T-cell Phenotype Research Articles

Association of T-Cell Phenotypes With Peri-Coronary Inflammation in People With and Without HIV and Without Cardiovascular Disease.

Persistent immune activation is linked to elevated cardiovascular diseases in people with HIV on antiretroviral therapy. The fat attenuation index (FAI) is a measure of peri-coronary inflammation that independently predicts cardiovascular disease risk in people without HIV. Whether FAI is associated with immune activation is unknown. Peripheral blood T-cell activation and homing phenotypes were measured in people with HIV (n=58) and people without HIV (n=16) without known cardiovascular disease who underwent coronary computed tomography angiography and had FAI measurements. A cross-sectional analysis of an observational cohort was performed. The primary aim was to evaluate associations of T-cell activation and phenotypes with the outcome variables, FAI values of the right coronary artery and left anterior descending artery, which were assessed using multivariable regression models adjusted for age, natal sex, race, low-density lipoprotein cholesterol, body mass index, and use of lipid-lowering medication. T cells from people with HIV showed greater activation, as measured by cluster of differentiation (CD) 38/human leukocyte antigen - DR isotype coexpression on CD4 central memory and terminally-differentiated effector memory subsets and on CD8 effector memory (TEM), than did cells from people without HIV. Expression of the chemokine receptor C-C Chemokine Receptor 2 was reduced on CD4 central memory and TEM and CD8 TEM and terminally-differentiated effector memory subsets in people with HIV. Among all participants, PD-1 (programmed cell death 1) in CD8 central memory was associated with worsened peri-coronary inflammation of the right coronary artery, whereas perforin/granzyme B on CD8 TEM was associated with improved peri-coronary inflammation of the right coronary artery and left anterior descending artery in adjusted analyses. When accounting for HIV serostatus, CD38/human leukocyte antigen - DR isotype coexpression on CD8 central memory, TEM, and terminally-differentiated effector memory cells was associated with more peri-coronary inflammation of the left anterior descending artery. The associations between T-cell activation with FAI are novel and suggest that T-cell activation may be an important driver of peri-coronary inflammation, occurring at an early stage of atherosclerosis, even before the development of clinical disease.

Phase I Trial of MCARH109, a G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D)-Targeted Chimeric Antigen Receptor T-Cell Therapy for Multiple Myeloma: An Updated Analysis.

MCARH109 is a first-in-class G protein-coupled receptor, class C, group 5, member D (GPRC5D)-targeted chimeric antigen receptor (CAR) T-cell therapy for patients with relapsed/refractory multiple myeloma. This phase I clinical trial included 17 patients and determined that MCARH109 is safe at a maximum tolerated dose of 150 × 106 CAR T cells. In this updated analysis, no new serious adverse events were reported at a median follow-up of 37 months. Overall, 12 (71%) of 17 patients responded, including seven (70%) of 10 patients previously treated with B-cell maturation antigen-targeted therapy. The median duration of response was 8.6 months (95% CI, 5.7 to not reached [NR]) with two patients sustaining a stringent complete response at the time of last follow-up, 32 months and 41 months, respectively. The median overall survival (OS) was NR and the 3-year OS estimate was 59% (95% CI, 40 to 88). Possible GPRC5D loss via immunohistochemistry was observed in 6 (60%) of 10 patients at relapse. High-dimensional spectral cytometry-based immune profiling associated an activated T-cell phenotype at apheresis with a response to MCARH109.

Machine learning evaluation of intensified conditioning on haematopoietic stem cell transplantation in adult acute lymphoblastic leukemia patients

