless of their CD19 expression. The anti-lymphoma efficacy of engager-CAR T cell therapy will be further evaluated in our well-established unique PDX model for MCL. In summary, we have successfully generated a CD20-directed bispecific T-cellengaging antibody and CD20-BiTE engager-T cells that could be combined with CAR T cell therapy, which was demonstrated effective in overcoming tumor antigen escape and resistance to current CD19-CAR T cell therapy in MCL.

PSGL-1: a novel immune checkpoint driving T-cell dysfunction in obstructive sleep apnea.

Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Recently, the adhesion receptor P-selectin glycoprotein-1 (PSGL 1) has been identified as a novel immune checkpoint, which are recognized major hallmarks in several types of cancer and have revolutionized cancer therapy. The expression of PSGL-1 and its ligands VISTA and SIGLEC-5 was assessed in the leucocytes of OSA patients and control subjects exploring the role of intermittent hypoxia (IH) using in vitro models. In addition, PSGL-1 impact on T-cells function was evaluated by ex vivo models. Data showed PSGL-1 expression is upregulated in the T-lymphocytes from patients with severe OSA, indicating a relevant role of hypoxemia mediated by intermittent hypoxia. Besides, results suggest an inhibitory role of PSGL-1 on T-cell proliferation capacity. Finally, the expression of SIGLEC-5 but not VISTA was increased in monocytes from OSA patients, suggesting a regulatory role of intermittent hypoxia. In conclusion, PSGL-1 might constitute an additional immune checkpoint leading to T-cell dysfunction in OSA patients, contributing to the disruption of immune surveillance, which might provide biological plausibility to the higher incidence and aggressiveness of several tumors in these patients.

Strong T-cell activation in response to COVID-19 vaccination in multiple sclerosis patients receiving B-cell depleting therapies.

Immunocompromised individuals, including multiple sclerosis (MS) patients on certain immunotherapy treatments, are considered susceptible to complications from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and specific vaccination regimens have been recommended for suitable protection. MS patients receiving anti-CD20 therapy (aCD20-MS) are considered especially vulnerable due to acquired B-cell depletion and impaired antibody production in response to virus infection and COVID-19 vaccination. Here, the humoral and cellular responses are analyzed in a group of aCD20-MS patients (n=43) compared to a healthy control cohort (n=34) during the first 6 months after a 2-dose cycle mRNA-based COVID-19 vaccination. Both IgG antibodies recognizing receptor binding domain (RBD) from CoV-2 spike protein and their blocking activity against RBD-hACE2 binding were significantly reduced in aCD20-MS patients, with a seroconversion rate of only 23.8%. Interestingly, even under conditions of severe B-cell depletion and failed seroconversion, a significantly higher polyfunctional IFNγ+ and IL-2+ T-cell response and strong T-cell proliferation capacity were detected compared to controls. Moreover, no difference in T-cell response was observed between forms of disease (relapsing remitting- vs progressive-MS), anti-CD20 therapy (Rituximab vs Ocrelizumab) and type of mRNA-based vaccine received (mRNA-1273 vs BNT162b2). These results suggest the generation of a partial adaptive immune response to COVID-19 vaccination in B-cell depleted MS individuals driven by a functionally competent T-cell arm. Investigation into the role of the cellular immune response is important to identifying the level of protection against SARS-CoV-2 in aCD20-MS patients and could have potential implications for future vaccine design and application.

Downregulation of TCF1 in HIV Infection Impairs T-cell Proliferative Capacity by Disrupting Mitochondrial Function.

