Phenotype Of CD4 Research Articles

Differences in the intrahepatic expression of immune checkpoint molecules on T cells and natural killer cells in chronic HBV patients

BackgroundPatients with chronic hepatitis B virus (HBV) infection are characterized by impaired immune response that fails to eliminate HBV. Immune checkpoint molecules (ICMs) control the amplitude of the activation and function of immune cells, which makes them the key regulators of immune response.MethodsWe performed a multiparametric flow cytometry analysis of ICMs and determined their expression on intrahepatic lymphocyte subsets in untreated and treated patients with HBV in comparison with non-pathological liver tissue.ResultsThe liver of untreated HBV patients exhibited a high accumulation of PD-1+CD8+ T cells, while the frequencies of 4-1BB+ T cells, 4-1BB+ natural killer (NK) cells, and TIM-3+CD8+ T cells were the highest in the chronic hepatitis phase. Our findings showed that the HBeAg status is linked to a distinct immune phenotype of intrahepatic CD8+ T cells and NK cells characterized by high expression of ICMs, particularly 4-1BB. Importantly, antiviral treatment partially restored the normal expression of ICMs. Finally, we described important differences in ICM expression between intrahepatic and circulating NK cells in HBV patients.ConclusionsOur study shows clear differences in the intrahepatic expression of ICMs on NK cells and T cells in chronic HBV patients depending on their clinical stage.

Phenotype-Led Identification of IL-10 Upregulators in Human CD4+ T-cells and Elucidation of Their Pharmacology as Highly Selective CDK8/CDK19 Inhibitors.

Therapeutics promoting the endogenous production of IL-10 have the potential to restore homeostasis in inflammatory disorders such as inflammatory bowel disease (IBD). Here we describe the identification of a series of IL-10 upregulators based on a pyrimidyl-piperidine scaffold through a high throughput phenotypic CD4+ T-cell multiplex assay. In vitro optimization of the initial hit yielded a lead with good potency and an in vitro clearance profile, compound 3-7, which additionally demonstrated efficacy in a murine endotoxin challenge PK-PD mechanistic model. Target deconvolution efforts identified compound 3-7 as a highly selective CDK8/19 inhibitor, and crystallographic studies unveiled its binding mode to the CDK8/Cyclin-C complex, characterized by an unusual water-mediated hydrogen bond to the kinase hinge region.

Metabolic, genetic and immunological features of relatives of type 1 diabetes patients with elevated insulin resistance.

Insulin resistance plays a pivotal role in the preclinical stages of type 1 diabetes (T1D). This study aims at exploring the genetic, metabolic, and immunological features associated with insulin resistance among individuals at risk of developing T1D. We retrospectively selected relatives of individuals with T1D from participants in the TrialNet Pathway to Prevention study. They were categorized into two groups: high-H (n = 27) and low-H (n = 30), based on the upper and lower quartiles of insulin resistance assessed using the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). Genetic predisposition was determined using the T1D Genetic Risk Score 1 (GRS1). Additionally, glucose control was evaluated through an oral glucose tolerance test and levels of metabolic hormones and inflammatory cytokines were measured in the serum. Flow cytometry analysis was employed to assess frequency and phenotype of islet-specific CD8 T cells. While GRS1 were similar between the low-H and high-H groups, high-H individuals displayed a distinct metabolic profile, characterized by compensatory hyperinsulinemia, even while maintaining normoglycemia. Circulating cytokine levels were similar between the two groups. However, immune profiling revealed a central memory and activated profile of GAD65-specific CD8 T cells, along with an increased frequency of insulin-specific CD8 T cells in high-H individuals. The enrichment in insulin-specific CD8 T cells was independent of body mass. These findings highlight the intricate interplay between insulin resistance, genetic factors, and immune activation in the context of T1D susceptibility, indicating potential connections between insulin resistance and immune responses specific to islet cells.

A novel designed anti-PD-L1/OX40 bispecific antibody augments both peripheral and tumor-associated immune responses for boosting anti-tumor immunity.