BackgroundThe advantage of intensified myeloablative conditioning (MAC) over standard MAC has not been determined in haematopoietic stem cell transplantation (HSCT) for adult acute lymphoblastic leukemia (ALL) patients.MethodsTo evaluate heterogeneous effects of intensified MAC among individuals, we analyzed the registry database of adult ALL patients between 2000 and 2021. After propensity score matching, we applied a machine-learning Bayesian causal forest algorithm to develop a prediction model of individualized treatment effect (ITE) of intensified MAC on reduction in overall mortality at 1 year after HSCT.ResultsAmong 2440 propensity score-matched patients, our model shows heterogeneity in the association between intensified MAC and 1-year overall mortality. Individuals in the high-benefit group (n = 1220), defined as those with ITEs greater than the median, are more likely to be younger, male, and to have higher refined Disease Risk Index (rDRI), T-cell phenotype, and grafts from related donors than those in the low-benefit group (n = 1220). The high-benefit approach (applying intensified MAC to individuals in the high-benefit group) shows the largest reduction in overall mortality at 1 year (risk difference [95% confidence interval], +5.94 percentage points [0.88 to 10.51], p = 0.011). In contrast, the high-risk approach (targeting patients with high or very high rDRI) does not achieve statistical significance (risk difference [95% confidence interval], +3.85 percentage points [−1.11 to 7.90], p = 0.063).ConclusionsThese findings suggest that the high-benefit approach, targeting patients expected to benefit from intensified MAC, has the capacity to maximize HSCT effectiveness using intensified MAC.

Expression of senescence-related CD161 promotes extranodal NK/T cell lymphoma by affecting T cell phenotype and cell cycle

PurposeThe intention of this work is to probe the role of senescence-related gene CD161 in extranodal NK/T cell lymphoma (ENKTL).MethodsThis study used H2O2 to establish three distinct in vitro oxidative stress aging models (NKL, SNT-8, and YT). Western blotting was employed to assess the levels of two iconic aging proteins, MMP1 and P53, and flow cytometry was utilized to investigate cell cycle and the expressions of CD4, CD8, and CD161. Cell viability was evaluated via the CCK-8 assay. The transcriptome analysis assessed the differential gene expression between the control and aging group of NKL. In vivo, we established a BALB/c mice aging tumor model. After 15 days, the mice were euthanized to harvest tumors. ELISA was employed to measure aging indicators in the mouse tissues. Flow cytometry was utilized to assess the levels of CD4, CD8, and CD161 in tumor samples. Hematoxylin-eosin (HE) staining was performed to evaluate the structure and cellular morphology of the tumor tissue.ResultsIn the NKL, SNT-8 and YT aging models, the levels of MMP1 and P53 proteins were significantly increased. Flow cytometry results indicated that all three cell types exhibited marked arrest in the G1 phase. Compared with the control group, the expressions of CD4 and CD161 in the aging group were significantly increased, while the expression of CD8 was decreased. Transcriptome analysis revealed 2,843 differentially expressed genes (DEGs) between the control and aging groups, with 2,060 up-regulated and 783 down-regulated genes identified. Following CD161 knockdown, cell viability of three cell types in the aging group was significantly reduced compared to the control group. The G1 phase of the cells was significantly interrupted. The expressions of CD4 and CD161 were significantly increased, and the expression of CD8 was decreased. However, in the aging + si-CD161 group, a partial alleviation of oxidative stress was observed with a reduction in CD161 expression levels. Animal experiments demonstrated that knockout of CD161 can inhibit tumor progression and partially mitigate oxidative stress.ConclusionsCD161 may inhibit ENKTL tumor development by regulating cell cycle and T-cell phenotype.

Abstract 4137500: The Histone Methyltransferase MLL1 Regulates Notch Signaling and T Cell Phenotype During Pathologic Abdominal Aortic Aneurysm Development

Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T H cells (Tregs) and pro-inflammatory T H 17 cells within the aortic wall. The mechanisms regulating CD4 + T H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T H cell phenotype by directly altering key transcription factors for T H 17 (RORγ) and Treg (FOXp3) differentiation in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T H 17 activity within AAA development and to determine key T H 17 cell signaling pathways that are altered Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3). Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4+ cells compared to non-aneurysmal control samples. Congruently, CD4 + T H cells isolated from the murine AngII-induced AAA model displayed a predominance of T H 17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4+ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4 + T H cell Notch signaling ( Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p<0.05) with decreased CD4+ T H 17 cell activity and reciprocal increase in Treg expression. Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.

T-Cell Phenotypes and Systemic Cytokine Profiles of People Living with HIV Admitted to Hospital with COVID-19.