BackgroundDespite the benefits of antiretroviral therapy (ART) for people with HIV, T-cell dysfunction cannot be fully restored. Metabolic dysregulation is associated with dysfunction of HIV-1-specific T-cells. Exploration of the factors regulating metabolic fitness can help reverse T-cell dysfunction and provide new insights into the underlying mechanism.MethodsIn this study, HIV-infected individuals and HIV-negative control individuals (NCs) were enrolled. T-cell factor (TCF)1 expression in cells was determined by quantitative reverse-transcriptase polymerase chain reaction and flow cytometry. Relevant microarray data from the GEO database were analyzed to explore the underlying mechanism. The effects of TCF1 on T-cell function and metabolic function were assessed in vitro.ResultsTCF7 mRNA expression in peripheral blood mononuclear cells was downregulated in rapid progressors compared with long-term non-progressors individuals and NCs. TCF1 expression on CD4+ and CD8+ T-cells was downregulated in treatment-naïve HIV-infected individuals compared with NCs. Interleukin (IL)2 production and proliferative capacity were impaired in TCF1 knockdown T-cells. Moreover, glycolytic capacity and mitochondrial respiratory function were decreased in TCF1 knockdown T-cells, and depolarized mitochondria were increased in TCF1 knockdown T-cells.ConclusionDownregulation of TCF1 in HIV infection impairs T-cell proliferative capacity by disrupting mitochondrial function. These findings highlight the metabolic regulation as a pivotal mechanism of TCF1 in the regulation of T-cell dysfunction.

Multiomic dynamic single-cell profiling of CAR T cell populations associated with efficacy

Abstract T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown efficacy for the treatment of hematologic malignancies. Although treatment with CAR T cell can result in high response rates, the properties of the cells that comprise the cellular infusion product, associated with clinical benefit are incompletely understood. We utilized a suite of high-throughput single-cell assays including single-cell RNA-sequencing (scRNA-seq); confocal microscopy; and Timelapse Imaging Microscopy In Nanowell Grids (TIMING). TIMING profiling of a cohort of 16 patients showed that persistent motility of T cells in the presence of tumor cells was associated with both serial killing capacity and polyfunctionality. Confocal microscopy on these same T cells revealed that persistent motility is linearly correlated with both mitochondrial volume and lysosomal number. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell proliferative capacity; interferon responses; and a distinct cluster of pathways related to actin cytoskeleton and migration. We employed a marker-free sorting strategy for enriching T cells with persistent motility. RNA-seq on sorted motile T cells showed an enrichment of the core motility signature. These motile T cells also demonstrated superior in vivo anti-leukemia efficacy in comparison to unsorted T cells. Lastly, we also confirmed the association with increased persistent motility and killing of CAR T cells across diverse CARs. In aggregate, our data identified that independent of CAR design or biomanufacturing, persistent motility serves as a selectable cell-intrinsic biomarker, desired in the bioactivity of expanded CAR+ T cells.

Design and Evaluation of TIM-3-CD28 Checkpoint Fusion Proteins to Improve Anti-CD19 CAR T-Cell Function.

Therapeutic targeting of inhibitory checkpoint molecules in combination with chimeric antigen receptor (CAR) T cells is currently investigated in a variety of clinical studies for treatment of hematologic and solid malignancies. However, the impact of co-inhibitory axes and their therapeutic implication remains understudied for the majority of acute leukemias due to their low immunogenicity/mutational load. The inhibitory exhaustion molecule TIM-3 is an important marker for the interaction of T cells with leukemic cells. Moreover, inhibitory signals from malignant cells could be transformed into stimulatory signals by synthetic fusion molecules with extracellular inhibitory receptors fused to an intracellular stimulatory domain. Here, we designed a variety of different TIM-3-CD28 fusion proteins to turn inhibitory signals derived by TIM-3 engagement into T-cell activation through CD28. In the absence of anti-CD19 CAR, two TIM-3-CD28 fusion receptors with large parts of CD28 showed strongest responses in terms of cytokine secretion and proliferation upon stimulation with anti-CD3 antibodies compared to controls. We then combined these two novel TIM-3-CD28 fusion proteins with first- and second-generation anti-CD19 CAR T cells and found that the fusion receptor can increase proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR T cells. These additionally armed CAR T cells showed excellent effector function. In terms of safety considerations, the fusion receptors showed exclusively increased cytokine release, when the CAR target CD19 was present. We conclude that combining checkpoint fusion proteins with anti-CD19 CARs has the potential to increase T-cell proliferation capacity with the intention to overcome inhibitory signals during the response against malignant cells.

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.

Torque Teno Virus Viral Load as a Marker of Immune Function in Allogeneic Haematopoietic Stem Cell Transplantation Recipients.