Bispecific antibodies (BsAbs) combining simultaneous PD-L1 blockade and conditional co-stimulatory receptor activation have been developed to improve immune checkpoint therapy response. However, several PD-L1-based BsAbs have encountered clinical challenges, including insufficient activity or unexpected toxicity. In this study, we propose OX40 as a more suitable target partner for PD-L1-based BsAb design compared to ongoing clinical partners (CD27 and 4-1BB). We present a novel Fc-silenced tetravalent PD-L1/OX40 BsAb (EMB-09), which efficiently blocks PD-1/PD-L1 interactions and induces PD-L1-dependent OX40 activation, leading to enhanced T cell activation. EMB-09 demonstrated improved anti-tumor activity compared to the anti-PD-L1 monoclonal antibody. Significantly, EMB-09 activated effector memory T cells in peripheral immune system, promoted the influx of stem-like CD8+ T cells into the tumor site, resulting in a more active phenotype of CD8+ tumor-infiltrating lymphocytes. In an ongoing first-in-human study in patients with advanced refractory solid tumors (NCT05263180), EMB-09 demonstrated a consistent pharmacodynamic response and early efficacy signals.

Transient Anti-TCRβ mAb Treatment Induces CD4+ T Cell Exhaustion and Prolongs Survival in a Mouse Model of Systemic Lupus Erythematosus.

T cells play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Chronic T cell receptor (TCR) signalling induces T cell exhaustion, characterised by reduced capacity to induce tissue damage. Here, we investigated the therapeutic potential of the anti-TCRβ (H57-597) monoclonal antibody (mAb) in a mouse model of SLE. Four-month-old MRL/lpr mice exhibiting SLE phenotypes received 5 weekly doses of anti-TCRβ mAb or phosphate-buffered saline (PBS) vehicle control. Subsequently, mouse survival was monitored daily. On day 1 post the final dose of treatment, SLE pathogenesis was determined using histological staining and spot urine test. T and B cell states in the brain, kidney, and secondary lymphoid organs were determined by flow cytometry. Transient treatment of anti-TCRβ mAb significantly prolonged the survival of MRL/lpr mice. Accordingly, MRL/lpr mice in the anti-TCRβ mAb group exhibited decreased proteinuria scores and minimal renal pathological damage compared to the PBS control group. Flow cytometric analysis revealed that anti-TCRβ mAb treatment resulted in a reduction in the frequencies of CD4+ T cells and CD138+B220lo/- plasma cells, plus an increase in Foxp3+ regulatory T cell frequency. Furthermore, CD4+ T cells from anti-TCRβ mAb treated mice exhibited elevated expression levels of PD-1 and TIM-3, with reduced IFN-γ production, indicative of an exhaustion-like phenotype. Therefore, transient administration of anti-TCRβ mAb treatment induces an exhaustion-like phenotype in CD4+ T cells, resulting in prolonged survival of MRL/lpr mice. Inducing autoreactive T-cell exhaustion holds promise as an attractive therapeutic approach for SLE.

CAR T cells secreting NGF-neutralizing scFv enhance efficacy in clear cell renal cell carcinoma by relieving immunosuppression through immunosympathectomy

BackgroundChimeric antigen receptor (CAR) T cells have demonstrated remarkable breakthroughs in treating hematologic malignancies, yet their efficacy in solid tumors is limited by the immunosuppressive microenvironment. Sympathetic nerves significantly contribute...

EZH2 inhibition enhances T cell immunotherapies by inducing lymphoma immunogenicity and improving T cell function.

T cell-based immunotherapies have demonstrated effectiveness in treating diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) but predicting response and understanding resistance remains a challenge. To address this, we developed syngeneic models reflecting the genetics, epigenetics, and immunology of human FL and DLBCL. We show that EZH2 inhibitors reprogram these models to re-express Tcell engagement genes and render them highly immunogenic. EZH2 inhibitors do not harm tumor-controlling Tcells or CAR-T cells. Instead, they reduce regulatory Tcells, promote memory chimeric antigen receptor (CAR) CD8 phenotypes, and reduce exhaustion, resulting in a decreased tumor burden. Intravital 2-photon imaging shows increased CAR-T recruitment and interaction within the tumor microenvironment, improving lymphoma cell killing. Therefore, EZH2 inhibition enhances CAR-T cell efficacy through direct effects on CAR-T cells, in addition to rendering lymphoma B cells immunogenic. This approach is currently being evaluated in two clinical trials, NCT05934838 and NCT05994235, to improve immunotherapy outcomes in B cell lymphoma patients.