Whether SARS-CoV-2 infection leads to a higher mortality and morbidity in people living with HIV (PLWH) in Africa remains inconclusive. In this study, we explored the differences in the T-cell phenotypes between people with and without HIV on the day of admission (V1) and ±7 days later (V2), as well as their cytokine/chemokine profiles on V1. Patients admitted with COVID-19 were recruited between May 2020 and December 2021 from the Steve Biko Academic and Tshwane District Hospitals in Pretoria, South Africa. Of 174 patients, 37 (21%) were PLWH. T-cell profiles were determined by flow cytometry, and cytokine levels were determined using a multiplex suspension bead array. PLWH were significantly younger than those without HIV, and were more likely to be female. In an adjusted analysis, PLWH had higher percentages of CD4+ central memory (CM) programmed cell death protein 1 (PD-1)+, CD8+ effector memory (EM)2, and CD8+ EM4 CD57+ cells, as well as higher concentrations of interleukin (IL)-35 at admission. PLWH with CD4+ T-cell counts of >200 cells/mm3 had altered CD4+ and CD8+ T-cell profiles, lower levels of systemic inflammation measured by plasma ferritin and PCT levels, and less severe disease. PLWH with CD4+ T-cell counts of <200 cells/mm3 on admission had higher concentrations of IL-6 and lower levels of IL-29. At V2, the percentages of CD4+ CM PD-1+ T-cells and CD8+ EM4 T-cells co-expressing CD57 and PD-1 remained higher in PLWH, while all other CD8+ EM populations were lower. Fewer CD8+ EM T-cells after ±7 days of admission may be indicative of mechanisms inhibiting EM T-cell survival, as indicated by the higher expression of IL-35 and the T-cell maturation arrest observed in PLWH. This profile was not observed in PLWH with severe immunodeficiency, highlighting the need for differentiated care in the broader PLWH population.

Murine Regulatory CD4+ T Cells Are Increased in Leukemic Spleens and Show High Co-Expression of Co-Regulatory Molecules CD39, CD73, PD1, and TIGIT.

Comprehensive characterization of AML-associated T cells during disease progression is essential to identify relevant immune escape mechanisms and new immunotherapeutic approaches. Investigating the processes that lead to an immunosuppressive environment under progression of AML is difficult in humans, because by the time of diagnosis the disease is often progressed far beyond the initial stages. Therefore, to investigate T-cell phenotypes during progression a C57BL/6 mouse model was used. The CD3+ T cells were characterized by performing multiparametric flow analyses at different time points (day 0 = healthy mice, day 7, day 14, and day 21). The study revealed that the spleen is highly infiltrated by reg CD4+ T cells at day 21 of AML progression. These spleen-infiltrating reg CD4+ T cells mainly showed an effector memory differentiation with high expression and co-expression of the checkpoint molecules TIGIT, PD-1, OX40, and the two ectoenzymes CD39 and CD73. Substantial expression of the checkpoint molecules was restricted to the central memory and effector memory compartments. Furthermore, functional evaluation of TIGIT was performed. Blocking TIGIT resulted in a significantly increased lysis of C1498 AML cells in cocultures with AML-primed CD3+ T cells. Together these data confirm that the expression of the checkpoint receptor TIGIT is relevant for dysfunction of AML-associated T cells and, thus, represents a suitable target for future immunotherapeutic approaches.

Torque teno virus: a potential marker of immune reconstitution in youths with vertically acquired HIV

Torque teno virus (TTV) viral load (VL), a component of the human virome, increases during immune suppression or dysregulation. This study aimed to explore TTV VL in youths living with vertically acquired HIV (YWVH) and its potential as an immunovirological marker. We performed an observational, retrospective study involving YWVH under antiretroviral treatment (ART) from the Spanish Cohort of HIV-infected children, adolescents, and vertically HIV-infected patients transferred to Adult Units (CoRISpe-FARO), compared to HIV-negative healthy donors (HD). Plasma TTV VL was assessed by qPCR. T-cell phenotype was analysed on cryopreserved peripheral blood mononuclear cells by flow cytometry. Correlations with baseline CD4 and CD8 and long-term virological evolution were examined. A total of 57 YWVH were compared with 23 HD. YWVH had a median CD4 T-cells of 736 cells/mm3 [IQR: 574–906], a median of 17 years [IQR: 14–20.5] since ART initiation, and 65 months [IQR: 39–116] under HIV-RNA virological control. TTV VL was higher among YWVH and in males compared with females (p < 0.05). Among YWVH, TTV VL correlated with CD4 and CD8 counts and the CD4/CD8 ratio (p = 0.002; r = − 0.39, p = 0.037; r = 0.277, p = 0.005; r = − 0.37 respectively). TTV VL correlated with activation expression markers (HLA-DR+/CD38+) on CD4 (p = 0.007, r = 0.39) and the soluble proinflammatory cytokine IL-6 (p = 0.006, r = 0.38).