Torque teno virus (TTV) has been proposed as a surrogate biomarker of T-cell function in allogeneic–haematopoietic–stem-cell transplantation (allo-HSCT). Conflicting data exists regarding the value of TTV to assess the degree of immunosuppression. The aim of the present study was to investigate the correlation between TTV viral load and immune function. Using samples from a prospective cohort composed of healthy-volunteers (HV) and allo-HSCT recipients at 6 months post-transplantation, we assessed the correlation between TTV viraemia and immune cell counts or T-cell proliferation capacity post-phytohaemagglutinin stimulation. TTV viraemia was detected in 68% of HV (n = 80) and 100% of allo-HSCT recipients (n = 41; p < 0.001); it was significantly higher in allo-HSCT recipients (3.9 vs. 2.1 Log copies/mL, p < 0.001). There was no correlation between T-cell function and CD3+T-cell count (rho: 0.002) suggesting that T-cell count can normalise without full functional recovery. Furthermore, no significant correlation was observed between TTV viraemia and absolute total/subset lymphocyte counts (rho: <0.13). The highest correlation was observed between TTV viral load and T-cell proliferation capacity (rho: −0.39). We therefore report an inverse correlation between T-cell function and TTV viraemia that is independent of T-cell count. Monitoring of TTV viraemia could be a fast suitable option to objectively assess the competence of immune function in at-risk populations.

Human Tolerogenic Dendritic Cells Regulate Immune Responses through Lactate Synthesis

Cell therapy is a promising strategy for treating patients suffering from autoimmune or inflammatory diseases or receiving a transplant. Based on our preclinical studies, we have generated human autologous tolerogenic dendritic cells (ATDCs), which are being tested in a first-in-man clinical trial in kidney transplant recipients. Here, we report that ATDCs represent a unique subset of monocyte-derived cells based on phenotypic, transcriptomic, and metabolic analyses. ATDCs are characterized by their suppression of Tcell proliferation and their expansion of Tregs through secreted factors. ATDCs produce high levels of lactate that shape Tcell responses toward tolerance. Indeed, Tcells take up ATDC-secreted lactate, leading to a decrease of their glycolysis. Invivo, ATDCs promote elevated levels of circulating lactate and delay graft-versus-host disease by reducing Tcell proliferative capacity. The suppression of Tcell immunity through lactate production by ATDCs is a novel mechanism that distinguishes ATDCs from other cell-based immunotherapies.

Immune checkpoint inhibitors reverse T-cell functional suppression in the bone marrow of a subset of AML patients

Abstract Acute Myeloid Leukemia is a blood cancer with poor prognosis despite decades of therapeutic development. Immune checkpoint inhibitors (ICIs) have been shown to be effective in many cancers, but their utility in AML has not been clearly established. To better understand the immune landscape of the AML microenvironment, we performed immunophenotyping on fresh bone marrow cells from AML patients using mass-spec based flow cytometry. In addition, we performed functional assays to evaluate T-cell proliferative capacity and determine whether AML bone marrow T cells can be modified by ICI treatment. Sixty two bone marrow samples from patients with AML were analyzed. We performed T-cell stimulation assays to measure proliferation and cytokine production in the presence and absence of ICIs. Samples were sub-categorized as being proliferators or non-proliferators based on the percent of T-cell division in response to TCR stimulation. Phenotypic analysis showed proliferation status correlated with a specific T-cell profile. Additionally, within the non-proliferator group, 70% overcame immune suppression and responded to at least one of the ICIs tested. In conclusion, we show that T-cell dysfunction is present in a subset of patients with AML. Nonetheless, this suppression can be reversed by ICIs in many cases, supporting the use of ICIs in AML therapy.