Immune Signatures of Solid Tumor Patients Treated With Immune Checkpoint Inhibitors: An Observational Study.

Our study aimed to comprehensively describe the features of peripheral blood multiple immune cell phenotypes in solid tumor patients during pretreatment and after immunotherapy, providing a more convenient approach for studying the prognosis of immunotherapy in different solid tumor patients. We prospectively recruited patients with advanced solid tumors from Peking Union Medical College Hospital (PUMCH) between February 2023 and April 2024. Using multicolor flow cytometry, our study comprehensively observed and described the signatures of peripheral blood lymphocyte subsets including activation, proliferation, function, naïve memory, and T cell exhaustion immune cell subsets in this population of pretreatment and after immunotherapy. Our study enrolled 59 advanced solid tumor patients with immunotherapy and 59 healthy controls were matched by age and gender. The results demonstrated a marked upregulation in the expression of lymphocyte activation markers CD38 and HLA-DR, as well as exhaustion and proliferation markers PD-1 and Ki67, in solid tumor patients compared to healthy controls. After immune checkpoint blockade (ICB) treatment, mainly the expression of Ki67CD4+T and HLA-DRCD38CD4+T, was significantly upregulated compared to pretreatment levels (p = 0.017, p = 0.019, respectively). We further found that gynecological tumors with better prognoses had higher baseline activation levels of CD4+ T cells compared to other solid tumors with poorer prognoses. Our study elucidated the characteristics of different lymphocyte subsets in the peripheral blood of solid tumor patients. Further research revealed changes in the phenotypes of different lymphocyte subsets after ICIs treatment, with the activated phenotype of CD4+ T cells playing a crucial role in the antitumor effect. This lays the groundwork for further exploration of prognostic biomarkers and predictive models for cancer patients with immunotherapy.

Antigen-specific T cell frequency and phenotype mirrors disease activity in DRB1*04:04+ rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is associated with high-risk HLA class II alleles known as the "RA shared epitope." Among prevalent shared epitope alleles, study of DRB1*04:04 has been limited. To define relevant epitopes, we identified citrullinated peptide sequences from synovial antigens that were predicted to bind to HLA-DRB1*04:04 and utilized a systematic approach to confirm their binding and assess their recognition by CD4 T cells. After confirming the immunogenicity of 13 peptides derived from aggrecan, cartilage intermediate layer protein (CILP), α-enolase, vimentin, and fibrinogen, we assessed their recognition by T cells from a synovial tissue sample, observing measurable responses to 8 of the 13 peptides. We then implemented a multicolor tetramer panel to evaluate the frequency and phenotype of antigen-specific CD4 T cells in individuals with anti-citrullinated protein antibody (ACPA)-positive RA and controls. In subjects with RA, CILP-specific T cell frequencies were significantly higher than those of other antigens. The surface phenotypes exhibited by antigen-specific T cells were heterogeneous, but Th1-like and Th2-like cells predominated. Stratifying based on disease status and activity, antigen-specific T cells were more frequent and most strongly polarized in RA subjects with high disease activity. In total, these findings identify novel citrullinated epitopes that can be used to interrogate antigen-specific CD4 T cells and show that antigen-specific T cell frequency is elevated in subjects with high disease activity.

CRISPR/Cas9-mediated RELA and RELC knockout in human regulatory T cells abrogates FOXP3 expression and suppressive function

Mutations in NF-κB-related molecules result in combined immunodeficiency characterized by recurrent infection. In this study, we aimed to investigate the association between mutations in NF-κB family members, RELA, RELB, and RELC, and regulatory T cell (Treg) function in humans. RELA, RELB, and RELC were knocked out using CRISPR/Cas9-mediated homologous recombination in CD4+/CD8+ T cells and Tregs isolated from healthy donors. The RELA, RELB, and RELC knockouts did not alter the phenotype or cytokine production profile of CD4+/CD8+ T cells or Jurkat cells. Similar to that observed in knockout mice, RELA or RELC knockout in MT-2 cells and freshly isolated Tregs reduced FOXP3 expression and the immune suppressive function of Tregs. Additionally, PD-L1 expression in effector T cells and Tregs decreased considerably following RELC knockdown. These findings demonstrated that the deletion of RELA or RELC resulted in the loss of Treg-like phenotype and function owing to the downregulation of FOXP3 expression.