T-Cell Dynamics Predicts Prognosis of Patients with Hepatocellular Carcinoma Receiving Atezolizumab Plus Bevacizumab.

Atezolizumab and bevacizumab show promise for treating hepatocellular carcinoma (HCC), but identifying responsive patients remains challenging, due to tumor heterogeneity. This study explores immune dynamics following this combination therapy. Between 2020 and 2023, 29 patients with advanced HCC who received atezolizumab plus bevacizumab at Severance Hospital, Seoul, were enrolled in this study. Peripheral blood mononuclear cells were analyzed using flow cytometry and statistical methods to assess immune alterations and identify biomarkers. Baseline characteristics showed a diverse HCC cohort with a mean age of 64 years and 82.8% male predominance. Absence of extrahepatic metastasis was associated with better overall survival. Immune responses revealed distinct CD4+ T-cell phenotypes between the 'partial response (PR) + stable disease (SD)' and 'progressive disease (PD)' groups, with an overall increase in CD8+ T-cell phenotypes. Patients with higher frequencies of CD8+PD-1+Ki-67+ T cells experienced significantly improved overall survival, while those with lower frequencies of CD4+Foxp3+PD-1+LAG3+ T cells also had notable survival benefits. These findings enhance the overall understanding of immune responses to this combination therapy, facilitating improved patient stratification and personalized therapeutic approaches for HCC.

Evaluation of the immune response of peripheral blood mononuclear cells cultured on Ti6Al4V-ELI polished or etched surfaces.

While titanium and its alloys exhibit excellent biocompatibility and corrosion resistance, their polished surfaces can hinder fast and effective osseointegration and other biological processes, such as angiogenesis, due to their inert and hydrophobic properties. Despite being commonly used for orthopedic implants, research focuses on developing surface treatments to improve osseointegration, promoting cell adhesion and proliferation, as well as increasing protein adsorption capacity. This study explores a chemical treatment intended for titanium-based implants that enhances tissue integration without compromising the mechanical properties of the Ti6Al4V substrate. However, recognizing that inflammation contributes to nearly half of early implant failures, we assessed the impact of this treatment on T-cell viability, cytokine production, and phenotype. Ti6Al4V with extra low interstitial (ELI) content discs were treated with hydrofluoric acid followed by a controlled oxidation step in hydrogen peroxide that creates a complex surface topography with micro- and nano-texture and modifies the chemistry of the surface oxide layer. The acid etched surface contains an abundance of hydroxyl groups, crucial for promoting bone growth and apatite precipitation, while also enabling further functionalization with biomolecules. While cell viability remained high in both groups, untreated discs triggered an increase in Th2 cells and a decrease of the Th17 subset. Furthermore, peripheral blood mononuclear cells exposed to untreated discs displayed a rise in various pro-inflammatory and anti-inflammatory cytokines compared to the control and treated groups. Conversely, the treated discs showed a similar profile to the control, both in terms of immune cell subset frequencies and cytokine secretion. The dysregulation of the cytokine profile upon contact with untreated Ti6Al4V-ELI discs, namely upregulation of IL-2 could be responsible for the decrease in Th17 frequency, and thus might contribute to implant-associated bacterial infection. Interestingly, the chemical treatment restores the immune response to levels comparable to the control condition, suggesting the treatment's potential to mitigate inflammation by enhancing biocompatibility.

Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand

Anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has evolved as a standard of care for various forms of relapsed/refractory B cell malignancies in major developed countries. However, access to industry-driven CAR-T cell therapy is limited in developing countries, partly due to the centralized manufacturing system. Here, we demonstrated the feasibility of the point-of-care (POC) manufacturing of anti-CD19 CAR-T cells from heavily pretreated patients and healthy graft donors at an academic medical center in Thailand using a closed semi-automated production platform, CliniMACS Prodigy, and established in-process quality control and release testing to ensure their identity, purity, sterility, safety, and potency. Nine out of the nine products manufactured were used in a pilot study (ISRCTN17901467). However, we did observe that starting Tcells with CD4/CD8 ratios of less than one-third had a high chance of manufacturing failure, which could be minimized by serum supplementation. Further analysis of Tcell phenotypes in the infused versus circulating CAR-T cells revealed the differentiation from early memory subtypes toward effector cells invivo. The POC manufacturing and quality control settings herein could be applied to other CAR-T cell products and may benefit other academics, especially those in developing countries, making CAR-T cells more accessible.

Stem-like exhausted CD8 T cells in pleural effusions predict improved survival in non-small cell lung cancer (NSCLC) and mesothelioma.

Anti-tumor CD8 T cells are important for immunity but can become 'exhausted' and hence ineffective. Tumor-infiltrating exhausted CD8+ T cells include less differentiated stem-like exhausted T (Texstem) cells and terminally exhausted T (Texterm) cells. Both subsets have been proposed as prognostic biomarkers in cancer patients. In this study, we retrospectively investigated their prognostic significance in patients with metastatic non-small cell lung cancer (NSCLC) and validated our findings in a mesothelioma cohort. Pre-treatment malignant pleural effusions (PEs) from 43 NSCLC (41 non-squamous, 2 squamous) patients were analyzed by flow cytometry. The percentages of Texstem and Texterm CD8 T cells were correlated with overall survival (OS) after adjusting for clinicopathological variables. Findings were validated using a mesothelioma cohort (n=49). Mass cytometry was performed on 16 pre-treatment PE samples from 5 mesothelioma and 3 NSCLC patients for T-cell phenotyping. Single-cell multi-omics analysis was performed on 4 pre-treatment PE samples from 2 NSCLC patients and 2 mesothelioma patients for analysis of the transcriptomic profiles, surface markers and T cell receptor (TCR) repertoire. Higher frequency of Texstem was associated with significantly increased OS [median 9.9 vs. 3.4 months, hazard ratio (HR) 0.36, 95% CI: 0.16-0.79, P=0.01]. The frequency of Texterm was not associated with OS. These findings were validated in the mesothelioma cohort (high vs. low Texstem, median OS 32.1 vs. 19.8 months, HR 0.31, 95% CI: 0.10-0.96, P=0.04). Detailed single-cell sequencing and mass cytometry profiling revealed that exhausted T cells from NSCLC expressed greater stem-likeness and less inhibitory markers than those from mesothelioma and that Texstem cells also contained 'bystander' virus-specific T cells. This study demonstrates that PE CD8 Texstem cell abundance is associated with better survival outcomes, and thus may be a useful prognostic biomarker.

Effects of switching from twice-daily tacrolimus to once-daily extended-release meltdose tacrolimus on cellular immune response.

LCP-Tacro [LCPT], a novel once-daily, extended-release formulation of tacrolimus, has a reduced Cmax with comparable AUC exposure, requiring a ∼30% dose reduction in contrast to immediate-release tacrolimus (IR-Tac). Once-daily LCPT in de novo kidney transplantation has a comparable efficacy and safety profile to that of IR-Tac with advantages in bioavailability and absorption. The present investigation intends to analyze the effects of conversion from IR-Tac to LCPT on phenotype and function of T-cells and B-cells. 16 kidney transplant patients treated by triple standard immunosuppression with a stable graft function undergoing a switch from IR-Tac to LCPT were included in this observational prospective study. We measured the main immune cell types and performed an in-depth characterization of B cell, dendritic cells and T cells including regulatory T cells of the patients before, 4 and 8 weeks after IR-Tac to LCPT conversion using multi-parameter fl ow cytometry. Additionally, we analyzed T cells by assessing third-party antigens (Tetanus Diphtheria, TD)-reactive T cells, which could be analyzed by restimulation with tetanus vaccine. Overall, we found no significant alterations following LCPT conversion for the most immune cell populations with a few cell populations showing transient quantitative increase. Thus, 4 weeks after conversion, more regulatory T cells could be measured in the patients with a significant shift from memory to naïve Tregs. Furthermore, we found a transient B cell expansion 4 weeks after conversion from IR-Tac to LCPT. There were no changes in the percentage of other basic immune cell types and the antigen-reactive T cells were also not altered after changing the medication to LCP-tacrolimus. Here, we demonstrate first insights into the immune system changes occurred under IR-Tac to LCPT conversion therapy in kidney transplant patients. While phenotypic and functional characteristics of the most immune cell populations did not change, we could observe an a transient expansion of regulatory T cells in peripheral blood following IR-Tac to LCTP conversion, which might additionally contribute to the overall immunosuppressive effect.

