Metabolic imbalance driving immune cell phenotype switching in autoimmune disorders: Tipping the balance of T- and B-cell interactions.

The interplay between the immune system and the metabolic state of a cell is intricate. In all phases of an immune response, the corresponding metabolic changes shall occur to support its modulation, in addition to the signalling through the cytokine environment and immune receptor stimulation. While autoimmune disorders may develop because of a metabolic imbalance that modulates switching between T-cell phenotypes, the effects that the interaction between T and B cells have on one another's cellular metabolism are yet to be understood in disease context. Here, we propose a perspective which highlights the potential of targeting metabolism to modulate T- and B-cell subtypes populations as well as T-B and B-T cell interactions to successfully treat autoimmune disorders. Specifically, we envision how metabolic changes can tip the balance of immune cells interactions, through definite mechanisms in both health and disease, to explain phenotype switches of B and T cells. Within this scenario, we highlight targeting metabolism that link inflammation, immunometabolism, epigenetics and ageing, is critical to understand inflammatory disorders. The combination of treatments targeting immune cells that cause (T/B) cell phenotype imbalances, and the metabolic pathways involved, may increase the effectiveness of treatment of autoimmune disorders, and/or ameliorate their symptoms to improve patients' quality of life.

Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome.

While the tumor immune microenvironment (TIME) of metastatic melanoma has been well characterized, the primary melanoma TIME is comparatively poorly understood. Additionally, although the association of tumor-infiltrating lymphocytes with primary melanoma patient outcome has been known for decades, it is not considered in the current AJCC melanoma staging system. Detailed immune phenotyping of advanced melanoma has revealed multiple immune biomarkers, including the presence of CD8+ T-cells, for predicting response to immunotherapies. However, in primary melanomas, immune biomarkers are lacking and CD8+ T-cells have yet to be extensively characterized. As recent studies combining immune features and clinicopathologic characteristics have created more accurate predictive models, this study sought to characterize the TIME of primary melanomas and identify predictors of patient outcome. We first phenotyped CD8+ T cells in fresh stage II primary melanomas using flow cytometry (n = 6), identifying a CD39+ tumor-resident CD8+ T-cell subset enriched for PD-1 expression. We then performed Opal multiplex immunohistochemistry and quantitative pathology-based immune profiling of CD8+ T-cell subsets, along with B cells, NK cells, Langerhans cells and Class I MHC expression in stage II primary melanoma specimens from patients with long-term follow-up (n = 66), comparing patients based on their recurrence status at 5 years after primary diagnosis. A CD39+CD103+PD-1- CD8+ T-cell population (P2) comprised a significantly higher proportion of intratumoral and stromal CD8+ T-cells in patients with recurrence-free survival (RFS) ≥5 years vs those with RFS <5 years (p = 0.013). Similarly, intratumoral B cells (p = 0.044) and a significantly higher B cell density at the tumor/stromal interface were associated with RFS. Both P2 and B cells localized in significantly closer proximity to melanoma cells in patients who remained recurrence-free (P2 p = 0.0139, B cell p = 0.0049). Our results highlight how characterizing the TIME in primary melanomas may provide new insights into how the complex interplay of the immune system and tumor can modify the disease outcomes. Furthermore, in the context of current clinical trials of adjuvant anti-PD-1 therapies in high-risk stage II primary melanoma, assessment of B cells and P2 could identify patients at risk of recurrence and aid in long-term treatment decisions at the point of primary melanoma diagnosis.

T cell phenotype switching in autoimmune disorders: Clinical significance of targeting metabolism.

Increasing efforts points to the understanding of how to maximize the capabilities of the adaptive immune system to fight against the development of immune and inflammatory disorders. Here we focus on the role of T cells as immune cells which subtype imbalance may lead to disease onset. Specifically, we propose that autoimmune disorders may develop as a consequence of a metabolic imbalance that modulates switching between T cell phenotypes. We highlight a Systems Biology strategy that integrates computational metabolic modelling with experimental data to investigate the metabolic requirements of T cell phenotypes, and to predict metabolic genes that may be targeted in autoimmune inflammatory diseases. Thus, we propose a new perspective of targeting T cell metabolism to modulate the immune response and prevent T cell phenotype imbalance, which may help to repurpose already existing drugs targeting metabolism for therapeutic treatment.

Identification of NSP3 (SH2D3C) as a Prognostic Biomarker of Tumor Progression and Immune Evasion for Lung Cancer and Evaluation of Organosulfur Compounds from Allium sativum L. as Therapeutic Candidates.

The multi-domain non-structural protein 3 (NSP3) is an oncogenic molecule that has been concomitantly implicated in the progression of coronavirus infection. However, its oncological role in lung cancer and whether it plays a role in modulating the tumor immune microenvironment is not properly understood. In the present in silico study, we demonstrated that NSP3 (SH2D3C) is associated with advanced stage and poor prognoses of lung cancer cohorts. Genetic alterations of NSP3 (SH2D3C) co-occurred inversely with Epidermal Growth Factor Receptor (EGFR) alterations and elicited its pathological role via modulation of various components of the immune and inflammatory pathways in lung cancer. Our correlation analysis suggested that NSP3 (SH2D3C) promotes tumor immune evasion via dysfunctional T-cell phenotypes and T-cell exclusion mechanisms in lung cancer patients. NSP3 (SH2D3C) demonstrated a high predictive value and association with therapy resistance in lung cancer, hence serving as an attractive target for therapy exploration. We evaluated the in silico drug-likeness and NSP3 (SH2D3C) target efficacy of six organosulfur small molecules from Allium sativum using a molecular docking study. We found that the six organosulfur compounds demonstrated selective cytotoxic potential against cancer cell lines and good predictions for ADMET properties, drug-likeness, and safety profile. E-ajoene, alliin, diallyl sulfide, 2-vinyl-4H-1,3-dithiin, allicin, and S-allyl-cysteine docked well into the NSP3 (SH2D3C)-binding cavity with binding affinities ranging from -4.3~-6.70 A and random forest (RF) scores ranging from 4.31~5.26 pKd. However, S-allyl-cysteine interaction with NSP3 (SH2D3C) is unfavorable and hence less susceptible to NSP3 ligandability. In conclusion, our study revealed that NSP3 is an important onco-immunological biomarker encompassing the tumor microenvironment, disease staging and prognosis in lung cancer and could serve as an attractive target for cancer therapy. The organosulfur compounds from A. sativum have molecular properties to efficiently interact with the binding site of NSP3 and are currently under vigorous preclinical study in our laboratory.

Inverted CD8 T-Cell Exhaustion and Co-Stimulation Marker Balance Differentiate Aviremic HIV-2-Infected From Seronegative Individuals.

HIV-2 is less pathogenic compared to HIV-1. Still, disease progression may develop in aviremic HIV-2 infection, but the driving forces and mechanisms behind such development are unclear. Here, we aimed to reveal the immunophenotypic pattern associated with CD8 T-cell pathology in HIV-2 infection, in relation to viremia and markers of disease progression. The relationships between pathological differences of the CD8 T-cell memory population and viremia were analyzed in blood samples obtained from an occupational cohort in Guinea-Bissau, including HIV-2 viremic and aviremic individuals. For comparison, samples from HIV-1- or dually HIV-1/2-infected and seronegative individuals were obtained from the same cohort. CD8 T-cell exhaustion was evaluated by the combined expression patterns of activation, stimulatory and inhibitory immune checkpoint markers analyzed using multicolor flow cytometry and advanced bioinformatics. Unsupervised multidimensional clustering analysis identified a cluster of late differentiated CD8 T-cells expressing activation (CD38+, HLA-DRint/high), co-stimulatory (CD226+/-), and immune inhibitory (2B4+, PD-1high, TIGIThigh) markers that distinguished aviremic from viremic HIV-2, and treated from untreated HIV-1-infected individuals. This CD8 T-cell population displayed close correlations to CD4%, viremia, and plasma levels of IP-10, sCD14 and beta-2 microglobulin in HIV-2 infection. Detailed analysis revealed that aviremic HIV-2-infected individuals had higher frequencies of exhausted TIGIT+ CD8 T-cell populations lacking CD226, while reduced percentage of stimulation-receptive TIGIT-CD226+ CD8 T-cells, compared to seronegative individuals. Our results suggest that HIV-2 infection, independent of viremia, skews CD8 T-cells towards exhaustion and reduced co-stimulation readiness. Further knowledge on CD8 T-cell phenotypes might provide help in therapy monitoring and identification of immunotherapy targets.

Do Combination Antiretroviral Therapy Regimens for HIV Infection Feature Diverse T-Cell Phenotypes and Inflammatory Profiles?

Immune abnormalities featuring HIV infection persist despite the use of effective combination antiretroviral therapy (cART) and may be linked to the development of noninfectious comorbidities. The aim of the present narrative, nonsystematic literature review is to understand whether cART regimens account for qualitative differences in immune reconstitution. Many studies have reported differences in T-cell homeostasis, inflammation, coagulation, and microbial translocation parameters across cART classes and in the course of triple vs dual regimens, yet such evidence is conflicting and not consistent. Possible reasons for discrepant results in the literature are the paucity of randomized controlled clinical trials, the relatively short follow-up of observational studies, the lack of clinical validation of the numerous inflammatory biomarkers utilized, and the absence of research on the effects of cART in tissues. We are currently thus unable to establish if cART classes and regimens are truly accountable for the differences observed in immune/inflammation parameters in different clinical settings. Questions still remain as to whether an early introduction of cART, specifically in the acute stage of disease, or newer drugs and novel dual drug regimens are able to significantly impact the quality of immune reconstitution and the risk of disease progression in HIV-infected subjects.

Distinct T-cell immunophenotypic signature in a subset of sarcoidosis patients with arthritis.

BACKGROUND: The objective of the study was to assess T-cell subsets in sarcoidosis patients with or without articular involvement. METHODS: Treatment-naïve patients were divided into Group A (articular) and Group B (non-articular) based on joint involvement. Flow cytometric analysis of T-cell subsets and pro-in˜flammatory cytokines were carried out in the peripheral blood. RESULTS: Patients in group A (n = 29, mean age 40 ± 10.1 years) were compared with group B (n = 18, 43 ± 12.2 years). T-cell subsets: the CD4/CD8 ratio was abnormal in two groups but had no significant difference (p = 0.63). Ratios of Th1/Treg, Th2/Treg and Th17/Treg were significantly increased in group A as compared to group B [p < 0.001] indicating polarisation of T-cell subsets. CD8 T-cells in group A had higher granzyme B expression (p = 0.03). B cells were increased in group A [p = 0.04]. Ratio of IFN-γ /IL10, IL-4/IL10, IL-17/IL10 in sera as well as culture supernatant were significantly higher in group A as compared to group B.

The difference of T cell phenotypes in end stage renal disease patients under different dialysis modality.

BACKGROUND: Impaired T cell immune function exists in end-stage renal disease (ESRD) patients. Dialysis treatment may lead to changes in T cell subsets. In the present study, we aimed to identify alterations of T cell phenotypes in ESRD patients, especially in those receiving peritoneal dialysis (PD), and analyze the potential associated factors. METHODS: In the present study, 110 PD patients and 110 age/gender-matched hemodialysis (HD) patients who met the inclusion criteria were studied. Pre-dialysis blood samples were obtained and analyzed by flow cytometry to detect the expression of CD45RO and CCR7. Univariate and multivariate regression analyses were used to determine the factors associated with the alteration of T cell phenotypes. RESULTS: In all dialysis patients, age was associated with the frequencies of both CD4+ and CD8+ naïve T cells, effector memory (EM) T cells and effector memory RA (EMRA) T cells but not central memory (CM) T cells. Dialysis modality was also associated with T cell subsets. Compared with HD patients, PD patients showed an increase in both CD4+ and CD8+ CM T cells and a reduction in both CD4+ and CD8+ EM and EMRA T cells. However, the number of CD4+ naïve T cells was lower and the number of CD8+ naïve T cells was higher in PD patients than those in HD patients. In PD patients, further multivariate analysis revealed that the frequency of CD4+ naïve T cells was positively associated with nPCR, while the frequency of CD8+ naïve T cells was negatively associated with age. CONCLUSION: In dialysis patients, the dialysis modality and age influence T cell subsets. There is a progression from naïve to effector T cells in HD patients compared with PD patients. In PD patients, different factors may influence the frequencies of CD4+ and CD8+ naïve T cells.

Longitudinal Changes in Cd4+, Cd8+ T Cell Phenotype and Activation Marker Expression Following Antiretroviral Therapy Initiation among Patients with Cryptococcal Meningitis.

Despite improvement in the prognosis of HIV/AIDS (human immunodeficiency virus/acquired immune deficiency syndrome) patients on antiretroviral therapy (ART), cryptococcal meningitis (CM) still causes 10-15% mortality among HIV-infected patients. The immunological impact of ART on the CD4+ and CD8+ T cell repertoire during cryptococcal co-infection is unclear. We determined longitudinal phenotypic changes in T cell subsets among patients with CM after they initiated ART. We hypothesized that ART alters the clonotypic phenotype and structural composition of CD4+ and CD8+ T cells during CM co-infection. For this substudy, peripheral blood mononuclear cells (PBMC) were isolated at four time points from CM patients following ART initiation during the parent study (ClinicalTrials.gov number, NCT01075152). Phenotypic characterization of CD4+ and CD8+ T cells was done using T cell surface marker monoclonal antibodies by flow cytometry. There was variation in the expression of immunophenotypic markers defining central memory (CD27+CD45R0+), effector memory (CD45R0+CD27-), immune activation (CD38+ and Human Leucocyte Antigen DR (HLA-DR+), and exhaustion (Programmed cell death protein one (PD-1) in the CD4+ T cell subset. In comparison to the CD4+ T cell population, the CD8+ central memory subset declined gradually with minimal increase in the effector memory subset. Both CD4+ and CD8+ T cell immune exhaustion and activation markers remained elevated over 12 weeks. The relative surge and decline in the expression of T cell surface markers outlines a variation in the differentiation of CD4+ T cells during ART treatment during CM co-infection.

A Mechanistic Computational Model Reveals That Plasticity of CD4+ T Cell Differentiation Is a Function of Cytokine Composition and Dosage.

CD4+ T cells provide cell-mediated immunity in response to various antigens. During an immune response, naïve CD4+ T cells differentiate into specialized effector T helper (Th1, Th2, and Th17) cells and induced regulatory (iTreg) cells based on a cytokine milieu. In recent studies, complex phenotypes resembling more than one classical T cell lineage have been experimentally observed. Herein, we sought to characterize the capacity of T cell differentiation in response to the complex extracellular environment. We constructed a comprehensive mechanistic (logical) computational model of the signal transduction that regulates T cell differentiation. The model's dynamics were characterized and analyzed under 511 different environmental conditions. Under these conditions, the model predicted the classical as well as the novel complex (mixed) T cell phenotypes that can co-express transcription factors (TFs) related to multiple differentiated T cell lineages. Analyses of the model suggest that the lineage decision is regulated by both compositions and dosage of signals that constitute the extracellular environment. In this regard, we first characterized the specific patterns of extracellular environments that result in novel T cell phenotypes. Next, we predicted the inputs that can regulate the transition between the canonical and complex T cell phenotypes in a dose-dependent manner. Finally, we predicted the optimal levels of inputs that can simultaneously maximize the activity of multiple lineage-specifying TFs and that can drive a phenotype toward one of the co-expressed TFs. In conclusion, our study provides new insights into the plasticity of CD4+ T cell differentiation, and also acts as a tool to design testable hypotheses for the generation of complex T cell phenotypes by various input combinations and dosages.

Influence of various medium environment to in vitro human T cell culture.

Nowadays, adoptive T cell immunotherapy is emerging as a novel and potent treatment for cancer. To prepare enough effective T cells for treatment use, their rapid expansion is favorable. Our study compared 6 commonly used cultural media for human T cells, including serum-containing media and serum-free media, namely RPMI 1640, IMDM, Gibco OpTmizer CTS T Cell Expansion SFM, Gibco AIM-V Medium CTS, LONZA X-VIVO 15, and StemSpan SFEM with or without Dynabeads Human T-Activator CD3/CD28, on in vitro T cell expansion, apoptosis, and immune phenotype. Our study results suggest that serum-free media provide better proliferation environment for T cells. Among the 3 serum-free media, we identify OpTmizer and AIM-V as better T cell culture environments compared with X-VIVO as T cells are proved to have higher viability in the first two media. Besides, we found that in vitro human T cells keep relatively resting status among non-CD3/CD28 groups, since they have weak proliferation and apoptosis abilities. The phenotypes of T cells in different cultural environments over time indicate T cells maturation during culture duration. These results provide a firm foundation of adoptive T cell immunotherapy.

Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes.

T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were in the range of 1-17 (median = 5), and in several patients, the number of clonotypes expanded within tumor lesions resembled that observed among autologous peripheral blood mononuclear cells. Moreover, several of these clonotypes were identical in TILs and PBMCs. Flow cytometry data demonstrated that the general differentiation status of CD8+ TILs differed from that of circulating CD8+ T cells. Furthermore, PD-1 and LAG-3 were expressed by a significantly higher percentage of CD8+ RCC-infiltrating lymphocytes as compared with PBMCs obtained from RCC patients or healthy individuals. Thus, CD8+ TILs display a differentiated phenotype and express activation markers as well as surface molecules associated with the inhibition of T-cell functions. However, TILs are characterized by a low amount of expanded T-cell clonotypes.