Adipose-Derived Mesenchymal Stem Cells from Arthritis Patients: Differential Modulation of CD4⁺ T Cell Activation and Cytokine Production.

BACKGROUND Adipose-derived stem cells (ASCs) from intra-articular adipose tissue of osteoarthritis (OA) and rheumatoid arthritis (RA) patients similarly regulate the proliferation of activated CD4⁺ T lymphocytes and exhibit comparable differentiation potential. This study aimed to assess the impact of ASCs from RA patients on CD4⁺ T cell activation and differentiation into Th17 and T regulatory (Treg) cells. MATERIAL AND METHODS Intra-articular adipose tissue samples were obtained from patients with RA and OA, who underwent knee replacement surgery. ASCs were isolated and cultured either with isolated CD4⁺ cells or with peripheral blood mononuclear cells. After culture, CD4⁺ T cell phenotype was evaluated by flow cytometry, and cytokine concentrations in culture supernatants were analyzed via ELISA. Blocking experiments were conducted to identify the soluble agents responsible for the immunomodulatory effects of ASCs. RESULTS RA- and OA-derived ASCs effectively modulated CD25 and CD69 expression on CD4⁺ cells. RA-derived ASCs failed to induce Tregs, decreased HLA-DR expression, and increased IL-35 production. RA- and OA-derived ASCs reduced TNF and IFN-γ production but increased IL-17 production. The immunomodulatory activities of ASCs were linked to the kynurenine pathway and prostaglandin E2. CONCLUSIONS This study indicates that ASCs modulate the phenotype of CD4⁺ T cells and influence the production of both pro-inflammatory and anti-inflammatory cytokines. However, ASCs from RA patients appear to have impaired immunomodulatory abilities, raising concerns about their therapeutic potential. Further research is needed to enhance our understanding of ASCs biology and their therapeutic utility.

Vaccine responses and hybrid immunity in people living with HIV after SARS-CoV-2 breakthrough infections

The COVID-19 pandemic posed a challenge for people living with HIV (PLWH), particularly immune non-responders (INR) with compromised CD4 T-cell reconstitution following antiretroviral therapy (CD4 count <350 cells per mm3). Their diminished vaccine responses raised concerns about their vulnerability to SARS-CoV-2 breakthrough infections (BTI). Our in-depth study here revealed chronic inflammation in PLWH and a limited anti-Spike IgG response after vaccination in INR. Nevertheless, the imprinting of Spike-specific B cells by vaccination significantly enhanced the humoral responses after BTI. Notably, the magnitude of cellular CD4 response in all PLWH was comparable to that in healthy donors (HD). However, the polyfunctionality and phenotype of Spike-specific CD8 T cells in INR differed from controls. The findings highlight the need for additional boosters with variant vaccines, and for monitoring ART adherence and the durability of both humoral and cellular anti-SARS-CoV-2 immunity in INR.

High-dimensional profiling of immune responses to kidney transplant reveals heterogeneous T helper 1 and B cell effectors associated with rejection

Rejection is a primary cause of allograft dysfunction after kidney transplantation. The diversity of immune subpopulations involved in the different endotypes of rejection remains to be delineated at single-cell resolution. In a cohort of 76 kidney transplant recipients, we conducted high-dimensional immune phenotyping of blood CD4 T and B cells, single-cell RNA and T/B cell receptor sequencing, and plasma cytokine profiling. Phenotypic, transcriptional, and clonal states of CD4T and B cells could significantly distinguish stable allograft states from rejection. Patients undergoing T cell-mediated rejection displayed accumulation of clonally expanded cytotoxic T helper (Th)1 cells and Th17-like cells, associated with predominant naive B cell responses. In contrast, antibody-mediated rejection was characterized by clonal expansion of Th1-polarized T follicular helper cells and effector T-bet+ memory B cells, both of which strongly expressed interleukin 12 and tumor necrosis factor-signaling pathways. Plasma cytokine analysis confirmed mixed Th1/Th17 and Th1/T follicular helper cell-driven inflammatory profiles distinguishing T cell-mediated rejection and antibody-mediated rejection, respectively. CD4T and B cell subpopulations and signatures were validated using bulk RNA-seq analysis of matched kidney allografts and using an independent single-cell RNA-seq data set. These data improve mechanistic understanding of the immune pathogenesis of rejection and support the development of more specific immunosuppressive therapies to treat allograft rejection.

Evaluation of CD96 Expression in Acute Myeloid Leukemia Patients: Relation to Prognosis and Induction Chemotherapy

Abstract Background In acute myeloid leukemia (AML), leukemic stem cells (LSCs) form a distinct group of leukemia cells showing stem cell-like features like self-renewal and proliferation. These potent cells significantly contribute to chemotherapy resistance, a major obstacle in AML therapy. LSCs express the characteristic phenotype CD34+/CD38- and have several specific markers, including CD33, CD96, CD123, CD90 and Death associated protein kinase (DAPK). Successful LSC eradication demands a combination of approaches, including targeting LSC-specific surface molecules. Objective To assess the expression of CD96 in AML patients at initial diagnosis and to correlate it with prognostic factors and response to induction chemotherapy. Materials and Methods This Case control study was conducted on 40 newly diagnosed AML patients in addition to 10 controls at Ain Shams University Hospitals between March 2022 and March 2023. Multicolor flowcytometry was employed to analyze CD96 expression in the CD34+/CD38- cell population of AML patients. The study further investigated the correlation between CD96 expression and both prognostic factors and response to induction chemotherapy. Results CD96 expression was significantly higher in AML patients group than control group (p-value = 0.008). CD96 was positive in 60% of AML patients and there was a highly significant association between CD96 expression and response to induction chemotherapy (p-value = &amp;lt;0.001). However, there was no statistically significant relation between CD96 expression with laboratory findings and standard prognostic factors. Conclusion Our study identified CD96 as a promising marker for LSCs in AML patients. Its frequent expression in the CD34+/CD38- LSC population and association with lower response rate to induction chemotherapy suggest its potential as a valuable tool for LSC identification, targeted therapy development and improved treatment outcome prediction in AML.

Differential expression of HIV target cells CCR5 and α4β7 in tissue resident memory CD4 T cells in endocervix during the menstrual cycle of HIV seronegative women.

Ovarian hormones are known to modulate the immune system in the female genital tract (FGT). We sought to define the impact of the menstrual cycle on the mucosal HIV target cell levels, and tissue-resident CD4 T cells. Here, we characterized the distribution, phenotype, and function of CD4 T cells with special emphasis on HIV target cells (CCR5+ and α4β7+) as well as tissue-resident memory (TRM; CD69+ and CD103+) CD4 T cells in FGT of cycling women. Peripheral blood and Endocervical cells (EC-collected from cytobrush) were collected from 105 healthy women and performed multicolor flow cytometry to characterize the various subsets of CD4 T cells. Cervicovaginal lavage (CVL) were collected for cytokine analysis and plasma were collected for hormonal analysis. All parameters were compared between follicular and luteal phase of menstrual cycle. Our findings revealed no significant difference in the blood CD4 T cell subsets between the follicular and luteal phase. However, in EC, the proportion of several cell types was higher in the follicular phase compared to the luteal phase of menstrual cycle, including CCR5+α4β7-cells (p=0.01), CD69+CD103+ TRM (p=0.02), CCR5+CD69+CD103+ TRM (p=0.001) and FoxP3+ CD4 T cells (p=0.0005). In contrast, α4β7+ CCR5- cells were higher in the luteal phase (p=0.0004) compared to the follicular phase. In addition, we also found that hormonal levels (P4/E2 ratio) and cytokines (IL-5 and IL-6) were correlated with CCR5+ CD4 T cells subsets during the follicular phase of the menstrual cycle. Overall, these findings suggest the difference in the expression of CCR5 and α4β7 in TRM CD4 T cell subsets in endocervix of HIV seronegative women between the follicular and luteal phase. Increase in the CCR5+ expression on TRM subsets could increase susceptibility to HIV infection during follicular phase of the menstrual cycle.