Automated manufacturing and characterization of clinical grade autologous CD20 CAR T cells for the treatment of patients with stage III/IV melanoma.

Point-of-care (POC) manufacturing of chimeric antigen receptor (CAR) modified T cell has expanded rapidly over the last decade. In addition to the use of CD19 CAR T cells for hematological diseases, there is a growing interest in targeting a variety of tumor-associated epitopes. Here, we report the manufacturing and characterization of autologous anti-CD20 CAR T cells from melanoma patients within phase I clinical trial (NCT03893019). Using a second-generation lentiviral vector for the production of the CD20 CAR T cells on the CliniMACS Prodigy®. We demonstrated consistency in cell composition and functionality of the products manufactured at two different production sites. The T cell purity was >98.5%, a CD4/CD8 ratio between 2.5 and 5.5 and transduction rate between 34% and 61% on day 12 (harvest). Median expansion rate was 53-fold (range, 42-65-fold) with 1.7-3.8×109 CAR T cells at harvest, a sufficient number for the planned dose escalation steps (1×105/kg, 1×106/kg, 1×107/kg BW). Complementary research of some of the products pointed out that the CAR+ cells expressed mainly central memory T-cell phenotype. All tested CAR T cell products were capable to translate into T cell activation upon engagement of CAR target cells, indicated by the increase in pro-inflammatory cytokine release and by the increase in CAR T cell amplification. Notably, there were some interindividual, cell-intrinsic differences at the level of cytokine release and amplification. CAR-mediated T cell activation depended on the level of CAR cognate antigen. In conclusion, the CliniMACS Prodigy® platform is well suited for decentralized POC manufacturing of anti-CD20 CAR T cells and may be likewise applicable for the rapid and automated manufacturing of CAR T cells directed against other targets. https://clinicaltrials.gov/study/NCT03893019?cond=Melanoma&term=NCT03893019&rank=1, identifier NCT03893019.

Characterization of Nasal Mucosal T Cells in Horses and Their Response to Equine Herpesvirus Type 1

Equine herpesvirus type 1 (EHV-1) enters through the upper respiratory tract (URT). Mucosal immunity at the URT is crucial in limiting viral infection and morbidity. Here, intranasal immune cells were collected from horses (n = 15) during an experimental EHV-1 infection. CD4+ and CD8+ T cells were the major intranasal cell populations before infection and increased significantly by day six and fourteen post-infection, respectively. Nasal mucosal T cells were further characterized in healthy horses. Compared to peripheral blood mononuclear cells (PBMC), mucosal CD8+ T-cell percentages were elevated, while CD4+ T-cell percentages were similar. A small population of CD4+CD8+ T cells was also recovered from mucosal samples. Within the URT tissue, CD4+ cells predominantly accumulated in the epithelial layer, while most CD8+ cells resided deeper in the mucosa or the submucosa below the basement membrane. In vitro stimulation of mucosal cells from healthy horses with (n = 5) or without (n = 5) peripheral T-cell immunity against EHV-1 induced IFN-γ production in nasal T cells upon polyclonal stimulation. However, after EHV-1 re-stimulation, mucosal T cells failed to respond with IFN-γ. This work provided the first characterization of mucosal T-cell phenotypes and functions in the URT of healthy horses and during EHV-1 infection.

Selective Reduction of Ca2+-Independent Phospholipase A2β (iPLA2β)-Derived Lipid Signaling From Macrophages Mitigates Type 1 Diabetes Development.