Effects of intermittent T-cell cluster disaggregation on proliferative capacity and checkpoint marker expression

Background/aim: T-cell immunotherapies are rapidly gaining grounds in clinical success. Presently, there is first-to-market knowledge on the translation of research scale methods to clinical and commercial scales. Improved understanding can lead to more consistent and efficient production, scaling, and eventual potency. T-cell checkpoint markers, proliferation, and T-cell cluster size and disaggregation are one set of parameters that have yet to be explored.Methods: We herein activated T-cells and assessed various mechanical dissociation frequencies in relation to expression of checkpoint markers (measured by flow cytometry).Results: We herein find increased T-cell proliferation capacity with increased dissociation frequency. We also find that with increased cluster size and duration, lower proliferation, and increased expression of checkpoint markers.Conclusions: These findings reveal new translation findings with respect to T-cell handling and production and suggest that T-cell disaggregation may be important to improved cell yields and phenotype.

Adjuvant combination therapy with gemcitabine and autologous γδ T-cell transfer in patients with curatively resected pancreatic cancer

Background aimsThe outcome for pancreatic cancer after surgery remains highly unsatisfactory, and development of more effective therapies is urgently needed. Therefore, we conducted a phase I clinical study of a novel combination of gemcitabine (GEM) and autologous γδ T-cell therapy for patients with curatively resected pancreatic cancer (University Hospital Medical Information Clinical Trials Registry identifier 000000931). MethodsFrom July 2008 to December 2012, 56 consenting patients were recruited. After preliminary testing of γδ T-cell proliferative capacity, 28 patients were eligible to receive combined GEM plus γδ T-cell therapy. ResultsDuring treatment, most of the adverse events observed were due to GEM, including myelosuppression and gastrointestinal disorders. No severe adverse events were obviously related to the γδ T-cell therapy. To evaluate clinical efficacy, patients receiving combined therapy (Group A, n = 28) were compared with those receiving GEM alone (Group B, n = 20). No significant differences were observed between the two groups in recurrence-free survival or overall survival. However, we found that, relative to progressing patients, more γδ T-cells were detectable in the blood of recurrence-free patients after only two injections (P < .0388) and more so five injections (P < .0175). Patients with >15% peripheral γδ T-cells after two injections and >20% after five injections had a chance of a more favorable clinical outcome. Accumulation of γδ T cells was positively related to the quality of the infused products, with those having >80% γδ T cells being optimal. DiscussionHigh quality of the γδ T-cell product is crucial to achieve a high percentage of γδ T cells in the blood and to achieve better clinical outcome.

Serine Is an Essential Metabolite for Effector T Cell Expansion

During immune challenge, T lymphocytes engage pathways of anabolic metabolism to support clonal expansion and the development of effector functions. Here we report a critical role for the non-essential amino acid serine in effector Tcell responses. Upon activation, Tcells upregulate enzymes of theserine, glycine, one-carbon (SGOC) metabolic network, and rapidly increase processing of serine into one-carbon metabolism. We show that extracellular serine is required for optimal Tcell expansion even in glucose concentrations sufficient to support Tcell activation, bioenergetics, and effector function. Restricting dietary serine impairs pathogen-driven expansion of Tcells invivo, without affecting overall immune cell homeostasis. Mechanistically, serine supplies glycine and one-carbon units for de novo nucleotide biosynthesis in proliferating Tcells, and one-carbon units from formate can rescue Tcells from serine deprivation. Our data implicate serine as a key immunometabolite that directly modulates adaptive immunity by controlling Tcell proliferative capacity.

Bisphenol A (BPA) aggravates multiple low-dose streptozotocin-induced Type 1 diabetes in C57BL/6 mice

Type 1 diabetes (T1D) is a T-cell-mediated autoimmune disorder characterized by destruction of insulin-producing pancreatic β-cells. Whereas epidemiological data implicate environmental factors in the increasing incidence of T1D, their identity remains unknown. Though exposure to bisphenol A (BPA) has been associated with several disorders, no epidemiologic evidence has linked BPA exposure and T1D. The goal of this study was to elucidate diabetogenic potentials of BPA and underlying mechanisms in the context of T-cell immunity, in a multiple low-dose streptozotocin (MLDSTZ)-induced autoimmune mouse T1D model. C57BL/6 mice were orally exposed to 1 or 10 mg BPA/L starting at 4 wk of age; diabetes was induced at 9 wk of age with STZ. T-cell composition, function, and insulitis levels were studied at Days 11 and 50 during diabetes development (i.e. post-first STZ injection). Results showed both BPA doses increased diabetes incidence and affected T-cell immunity. However, mechanisms of diabetogenic action appeared divergent based on dose. Low-dose BPA fits a profile of an agent that exhibits pro-diabetogenic effects via T-cell immunomodulation in the early stages of disease development, i.e. decreases in splenic T-cell subpopulations [especially CD4+ T-cells] along with a trend in elevation of splenic T-cell formation of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6). In contrast, high-dose BPA did not affect T-cell populations and led to decreased levels of IFN-γ and TNF-α. Both treatments did not affect insulitis levels at the disease early stage, but aggravated it later on. By the study end, besides decreasing T-cell proliferative capacity, low-dose BPA did not affect other T-cell-related parameters, including cytokine secretion, comparable to the effects of high-dose BPA. In conclusion, this study confirmed BPA as a potential diabetogenic compound with immunomodulatory mechanisms of action – in the context of T-cell immunity – that seemed to be dose dependent in the early immunopathogenesis of a MLDSTZ-induced model of T1D.

Functional Impairment of Murine Dendritic Cell Subsets following Infection with Infective Larval Stage 3 of Brugia malayi.

Filarial parasites cause functional impairment of host dendritic cells (DCs). However, the effects of early infection on individual DC subsets are not known. In this study, we infected BALB/c mice with infective stage 3 larvae of the lymphatic filarial parasite Brugia malayi (Bm-L3) and studied the effect on fluorescence-activated cell sorter (FACS)-sorted DC subsets. While myeloid DCs (mDCs) accumulated by day 3 postinfection (p.i.), lymphoid DCs (LDCs) and CD8+ plasmacytoid DCs (pDCs) peaked at day 7 p.i. in the spleens and mesenteric lymph nodes (mLNs) of infected mice. Increased tumor necrosis factor alpha (TNF-α) but reduced interleukin 12 (IL-12) and Toll-like receptor 4 (TLR4), -6, and -9 and reciprocal secretion of IL-4 and IL-10 were also observed across all DC subsets. Interestingly, Bm-L3 increased the expression of CD80 and CD86 across all DC subsets but decreased that of major histocompatibility complex class II (MHC-II) on mDCs and pDCs, resulting in their impaired antigen uptake and presentation capacities, but maximally attenuated the T-cell proliferation capacity of only mDCs. Furthermore, Bm-L3 increased phosphorylated p38 (p-p38), but not p-ERK, in mDCs and LDCs but downregulated them in pDCs, along with differential modulation of protein tyrosine phosphatases SHP-1, TCPTP, PTEN, and PTP1B across all DC subsets. Taken together, we report hitherto undocumented effects of early Bm-L3 infection on purified host DC subsets that lead to their functional impairment and attenuated host T-cell response.

Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency

AbstractAdenosine deaminase (ADA) deficiency is a rare, autosomal-recessive systemic metabolic disease characterized by severe combined immunodeficiency (SCID). The treatment of choice for ADA-deficient SCID (ADA-SCID) is hematopoietic stem cell transplant from an HLA-matched sibling donor, although <25% of patients have such a donor available. Enzyme replacement therapy (ERT) partially and temporarily relieves immunodeficiency. We investigated the medium-term outcome of gene therapy (GT) in 18 patients with ADA-SCID for whom an HLA-identical family donor was not available; most were not responding well to ERT. Patients were treated with an autologous CD34+-enriched cell fraction that contained CD34+ cells transduced with a retroviral vector encoding the human ADA complementary DNA sequence (GSK2696273) as part of single-arm, open-label studies or compassionate use programs. Overall survival was 100% over 2.3 to 13.4 years (median, 6.9 years). Gene-modified cells were stably present in multiple lineages throughout follow up. GT resulted in a sustained reduction in the severe infection rate from 1.17 events per person-year to 0.17 events per person-year (n = 17, patient 1 data not available). Immune reconstitution was demonstrated by normalization of T-cell subsets (CD3+, CD4+, and CD8+), evidence of thymopoiesis, and sustained T-cell proliferative capacity. B-cell function was evidenced by immunoglobulin production, decreased intravenous immunoglobulin use, and antibody response after vaccination. All 18 patients reported infections as adverse events; infections of respiratory and gastrointestinal tracts were reported most frequently. No events indicative of leukemic transformation were reported. Trial details were registered at www.clinicaltrials.gov as #NCT00598481.

Host lung immunity is severely compromised during tropical pulmonary eosinophilia: role of lung eosinophils and macrophages.

Eosinophils play a central role in the pathogenesis of tropical pulmonary eosinophilia, a rare, but fatal, manifestation of filariasis. However, no exhaustive study has been done to identify the genes and proteins of eosinophils involved in the pathogenesis of tropical pulmonary eosinophilia. In the present study, we established a mouse model of tropical pulmonary eosinophilia that mimicked filarial manifestations of human tropical pulmonary eosinophilia pathogenesis and used flow cytometry-assisted cell sorting and real-time RT-PCR to study the gene expression profile of flow-sorted, lung eosinophils and lung macrophages during tropical pulmonary eosinophilia pathogenesis. Our results show that tropical pulmonary eosinophilia mice exhibited increased levels of IL-4, IL-5, CCL5, and CCL11 in the bronchoalveolar lavage fluid and lung parenchyma along with elevated titers of IgE and IgG subtypes in the serum. Alveolar macrophages from tropical pulmonary eosinophilia mice displayed decreased phagocytosis, attenuated nitric oxide production, and reduced T-cell proliferation capacity, and FACS-sorted lung eosinophils from tropical pulmonary eosinophilia mice upregulated transcript levels of ficolin A and anti-apoptotic gene Bcl2,but proapoptotic genes Bim and Bax were downregulated. Similarly, flow-sorted lung macrophages upregulated transcript levels of TLR-2, TLR-6, arginase-1, Ym-1, and FIZZ-1 but downregulated nitric oxide synthase-2 levels, signifying their alternative activation. Taken together, we show that the pathogenesis of tropical pulmonary eosinophilia is marked by functional impairment of alveolar macrophages, alternative activation of lung macrophages, and upregulation of anti-apoptotic genes by eosinophils. These events combine together to cause severe lung inflammation and compromised lung immunity. Therapeutic interventions that can boost host immune response in the lungs might thus provide relief to patients with tropical pulmonary eosinophilia.

PD-1+Tim-3+ CD8+ T Lymphocytes Display Varied Degrees of Functional Exhaustion in Patients with Regionally Metastatic Differentiated Thyroid Cancer.

Regional metastatic differentiated thyroid cancer (mDTC) provides a unique model in which to study the tumor-immune interface. These lymph node metastases persist for years, generally without progression to distant metastases. Although the immune system likely impedes disease progression, it is unsuccessful in eliminating disease. Our previous studies revealed that programmed death-1 (PD-1)(+) T cells were enriched in tumor-involved lymph nodes (TILN). Tumor-associated leukocytes and tumor cells were collected from grossly involved lymph nodes from 12 patients to further characterize the phenotype and functional potential of mDTC-associated PD-1(+) T cells. PD-1(+)CD4(+) and PD-1(+)CD8(+) T cells were enriched in 8 of 12 TILN samples. PD-1(+) T cells coexpressed Tim-3 and CD69 and failed to downregulate CD27. CD8(+) T cells, but not CD4(+) T cells, from these samples were variably deficient in their ability to produce effector cytokines when compared with control TILNs that lacked resident PD-1(+) T cells. PD-1(+)CD8(+) T cells were capable of exocytosis but lacked intracellular perforin. Surprisingly, T-cell proliferative capacity was largely maintained in all samples. Thus, although PD-1 expression by mDTC-associated CD8(+) T cells was associated with dysfunction, exhaustion was not complete. Notably, molecular markers of exhaustion did not translate to dysfunction in all samples or in CD4(+) T cells. Regulatory T cells (Treg), PD-L1, and galectin-9 were commonly found in mDTC and likely contributed to the initiation of T-cell exhaustion and disease progression. Therapies that release the effects of PD-1 and Tim-3 and reduce the suppressive effects of Tregs may encourage tumor elimination in patients with mDTC.