CXCR4 orchestrates the TOX-programmed exhausted phenotype of CD8+ T cells via JAK2/STAT3 pathway

Evidence from clinical trials suggests that CXCR4 antagonists enhance immunotherapy effectiveness in several cancers. However, the specific mechanisms through which CXCR4 contributes to immune cell phenotypes are not fully understood. Here, we employed single-cell transcriptomic analysis and identified CXCR4 as a marker gene in Tcells, with CD8+PD-1high exhausted T (Tex) cells exhibiting high CXCR4 expression. By blocking CXCR4, the Tex phenotype was attenuated invivo. Mechanistically, CXCR4-blocking Tcells mitigated the Tex phenotype by regulating the JAK2-STAT3 pathway. Single-cell RNA/TCR/ATAC-seq confirmed that Cxcr4-deficient CD8+ Tcells epigenetically mitigated the transition from functional to exhausted phenotypes. Notably, clinical sample analysis revealed that CXCR4+CD8+ Tcells showed higher expression in patients with a non-complete pathological response. Collectively, these findings demonstrate the mechanism by which CXCR4 orchestrates CD8+ Tex cells and provide a rationale for combining CXCR4 antagonists with immunotherapy in clinical trials.

Mtb specific HLA-E restricted T cells are induced during Mtb infection but not after BCG administration in non-human primates and humans.

Novel vaccines targeting the world's deadliest pathogen Mycobacterium tuberculosis (Mtb) are urgently needed as the efficacy of the Bacillus Calmette-Guérin (BCG) vaccine in its current use is limited. HLA-E is a virtually monomorphic unconventional antigen presentation molecule and HLA-E restricted Mtb specific CD8+ T cells can control intracellular Mtb growth, making HLA-E a promising vaccine target for Mtb. In this study, we evaluated the frequency and phenotype of HLA-E restricted Mtb specific CD4+/CD8+ T cells in the circulation and bronchoalveolar lavage fluid of two independent non-human primate (NHP) studies and from humans receiving BCG either intradermally or mucosally. BCG vaccination followed by Mtb challenge in NHPs did not affect the frequency of circulating and local HLA-E/Mtb CD4+ and CD8+ T cells, and we saw the same in humans receiving BCG. HLA-E/Mtb T cell frequencies were significantly increased after Mtb challenge in unvaccinated NHPs, which was correlated with higher TB pathology. Together, HLA-E/Mtb restricted T cells are minimally induced by BCG in humans and rhesus macaques (RMs) but can be elicited after Mtb infection in unvaccinated RMs. These results give new insights into targeting HLA-E as a potential immune mechanism against TB.

In vitro-irradiated cancer vaccine enhances anti-tumor efficacy of radiotherapy and PD-L1 blockade in a syngeneic murine breast cancer model

Background and PurposeLocal radiotherapy (RT) exerts immunostimulatory effects by inducing immunogenic cell death. However, it remains unknown whether in vitro–irradiated tumor cells can elicit anti-tumor responses and enhance the efficacy of local RT and immune checkpoint inhibitors when injected in vivo. Methods and MaterialsWe tested the “in vitro–irradiated cancer vaccine (ICV)”, wherein tumor cells killed by varying doses of irradiation and their supernatants are intravenously injected. We examined the efficacy of combining local RT (24 Gy in three fractions), PD-L1 blockade, and the ICV in a murine breast cancer model. The immune cell profiles were analyzed via flow cytometry and immunohistochemistry. The cytokine levels were measured by multiplex immunoassays. ResultsThe ICV significantly increased the effector memory phenotype and interferon-γ production capacity in splenic CD8+ T cells. The in vitro–irradiated products contained immune response-related molecules. When combined with local RT and PD-L1 blockade, the ICV significantly delayed the growth of irradiated and non-irradiated tumors. The triple combination therapy increased the proportions of CD8+ T cells and effector memory CD8+ T cells while decreasing the proportion of CTLA-4+ exhausted CD8+ T cells within tumor microenvironment. Additionally, plasma level of interferon-γ and proliferation of effector T cells in the spleen and tumor-draining lymph nodes were significantly increased by the triple combination therapy. ConclusionsThe ICV enhanced the therapeutic efficacy of local RT and PD-L1 blockade by augmenting anti-tumor immune responses. Our findings suggest a therapeutic potential of in vitro–irradiation products of tumor cells.

Single-cell RNA sequencing comparison of CD4+, CD8+ and TCR-γδ+ cutaneous T-cell lymphomas reveals subset-specific molecular phenotypes.

Malignant clones of primary cutaneous T-cell lymphomas (CTCL) can show a CD4, CD8 or TCR-γδ phenotype, but their individual impact on tumor biology and skin lesion formation remains ill-defined. To perform a comprehensive molecular characterization of CD4+ vs. CD8+ and TCR-γ/δ+ CTCL lesions. We performed scRNA-seq of 18 CTCL skin biopsies to compare classic CD4+ advanced-stage mycosis fungoides (MF) with TCR-γ/δ+MF and primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (Berti's lymphoma). Malignant clones of TCR-γ/δ+MF and Berti's lymphoma showed similar clustering patterns distinct from CD4+MF, along with increased expression of cytotoxic markers such as NKG7, CTSW, GZMA, and GZMM. Only advanced-stage CD4+MF clones expressed central memory T-cell markers (SELL, CCR7, LEF1), alongside B1/B2 blood involvement, whereas TCR-γ/δ+MF and Berti's lymphoma harbored a more tissue-resident phenotype (CD69, CXCR4, NR4A1) without detectable cells in the blood. CD4+MF and TCR-γ/δ+MF skin lesions harbored strong type 2 immune activation across myeloid cells, while Berti's lymphoma was more skewed towards type 1 immune responses. Both CD4+MF and TCR-γ/δ+MF lesions showed upregulation of keratinocyte hyperactivation markers such as S100As and KRT16 genes. This increase was entirely absent in Berti's lymphoma, possibly reflecting an aberrant keratinocyte response to invading tumor cells, that could contribute to the formation of the typical ulcero-necrotic lesions within this entity. Our scRNAseq profiling study reveals specific molecular patterns associated with distinct CTCL subtypes.

Features of T-cell and humoral immunity of patients with acute coronary syndrome, with and without COVID-19, depending on/peripheral blood B-lymphocytes with the phenotype CD3–CD19+CD5+ level

The aim of the work was to assess the T-cell and humoral immune arms in patients with acute coronary syndrome (ACS) recovered from or not exposed to COVID-19. 86 men with ACS aged 40 to 65 years old, who suffered or not exposed to COVID-19, which required stenting of coronary arteries, were examined. Depending on the peripheral blood B-lymphocyte CD3–CD19+CD5+ level and the former COVID-19 history, all patients were divided into 6 groups. Of the previously COVID-19 exposed subjects CD3–CD19+CD5+ lymphocytes were at: reduced (group 1), normal (group 2), increased (group 3) level. COVID-19 unexposed subjects had CD3–CD19+CD5+ lymphocyte level: reduced (group 4), normal (group 5), increased (group 6). The most severe clinical manifestations were observed in group 1: with a larger rate of stent thrombosis and increased mortality. In absolute numbers, T-lymphocytes were significantly lower in group 1 compared to other groups, except group 4. The lowest both relative and absolute T-helper cell counts were recorded in covid-19 patients, with reduced CD3–CD19+CD5+ cell level. NK-lymphocytes both in relative and absolute level were significantly (p 0.05) higher in patients who suffered from COVID-19 as compared to those in patients unexposed to previous COVID-19. Percentage of late-activation T-regulatory cells was minimal in patients with former COVID-19 along with high B-lymphocyte CD3–CD19+CD5+ level, which significantly (p 0.05) differed from those in subjects lacking former COVID-19 history. In addition, significantly lower B-lymphocyte CD3–CD19+CD5– level was found in group 1 and 4. The IgM level peaked in group 5 — subjects lacking former COVID-19 history and having normal CD3–CD19+CD5+ cell level that significantly differed in comparison with that of in group 1, 2, 3. At the same time, the maximum value of IgG level was recorded in COVID-19 patients with normal CD3–CD19+CD5+ cells, and significantly differed from those found in group 5 and 6. C1-inhibitor level was higher in group 3 that significantly differed from that in other groups. SARS-CoV-2-specific IgG and IgM levels were significantly higher in COVID-19-positive vs. COVID-19-negative patients. Conclusion. In patients who have suffered COVID-19 with low B-lymphocyte CD3–CD19+CD5+ level, a significantly compromised immune protection was noted in comparison with other groups: a decline in total T-lymphocyte, T-helper, early- and late-activation T-lymphocyte, T-regulatory cell, B-lymphocyte CD3–CD19+CD5– levels, which was associated with a more severe disease course characterized by stent thrombosis and large mortality.