Type 1 diabetes (T1D) is a consequence of autoimmune destruction of β-cells and macrophages (MΦ) have a central role in initiating processes that lead to β-cell demise. We reported that Ca2+-independent phospholipase A2β (iPLA2β)-derived lipid (iDL) signaling contributes to β-cell death. As MΦ express iPLA2β, we assessed its role in T1D development. We find that selective reduction of myeloid-iPLA2β in spontaneously diabetes-prone nonobese diabetic (NOD) mice (a) deceases proinflammatory eicosanoid production by MΦ, (b) favors anti-inflammatory (M2-like) MΦ phenotype, and (c) diminishes activated CD4+ and CD8+ T-cells phenotype in the pancreatic infiltrate, prior to T1D onset. These outcomes are associated with a significant reduction in T1D. Further, inhibition of select proinflammatory lipid signaling pathways reduces M1-like MΦ polarization and adoptive transfer of M2-like MΦ reduces NOD T1D incidence, suggesting a mechanism by which iDLs impact T1D development. These findings identify MΦ-iPLA2β as a critical contributor to TID development and potential target to counter T1D onset.

Abstract A050: MDSC-derived transmembrane TNF is a central regulator of stromal inflammation and T-cell dysfunction in pancreatic cancer

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by stromal inflammation and T-cell dysfunction. Neutrophilic/granulocytic myeloid-derived suppressor cells (gMDSC) infiltrate early in PDAC development and contribute to a highly immunosuppressive tumor microenvironment (TME). We have previously shown that gMDSC-derived tumor necrosis factor (TNF) is a novel regulator of therapeutic resistance in the PDAC TME, and pharmacological targeting of transmembrane (tm)TNF-TNFR2 signaling can reprogram the TME to improve chemosensitivity. Here we dissect the functional consequences of gMDSC-derived tmTnf on cancer-associated fibroblast (CAF) skewness and CD8+ T-cell function. METHODS: To assess global effects of tmTNF in orthotopically and subcutaneously injected KPC models of PDAC, we compared T-cell/CAF phenotypes in littermate vs transgenic mice overexpressing exclusively tmTnf (tmTnfOE)—in which site-directed mutagenesis of TNF cleavage sites renders tmTNF uncleavable, resulting in systemically overexpressed tmTNF signaling. To investigate effects of gMDSC-derived tmTnf in vitro, J774M cells that phenocopy gMDSCs were engineered to overexpress exclusively tmTnf (J-tmTnfOE) or wild-type Tnf (J-TnfWT) following CRISPR/cas9 silencing of endogenous Tnf (J-TnfKO). These cells were co-cultured with dual reporter CAFs (Il6GFP=inflammatory iCAF; Acta2dsRed=myofibroblastic myCAF) and CD8+ T-cells followed by cell trace violet proliferation assay, flow cytometry phenotyping, and IFNγ ELISA. RESULTS: Compared to littermate controls, flank and orthotopic KPC tumor-bearing tmTnfOE mice displayed accelerated tumor kinetics and volume, respectively, as well as increase in stromal fibrosis and collagen deposition (Trichrome/Sirius Red). Flow cytometric phenotyping revealed increased proportion of iCAF (PDPN+Ly6C+) vs myCAF (PDPN+Ly6C-) in tmTNFOE vs littermate mice. Moreover, intratumoral gMDSCs isolated from tmTNFOE mice augmented CAF-Il6GFP:Acta2dsRed ratios ex vivo. CD8+ T-cell phenotyping revealed higher proportions of CD39+Ly108- dysfunctional and KLRG1+CD127- short-lived effector T-cells (SLEC), with concurrent reduction in CD127+KLRG1- memory progenitor effector T-cells (MPEC), in tmTnfOE compared with littermate tumors. Using confocal microscopy in vitro, we confirmed that transmembrane sequestration of Tnf was more pronounced in J-tmTNFOE vs J-TnfWT, with complete loss of subcellular Tnf in J-TnfKO cells. To define the precise contribution of gMDSC-tmTNF on CAF/T-cell skewness, co-culture of J-tmTnfOE with dual-reporter CAFs resulted in striking induction in CAF-Il6GFP compared with J-TnfKO-CAF co-cultures. Moreover, J-tmTnfOE significantly restrained CD8+ T-cell proliferation, MPEC differentiation, and IFN-γ release relative to J-TnfKO. CONCLUSIONS: gMDSC-tmTnf is a central regulator of inflammatory CAF polarization and CD8+ T-cell dysfunction in PDAC. Selective pharmacologic or genetic strategies to disrupt gMDSC-tmTNF derived signaling in the TME may improve chemoimmunotherapy sensitivity in PDAC. Citation Format: Andrew M Adams, Haleh Amirian, Christine I Rafie, Karthik Rajkumar, Erin Dickey, Harper M Marsh, Manan Patel, Siddharth Mehra, Nagaraj Nagathihalli, Nipun Merchant, Yoon S Yap, Luisa Cimmino, Erietta Stelekati, Anna Bianchi, Jashodeep Datta. MDSC-derived transmembrane TNF is a central regulator of stromal inflammation and T-cell dysfunction in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A050.

Different Antigen-Specific CD4+ and CD8+ T-Cell Response against HCMV Proteins in Pregnant Women with Primary Infection and in Control Subjects with Remote Infection.

Background/Objectives: Human cytomegalovirus (HCMV) is the most frequent cause of congenital infections. The HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection in pregnancy. In this study, the antigen-specific T-cell response against different HCMV proteins (IE-1, pp65, gB, gHgLpUL128L) was investigated in pregnant women with primary infection and in control subjects with remote infection to identify possible components of a vaccine. Methods: Blood samples from 35 pregnant women with HCMV primary infection and 30 HCMV-seropositive healthy adult subjects with remote infection were tested. The antigen-specific T-cell response was measured using cytokine intracellular staining after stimulation with IE-1, pp65, gB and gHgLpUL128L peptides pool. Results: The pp65-specific CD4+ T-cell response was higher in pregnant women with HCMV primary infection at the late time point and in control subjects with remote infection, while the pregnant women at the early time point showed a higher gB-specific CD8+ T-cell response. Regarding the CD4+ and CD8+ T-cell phenotypes, we observed that HCMV-specific CD4+ and CD8+ T cells expressing CD45RA+ remained constant in pregnant women with primary infection at the early and late time points and in subjects with remote infection, while HCMV-specific CD4+ and CD8+ T cells expressing IL-7R+ or producing IL-2 were higher in control subjects with remote infection than in pregnant women with HCMV primary infection. Conclusions: The T-cell response was higher against gB in the early phase of infection and against pp65 in the late phase. Therefore, these proteins should be taken into consideration as candidates for a vaccine.

Dendritic Cells Pulsed with HAM/TSP Exosomes Sensitize CD4 T Cells to Enhance HTLV-1 Infection, Induce Helper T-Cell Polarization, and Decrease Cytotoxic T-Cell Response.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive demyelinating disease of the spinal cord due to chronic inflammation. Hallmarks of disease pathology include dysfunctional anti-viral responses and the infiltration of HTLV-1-infected CD4+ T cells and HTLV-1-specific CD8+ T cells in the central nervous system. HAM/TSP individuals exhibit CD4+ and CD8+ T cells with elevated co-expression of multiple inhibitory immune checkpoint proteins (ICPs), but ICP blockade strategies can only partially restore CD8+ T-cell effector function. Exosomes, small extracellular vesicles, can enhance the spread of viral infections and blunt anti-viral responses. Here, we evaluated the impact of exosomes isolated from HTLV-1-infected cells and HAM/TSP patient sera on dendritic cell (DC) and T-cell phenotypes and function. We observed that exosomes derived from HTLV-infected cell lines (OSP2) elicit proinflammatory cytokine responses in DCs, promote helper CD4+ T-cell polarization, and suppress CD8+ T-cell effector function. Furthermore, exosomes from individuals with HAM/TSP stimulate CD4+ T-cell polarization, marked by increased Th1 and regulatory T-cell differentiation. We conclude that exosomes in the setting of HAM/TSP are detrimental to DC and T-cell function and may contribute to the progression of pathology with HTLV-1 infection.

Chemotherapy drives tertiary lymphoid structures that correlate with ICI-responsive TCF1+CD8+ T cells in metastatic ovarian cancer.

Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICIs) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. We harnessed a variety of transcriptomic, spatial and functional assays to characterize the differential impact of neo-adjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to NACT-naïve HGSOC samples from 5 independent patient cohorts. We found neo-adjuvant chemotherapy (NACT)-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD-1+ CD8+ T cells over their ICI-insensitive TIM-3+PD-1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD-1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC.