Subpopulation Of CD4 Research Articles

Circulating tumor-reactive KIR+CD8+ T cells suppress anti-tumor immunity in patients with melanoma.

Effective anti-tumor immunity is driven by cytotoxic CD8+ T cells with specificity for tumor antigens. However, the factors that control successful tumor rejection are not well understood. Here we identify a subpopulation of CD8+ T cells that are tumor-antigen-specific and can be identified by KIR expression but paradoxically impair anti-tumor immunity in patients with melanoma. These tumor-antigen-specific KIR+CD8+ regulatory T cells target other tumor-antigen-specific CD8+ T cells, can be detected in both the tumor and the blood, have a conserved transcriptional program and are associated with a poor overall survival. These findings broaden our understanding of the transcriptional and functional heterogeneity of human CD8+ T cells and implicate KIR+CD8+ regulatory T cells as a cellular mediator of immune evasion in human cancer.

Central Memory CD4 T Cells from Persons with HIV Accumulate DNA Content Defects During Proliferative Response.

Central memory (TCM) cells are a subpopulation of CD4 T cells that sustain overall CD4 T cell counts in HIV infection. The mechanisms underlying their eventual demise, which leads to loss of CD4 T cell counts, are not known. To understand their proneness to death despite their increased movement to proliferation, we examined cell division together with possible cell accumulation in different phases of the cell cycle. Purified circulating TCM cells from untreated people living with HIV (PLWH) (n = 9) and healthy controls (n = 10) were stimulated in vitro using anti-CD3/CD28 agonistic antibodies plus IL-2 and cultured for 4 days. Cell viability, DNA content, proliferation, and cyclin A and cyclin B expression were measured. We found that PLWH TCM cells more frequently had a DNA content lower than G0/G1, compared with controls (p = .043). These cells accumulated with each division. The proportion of cells with sub-G0/G1 DNA content that were cycling (expressing cyclin A) was greater in the PLWH group (p = .003). The percentage of TCM cells expressing cyclin A+ among those in G0/G1 and was also greater in the PLWH group (p = .043), suggesting arrest before G2/M. While TCM cells from PLWH can proliferate, during this process some of them accumulate defects in DNA content that are incompatible with viability, suggesting that they could be intrinsically prone to cell cycle-dependent death. This provides a possible mechanism underlying the increased TCM cell turnover in HIV infection.

Flow Cytometric Immunophenotyping: Minimal Differences in Fresh and Cryopreserved Peripheral Blood Mononuclear Cells versus Whole Blood.

Background/Objectives: Flow cytometry is a convenient tool in immunophenotyping for monitoring the status of immunological conditions and diseases. The aim of this study was to investigate the effect of isolation and cryopreservation by flow cytometric analysis on subpopulations of CD4+ T helper (Th), T regulatory (Treg), CD8+ T cytotoxic (Tc), CD56+ NK, CD19+ B and monocytes. Freshly isolated and cryopreserved peripheral blood mononuclear cells (PBMCs) were compared to fresh whole blood. Methods: Peripheral blood was collected from healthy donors and prepared for flow cytometric analysis using the same panels of antibodies throughout the study. Results: Comparisons between fresh (F)- and cryopreserved (C)-PBMCs showed no major differences in percentages of CD4+, Th1, Th2 and CD4+CD25+CD127low Treg cells. No differences in percentage of CD8+ or subpopulations of naive/stem, central or effector memory cells were observed between F- and C-PBMCs. The percentage of CD56+ NK cells, CD19+ B cells or classical and nonclassical monocytes did not differ between F-and C-PBMCs either. On the contrary, whole blood had lower percentages of Th and NK cells but higher percentages of Th1, Th17, Th1Th17, Tregs, Tc and B cells compared to C-PBMCs, while it had a higher proportion of Tc compared to F-PBMCs. Conclusions: Flow cytometric immunophenotyping minimally differs between freshly isolated and cryopreserved PBMCs. This implies the possibility of cryostorage of cohorts for later analysis. Importantly, care must be taken when comparing results from whole blood with isolated and cryopreserved PBMCs. Collectively, these results can contribute to the standardization of flow cytometric protocols in both clinical and research settings.

Severe myelotoxicities related to Th1, Th2, Th17 and Treg CD4+ T cells subpopulations and their cytokine profiles in Mexican children with acute lymphoblastic leukemia during induction of chemotherapy

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer in the world, and the myelotoxicities due to chemotherapy can lead to severe infections and hemorrhages with high morbidity and mortality. The immune response has implications in the severity of cancer or survival. This immune response is associated with the predominant subpopulations CD4+ T cells and their effector cytokines. However, it is not known if the immune responses are associated with severe myelotoxicities and mortality in children with ALL. Methods To evaluate the contributions of immunological factors on ALL severe myelotoxicities, we identified the Th1, Th2, Th9, Th17, Th22 and Tregs subpopulations of CD4+ T cells according to their chemokine receptor expression patterns in peripheral blood mononuclear cells by flow cytometry. We evaluated also their cytokines levels by Milliplex in serum from 10 children with ALL at diagnosis (day 0), during induction (day 19) and at the end of induction (day 30) between April 2022 and July 2023. Clinical characteristics such age, sex, type and risk of ALL, hemoglobin and platelets levels, absolute neutrophil count, grade of severity and type of myelotoxicities, infections, and hemorrhages were collected. Clinical outcomes as hospitalization care requirements, ICU admission, leukemia remission or relapsed and mortality were followed and documented. Results The age of children was 8.9 years, (SD,5.6; range 2-16), 60% were male sex, all children had pre- B ALL diagnosis, and 80% were intermediate risk-ALL. Th1 cells were predominant at diagnosis (8.1%) and at the end of induction (7.8%), and Th2 cells at day 19 (7.4%), whereas Th17 were significantly decreased at day 30 (2.6%) (p<0.05). Four patients presented bloodstream infection, two by Gram-positive bacteria (Micrococcus, and Streptococcus), one by Klebsiella pneumoniae antimicrobial resistant, and one by Trichosporum asahii. Severe myelotoxicities were presented in 20% of the patients at day19, and in 30% at day 30. Remission was documented in all patients at day 30 and one was identified with early relapse in the follow-up. Three patients (30%) presented poor outcomes, requiring care in ICU and one died (10%) due to fungal infection. Treg cells were significantly predominant in the children who died by fungal infection (p<0.05), with IL-10 level of 84.6 pg/mL but not detectable IL-35 levels. Conclusion Th17 and Treg subpopulations of CD4+ T cells can play an essential role in immune microenvironment during induction related with the severity of the myelotoxicities and infections, and cytokine IL-10 could be used as marker of infection outcome.

CD8+ Tumor-Infiltrating Lymphocytes in Head and Neck Cancer: A Review.

CD8+ tumor-infiltrating lymphocytes (TILs) are increasingly used in oncology as a prognostic and predictive tool to guide patient management. This review summarizes current literature on CD8+ TILs in head and neck squamous cell carcinoma (SCC). Published meta-analyses and clinical trials evaluating CD8+ TILs were analyzed. Consistent positive associations between elevated CD8+ TILs and overall survival have been observed across head and neck sites. CD8+ TILs have been found to predict response to treatment, most commonly immunotherapy, but also chemoradiation. Numerous trials have shown that increased CD8+ TIL frequencies in pretreatment biopsies could identify patients likely to respond to neoadjuvant therapies. CD8+ TIL infiltration has also been elevated in responders both during and after treatment. However, wider adoption of CD8+ TIL quantification as a biomarker has been limited by the need for clinical validation and universal measurement guidelines for head and neck SCC, as there are for other malignancies. Measurement variability includes which tumor compartment is sampled, how TILs are quantified, and which cutoffs are clinically relevant. For several head and neck SCC, measurement of CD8+ TILs in the central or intratumoral compartment, followed by the stromal compartment, has been most consistently associated with survival. Future studies are needed to evaluate subpopulations of CD8+ TILs and biomarker-based treatment selection.

Expression of immune checkpoint markers in peripheral blood lymphocytes of women with fibroadenoma of the breast

Changes of the immune state in patients with fibroadenoma (FA) or other benign lesions of the breast, as well as the involvement of immune checkpoints in pathogenesis of these lesions, remain underexplored. The aim of this study was to compare the expression level of the key immune checkpoint markers CD279/PD-1, CD274/PD-L1, CD366/TIM3, and CD223/LAG3 in total circulating lymphocytes, T cell population as well as in its CD4 and CD8 subsets in peripheral blood from women with breast FA and healthy controls. Blood samples were taken from 12 women diagnosed with FA of the breast (aged 23-54 years, FA group) and 15 healthy women (aged 22-52 years, control group). Sample uptake was performed immediately before surgery, and samples were further analyzed by multicolor flow cytometry using monoclonal antibodies CD3-VioBlue, CD4/CD8-FITC, PD1-PE, PD-L1-PerCP-Cy.5.5, and TIM3/LAG3-APC. Each sample was incubated with 4 antibody combinations: CD3/CD4/PD1/PD-L1/TIM3, CD3/CD4/PD1/PD-L1/LAG3, CD3/CD8/PD1/PD-L1/TIM3, and CD3/CD8/PD1/PD-L1/LAG3. First, mono-expression of each of the 4 immune checkpoint markers was evaluated in the lymphocyte gate from both investigated groups. In FA samples, a significant increase in PD-L1 expression (assessed as percent of positive cells and fluorescence intensity change) was observed. Regarding expression of immune checkpoints in CD3+ T cells, along with significantly increased %PD-L1+, elevated numbers of PD1+T cells were detected. As for the differences in immune checkpoint expression changes between CD4+ and CD8+ T cell subsets, FA patient group demonstrated a more prominent increase in the amount of CD8+PD1+T cells relative to CD4 subset. The profiles of PD-L1 changes in CD4 and CD8 subpopulations were comparable showing, in both cases, a significant increase in FA sample group. We also analyzed changes in co-expression of any two immune checkpoint markers in CD4+ and CD8+ T cell subsets. The most noticeable was as increase in the prevalence of PD1+PD-L1+ phenotype in both T cell subpopulations from FA patients compared to the controls. With respect to co-expression of 3 checkpoint markers in CD4+ and CD8+ T cells, a significant increase in the percentage of PD1+PD-L1+TIM3+ cells among CD4 T helpers was found in FA. Thus, specific changes of T cell phenotype related to (co-)expression of immune checkpoint regulators may occur in systemic circulation of women with breast FA.

Dynamic Peripheral T-Cell Analysis Identifies On-Treatment Prognostic Biomarkers of Atezolizumab plus Bevacizumab in Hepatocellular Carcinoma

Introduction: Variability in response to atezolizumab plus bevacizumab (AB) treatment of hepatocellular carcinoma (HCC) underscores the critical need for the development of effective biomarkers. We sought to identify peripheral blood biomarkers reflecting response to AB treatment. Methods: We analyzed dynamic changes in peripheral blood mononuclear cells from a prospective, multicenter cohort of 65 patients with HCC, using flow cytometry to evaluate the T-cell population before and 3 weeks after the first AB treatment. Results: We found a unique response of the CD8+ T cells in terms of both frequency and phenotype, in contrast to CD4+ T cells and regulatory T cells. Notably, CD8+ T cells showed significant changes in expression of Ki-67 and T-cell immunoreceptors with Ig and ITIM domains (TIGIT). These distinct responses were observed particularly in the programmed cell death receptor-1 (PD-1)+ subpopulation of CD8+ T cells. Interestingly, the baseline differentiation status of PD-1+CD8+ T cells, particularly the central memory T-cell subset, correlated positively with greater proliferation (higher Ki-67 expression) of PD-1+CD8+ T cells after treatment. Moreover, effector memory cells expressing CD45RA correlated negatively with the increase in TIGIT+/PD-1+CD8+ T cells. The increase in TIGIT+/CD8+ T cells was associated with the development of immune-related adverse events, whereas increase in Ki-67+/PD-1+CD8+ T cells was associated with the better objective response rate. Importantly, dynamic shifts of Ki-67+/PD-1+CD8+ T cells and TIGIT+/CD8+ T cells significantly predicted progression-free survival and overall survival, as confirmed by multivariate analysis. Conclusion: These findings highlight the potential of dynamic changes in CD8+ T cells as an on-treatment prognostic biomarker. Our study underscores the value of peripheral blood profiling as a noninvasive and practical method for predicting the clinical outcomes of AB treatment in patients with HCC.

KLF2 determines the susceptibility of T cells to immunoregulatory NK cells.

Natural killer (NK) cells suppress cellular and humoral immune responses via killing of T cells, resulting in diminished vaccine responses in mice and humans. Efforts to overcome this roadblock and achieve optimal immunity require an improved understanding of the molecular mediators facilitating NK cell-targeting of discrete subsets of CD4 T cells. We employed single-cell forensic victimology and CRISPR-Cas9 editing to delineate a transcriptional program uniquely responsible for the susceptibility of a subpopulation of CD4 T cells to perforin-dependent immunoregulation by NK cells. The unique vulnerability of these CD4 T cells relative to other subsets of CD4 T cells was not associated with a pattern of NK-cell-receptor ligand expression that would favor activation of NK cells. Instead, susceptible CD4 T cells were skewed toward follicular helper T cell (Tfh) differentiation and exhibited intermediate expression of Klf2 and a related suite of KLF2-target genes (e.g. S1pr1) involved in cell migration and spatial positioning. NK-cell dependent suppression of the subset of Tfh exhibiting intermediate expression of KLF2 and S1PR1 was confirmed with single-cell proteomics. CRISPR targeting of KLF2 in CD4 T cells prevented suppression by NK cells. Thus, KLF2 regulation of spatial positioning of T cells is a key determinant of NK-cell immunoregulatory function and a possible target for strategies to enhance vaccine efficacy.

The constant domain of CRTAM is essential for high-affinity interaction with Nectin-like 2

CRTAM (Class-I MHC restricted T cell-associated molecule) is a member of the Nectin-like family, composed of two extracellular domains, one constant domain (IgC) and another variable domain (IgV), expressed in activated CD8 T cells, epithelial cells, natural killer (NK) cells, and in a subpopulation of CD4 T cells. CRTAM recognizes the ligand Nectin-like 2 (Necl2) through the IgV domain. However, the role of the IgC domain during this ligand recognition has yet to be understood. In this study, we show the purification of soluble-folded Ig domains of CRTAM, and we demonstrate that the IgC domain forms a homodimer in solution via hydrophobic interactions. By surface plasmon resonance (SPR) analysis, we also demonstrate that CRTAM binds to Necl2 with an affinity of 2.16 nM. In conclusion, CRTAM's IgC is essential for a high-affinity interaction with Necl-2.

Characterization of novel CD8+ regulatory T cells and their modulatory effects in murine model of inflammatory bowel disease

Dysregulation of mucosal immune system has been proposed to be critical in the pathogenesis of inflammatory bowel diseases (IBDs). Regulatory T cells (Tregs) play an important role in regulating immune responses. Tregs are involved in maintaining intestinal homeostasis and exerting suppressive function in colitis. Our previous studies showed that a novel forkhead box protein P3 (Foxp3) negative Tregs (Treg-of-B cells), induced by culturing naïve CD4+ T cells with B cells, could protect against colitis and downregulate T helper (Th) 1 and Th17 cell cytokines in T cell-mediated colitis. In the present study, we aimed to induce Treg-of-B cells in the CD8+ T-cell population and investigate their characteristics and immunomodulatory functions. Our results showed that CD8+ Treg-of-B cells expressed Treg-associated markers, including lymphocyte-activation gene-3 (LAG3), inducible co-stimulator (ICOS), programmed death-1 (PD-1), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), tumor necrosis factor receptor superfamily member-4 (TNFRSF4, OX40), and tumor necrosis factor receptor superfamily member-18 (TNFRSF18, GITR), but did not express Foxp3. CD8+ Treg-of-B cells produced higher concentration of inhibitory cytokine interleukin (IL)-10, and expressed higher levels of cytotoxic factor granzyme B and perforin after stimulation, compared to those of CD8+CD25- T cells. Moreover, CD8+ Treg-of-B cells suppressed T cell proliferation in vitro and alleviated colonic inflammation in chronic dextran sulfate sodium (DSS)-induced colitis. In conclusion, our study identified a novel subpopulation of CD8+ Tregs with suppressive effects through cell contact. These CD8+ Treg-of-B cells might have therapeutic potential for IBDs.

Divergent immune profiles in distinct populations - A vietnamese-german comparison

The human immune system exhibits fascinating diversity, sculpted by an intricate interplay of genetic and environmental influences. This study delves into these complexities by comparing the immunological landscapes of healthy individuals from distinct backgrounds: 40 Vietnamese and 24 German participants. Our comprehensive analysis, encompassing 42 lymphocyte populations and 17 cytokines, reveals profound differences in immune profiles between these two populations. Utilizing multicolor flow cytometry and advanced analytical platforms, we conducted a comprehensive characterization of the cellular and molecular components of individual immune systems. Statistical analyses revealed highly significant differences (p < 0.05) between Vietnamese and German cohorts in 33 out of 42 lymphocyte populations and 15 out of 17 cytokines. These disparities encompassed a wide range of immune cell subsets, including T, B and NK cells and involved both activating and inhibitory immune regulators. Healthy Vietnamese subjects exhibited significantly higher numbers of B, T and NK cells compared to their German counterparts. Vietnamese participants displayed higher proportions of plasma cells, immature B cells, and B cells with low CD21 expression (CD21low), which have a phenotypic characteristic of chronic stimulation. Of the 16 subpopulations of CD4+ and CD8+ T cell subpopulations, 11 were significantly elevated in Vietnamese compared to German subjects. Additionally, Vietnamese participants expressed higher proportions of markers for functional and activating NK receptors, indicating the presence of highly cytotoxic NK cells in this population. Almost all of the 17 cytokines examined were significantly lower in Vietnamese subjects. These results demonstrate statistically significant variations in immunological profiles between healthy individuals from Eastern and Western populations. Our findings suggest that caution should be exercised when applying Western reference health profiles to Eastern subjects in clinical settings. This comprehensive analysis underscores the importance of considering population-specific immune profiles in clinical and research contexts, particularly when evaluating immunological parameters across diverse ethnic groups.

The potential role of CD8+ cytotoxic T lymphocytes and one branch connected with tissue-resident memory in non-luminal breast cancer.

Advances in understanding the pathological mechanisms of breast cancer have resulted in the emergence of novel therapeutic strategies. However, triple-negative breast cancer (TNBC), a molecular subtype of breast cancer with a poor prognosis, lacks classical and general therapeutic targets, hindering the clinical application of several therapies to breast cancer. As insights into the unique immunity and molecular mechanisms of TNBC have become more extensive, immunotherapy has gradually become a valuable complementary approach to classical radiotherapy and chemotherapy. CD8+ cells are significant actors in the tumor immunity cycle; thus, research on TNBC immunotherapy is increasingly focused in this direction. Recently, CD8+ tissue-resident memory (TRM) cells, a subpopulation of CD8+ cells, have been explored in relation to breast cancer and found to seemingly play an undeniably important role in tumor surveillance and lymphocytic infiltration. In this review, we summarize the recent advances in the mechanisms and relative targets of CD8+ T cells, and discuss the features and potential applications of CD8+ TRM cells in non-luminal breast cancer immunotherapy.

Single-cell analysis and machine learning identify psoriasis-associated CD8+ T cells serve as biomarker for psoriasis.

Psoriasis is a chronic inflammatory skin disease, the etiology of which has not been fully elucidated, in which CD8+ T cells play an important role in the pathogenesis of psoriasis. However, there is a lack of in-depth studies on the molecular characterization of different CD8+ T cell subtypes and their role in the pathogenesis of psoriasis. This study aims to further expound the pathogenesy of psoriasis at the single-cell level and to explore new ideas for clinical diagnosis and new therapeutic targets. Our study identified a unique subpopulation of CD8+ T cells highly infiltrated in psoriasis lesions. Subsequently, we analyzed the hub genes of the psoriasis-specific CD8+ T cell subpopulation using hdWGCNA and constructed a machine-learning prediction model, which demonstrated good efficacy. The model interpretation showed the influence of each independent variable in the model decision. Finally, we deployed the machine learning model to an online website to facilitate its clinical transformation.

#1167 Examination of subsets of CD4+ lymphocyte subpopulations in long-surviving renal transplant patients

Abstract Background and Aims Subpopulations of CD4+ cells, play a major role in mediation of rejection and tolerance to a renal transplant. This study examined subsets of CD4+ T cells and CD4+CD25+CD127lo Treg in blood of renal graft recipients with grafts surviving &amp;gt;10 years for changes in lymphocyte subpopulations that may indicate transplant tolerance and potentially identify patients who could reduce immunosuppression. Based on CD45RA and CD25/Foxp3 expression, Miyara et al identified five populations (Pop) within CD4+ cells. Pop I, II and III are T regulatory cells (Treg); Pop I as naïve (CD45RA+Foxp3+) Treg, Pop II as activated (CD45RA-Foxp3hi) Treg, and Pop III as cytokine secreting (CD45RA-Foxp3+) cells which also includes activated effector cells. Pop IV are activated T cells (CD45RA-Foxp3−), and Pop V are naïve T cells (CD45RA+Foxp3−). Here, we examined three Treg populations (Pop I-III) within CD4+CD25+CD127lo Treg, and Pop IV and V within CD4+ T cells. To further characterise these subpopulations, we examined chemokine receptor expression (CXCR3, CCR4, CCR6, and CCR7) to identify T helper phenotype (Th-like) of Treg populations. Th1-like Treg were identified as CCR4+CXCR3+CCR6−, Th2-like as CCR4+CXCR3-CCR6−, Th17-like as CCR4+CXCR3-CCR6+, and Th1/Th17-like as CCR4+CXCR3+CCR6+. We also examined expression of activated Treg effector molecules CD39, Class II MHC, and PD-1. Method Peripheral blood mononuclear cells (PBMC) were isolated from fresh blood collected from healthy volunteers (HV) (n = 44) and long-term stable renal transplant patients with grafts surviving &amp;gt;10 yrs (RT) (n = 25). Panels of monoclonal antibodies for Treg (CD4/CD25/CD127/CD45RA/Foxp3), chemokine receptors (CXCR3, CCR4, CCR6, CCR7), and Treg activation and suppression-related molecules (CD39/HLA-DR/PD-1) were also used. Data was acquired on BD FACSCanto II using BD FACSDiva software (v8.0) and analysed using FlowJo. Lymphocyte populations were examined after FSC vs SSC gating and doublets exclusion. T cell and Treg populations were analysed within CD4+ and CD4+CD25+CD127lo T cells. Results RT had lower lymphocyte counts than HV (p = 0.0156) but had a similar percentage of CD4+ T cells. Treg were significantly lower in RT compared to HV (p = 0.0012). RT patients had less naïve Treg (Pop I, p = 0.0083) and naïve effector T cells (Pop V, p = 0.0003), but had higher activated effector T cells (Pop IV, p = 0.0002) than HV. There were no differences in activated Treg Pop II or III between the two groups. There were significantly less cells expressing CXCR3 in RT than HV in Pop I (p = 0.0120), Pop II (p &amp;lt; 0.0001), Pop III (p &amp;lt; 0.0001) and Pop IV (p = 0.0047). Similarly, RT had fewer cells expressing CCR6 compared to HV in Pop II (p = 0.0019), Pop III (p = 0.0015), and Pop V (p = 0.0017). RT had lower proportions of CCR7 expressing cells in Pop I (p = 0.0035), Pop IV (p = 0.0097), and Pop V (p = 0.0002), but higher in Pop II (p = 0.0041) compared to HV. There were similar percentages of cells with Th1 and Th17-like phenotypes in both groups. However, RT had significantly more Th2-like Treg in Pop II (p = 0.0033) and Pop III (p = 0.0100), and significantly less Th1/Th17-like Treg in Pop II (p = 0.0005) and Pop III (p = 0.0017) compared to HV. There were little to no differences in the Treg activation and suppression-related molecules within subpopulations of Treg. Conclusion Long-surviving RT patients had less naïve Treg and naïve effector cells, but increased proportions of activated effector cells. There were little to no differences in the expression of Treg activation or suppression-related molecules in Treg subpopulations. Unexpectedly, activated Treg in RT had significantly higher proportions with a Th2 phenotype and significantly less Treg with a Th1/Th17 phenotype. Whether this is a consequence of immunosuppression, or a biological shift associated with tolerance, remains to be resolved. Further studies are required to investigate the relevance of these changes in identifying induction of transplant tolerance.

#2899 Role of the Th17.1 cells in IgA nephropathy

Abstract Background and Aims T helper (Th) cells play a crucial role in glomerulonephritis, including IgA nephropathy (IgAN). Recently identified Th17.1 cells, which are elevated in sarcoidosis, lupus, and Takayasu arteritis, express chemokine receptors CCR6 and CXCR3. These receptors are implicated in inflammatory cell infiltration and fibrosis in glomerulonephritis models. Notably, Th17.1 cells express MDR-1 (multidrug resistance protein) and the drug efflux protein p-glycoprotein, associated with corticosteroid resistance. However, the exact role of Th17.1 cells in IgAN remains unclear. Intriguingly, Th17.1 cells produce INF-γ and IL-17, cytokines implicated in IgAN pathogenesis. Method In this retrospective cross-sectional study, we examined circulating Th17.1 cells in 18 confirmed IgAN patients. Antibodies such as anti-CXCR3-FITC and anti-CCR6-PCy7 (Biolegend, U.S.A.) were used to stain Th subpopulations. We also used combinations of the following antibodies: anti-CD45-FITC, anti-CD4-PE, anti-CD8-ECD, anti-CD3-PCy5, anti-CD56-PCy5, anti-CD3-PCy7, anti-CD19-ECD, anti-CD127-FITC, anti-CD25-PCy5, and anti-CD4-PCy7 (Coulter Immunotech, France) to evaluate T, B, NK, and Treg cells. Multicolor flow cytometry was performed using a Navios cytometer, and data were analyzed using Kaluza software (Beckman Coulter, U.S.A.). Th effector subpopulations of CD4+ lymphocytes were identified based on chemokine receptor expression: Th17.1 (CXCR3+CCR6+). Th17.1 cells were quantified as a percentage of CD4+ lymphocytes. Th1, Th2, and Treg cells were also evaluated. We explored correlations between Th17.1 cell counts, estimated glomerular filtration rate (eGFR, mL/min/1.73 m²), and proteinuria (24-hour collection). Among the patients, 5 were classified as refractory (progressive eGFR decline &amp;gt; 5 mL/min/1.73 m² and 24-hour proteinuria &amp;gt; 1g despite steroid and immunosuppressant therapy). Results The study included 18 patients. The mean (95% CI) Th17.1 cell count was 20% (16-23), and the 24-hour proteinuria was 2.1g (1.4-2.7). A significant direct correlation was observed between Th17.1 cells and proteinuria (g/L, adjusted for 24 hours) (r = 0.50, P = 0.03). Refractory patients exhibited higher Th17.1 levels compared to others (25% [95% CI: 17-33] vs. 18% [95% CI: 14-21], P = 0.02) (Fig. 1). Patients with proteinuria &amp;lt; 0.5g/24 h had lower Th17.1 cell counts compared to those with higher proteinuria (16% [95% CI: 12-20] vs. 21% [95% CI: 17-25], P = 0.06. No correlation was found between eGFR, proteinuria, and Th1, Th2, Th17, or Treg cells. Conclusion These findings suggest that Th17.1 cells may contribute to the pathogenesis of IgAN and could potentially indicate the response to steroids or immunosuppressants in treatment. While the direct involvement of Th17.1 cells in kidney tissue remains unexplored, further investigation is warranted due to its therapeutic and prognostic implications.

Immunophenotypic profiling reveals novel T cell subset associated with “tolerance” in pediatric transplant recipients

Abstract Achieving allograft health remains a challenge in pediatric organ transplantation. Recently. we performed multilineage, single-cell analysis of the immune cell composition in pediatric solid-organ transplant recipients who received a heart, liver, or kidney allograft (n=52); 24 children had stable graft function while 28 children had a rejection episode. Hierarchical clustering of the mean cell type proportion across allograft types and health states demonstrated a distinct subpopulation of CD4 T cells (CD45RA-,CD25+,CD5+,CD38-) was significantly (p&amp;lt;0.05) associated with stable graft function and were termed TOT cells. The cosine similarity analysis of proteomics data showed that TOT cells are phenotypically most like Tregs and least like CD4+ TEMRA. To characterize the TOT cells, we developed an 8-marker flow cytometry panel to specifically sort Treg and TOT populations and performed 10x single-cell RNA-seq and TCR-seq with subsequent analysis in R and Loupe Browser. Differentially expressed genes that were specifically associated with TOT cells included ANXA1, IL7R, TCF7, THEMIS, NELL2, KLRB1 and CD40LG while Treg expressed FOXP3, IKZF2, CTLA4, IL2RA, and RTKN2, consistent with their known function in immune suppression. TCR analysis indicates unique expansions of T cell clonotypes in TOT cells not seen in Treg. Further investigation into the immunoregulatory capacity of TOT cells could be instrumental in developing new strategies to prolong graft survival.

Relationship of thymic area with clinical-epidemiological variables and values of T-lymphocyte subpopulations in peripheral blood of children with recurrent infections

BackgroundRecurrent infections in childhood are the main cause of remission to the immunology service. T lymphocytes generated in the thymus are essential for fighting infection, making the thymus area an important predictor of the immune system’s competence. This study aimed to identify the possible relationship of the thymic area with clinical-epidemiological variables and values of subpopulations of T lymphocytes in the peripheral blood of children with recurrent infections.MethodsWe conducted applied research using a transversal analytical design at the National Medical Genetics Center (Havana, Cuba), from January to August 2022. The study covered 73 children of which we analyzed clinical-epidemiological variables and the size of the thymus through ultrasound. Furthermore, we determined the relative and absolute values of the subpopulations of T cells using flow cytometry.ResultsOf the children studied, 65.8% had thymic hypoplasia. The children who breastfed for less than 6 months showed four times the risk of developing moderate-severe thymus hypoplasia (OR = 3.90, 95% CI: 1.21–12.61). A direct relationship was found between the area of the thymus and the child’s size (r = 0.238, p = 0.043) and weight (r = 0.233, p = 0.047). The relative values of CD3+ T lymphocytes decreased in the cases of mild hypoplasia (p = 0.018) and moderate-severe hypoplasia (p = 0.049). The thymus area was associated with the absolute cell count of CD8+ effector memory T cells (rs = −0.263, p = 0.024) and of the central memory T cells (r = −0.283, p = 0.015).ConclusionsBreastfeeding for less than 6 months, as well as the weight and size of the child, are related to their thymus area. The subpopulation values of T lymphocytes detected suggest that patients with thymic hypoplasia develop a contraction of CD3+ T cells, which can make them more vulnerable to infectious processes. This finding was combined with an expansion of the memory compartments of the subpopulations of CD8+ T cells, suggesting a greater susceptibility to intracellular viral and bacterial infections in these cases.

Nail Telocytes: Identification, Potential Physiological Function, and Role in Pathology: A Reappraisal of the So-Called Onychofibroblasts/Onychodermis.

Some authors have suggested that the fibroblasts of the nail mesenchyme (onychofibroblasts) can be distinguished from skin fibroblasts by their high expression of CD10. My 2015 study documented the presence of a relatively sparse CD34 + /CD10 + dendritic subpopulation in the dermis and hypodermis of the matrix. For some time now, my hypothesis has been that these interstitial dendritic mesenchymal cells of the matrix correspond to telocytes. Telocytes have been described as peculiar interstitial dendritic cells present in the mesenchymal tissue of numerous organs, including the skin, but their presence and characteristics in the nail unit have not been explored. This study was undertaken to more comprehensively investigate the existence and characteristics of nail telocytes. A series of 20 normal adult nail units were examined with a combination of morphological and immunohistochemical analyses. The matrix dermis contained a sparse subpopulation of CD34 + /CD10 + elongated telocytes with a higher density in the lunular region and, at this distal level, a change in their immunohistochemical profile, resulting in a progressive loss of CD34 expression. The matrix hypodermis showed CD34 + /CD10 + telocytes in their classical elongated aspect, which acquired, especially in the distal fibromyxoid area of the thumb, an oval to round morphology with multiple intracytoplasmic vacuoles. The characteristic dynamic immunophenotypic profile of the dermal telocytes with a progressive distal loss of the defining molecule CD34 was equally observed in the distal hypodermis. The nail bed dermis was thick with a dense fibrous connective tissue. A reticular network of CD34 - /CD10 + telocytes was present in the superficial dermis of the proximal nail bed. The mesenchymal cells of the deep part of the proximal nail bed dermis and the entire distal nail bed dermis were CD34 - /CD10 - . The adult nail mesenchyme is composed of 3 microanatomically distinct regions. Only the thumb has a distal hypodermis rich in mucinous material. The population of telocytes is relatively sparse compared with the fibroblastic population of the entire nail mesenchyme. The concept of onychodermis/onychofibroblasts is not valid. Nail telocytes have a dynamic immunohistochemical profile depending on whether they are located proximally or distally. The CD34 + /CD10 + profile correlates with the onychogenic epithelial region, while the CD34 - /CD10 + profile correlates with a spatial rearrangement of the nail epidermal bed.

Імунологічні особливості у вагітних із великими акушерськими синдромами

Aim of the research – to evaluate the adaptive capabilities of the immunological system in pregnant women who had complications from the group of major obstetric syndromes (МОS) for the further development of an algorithm for the prevention of this complication. Materials and methods. The main study group (MG) - 99 pregnant women who had a complication from the group of MOS, which was clinically manifested by placental insufficiency (PI); control group (CG) - 50 practically healthy pregnant women with a healthy reproductive history and an uncomplicated course of this pregnancy. The number (x109/l) and relative (%) of lymphocyte subpopulations CD3+ (T-lymphocytes), CD4+ (helper-inducers), CD8+ (cytotoxic suppressors), CD56+ (natural killers), CD19+ (B-lymphocytes) were determined; the presence of antiphospholipid antibodies (APA); total hemolytic activity of the classical way of complement activation (CH50); levels of IgG, IgM, IgA. Statistical processing of research results was carried out using standard Microsoft Excel 5.0 and Statistical 6.0 programs. Results. In the I and II trimesters of pregnancy in MG and CG, a tendency to decrease was observed, СM4+ (51.8±2.5% and 48.9±1.6; 58.4±2.3% and 55.6±2.6%), and СD8+ (24.6±2.4% and 21.2±1.2%; 31.3±2.5% and 30.9±2.7%), which led to an increase in the immunoregulatory index (СD4+/СD8+) in MG (р&lt;0.05). The level of СD19+, during gestation there was an increase in their relative number in MG (13.6±0.98%; 16.4±2.03% and 21.1±2.01%) (p&lt;0.05); a predominance of the percentage of CD56+ lymphocytes, which have the properties of natural killers, was observed in pregnant MG (12.3±1.7%; 15.1±1.7%; 13.9±1.73% vs. 8.6±1.4%; 8.1±1.18%; 7.2±0.98%; p&lt;0.05). Hemolytic activity of the classical pathway of complement activation (CH50) in MG pregnant women was 1.5 times higher compared to CG pregnant women (p&lt;0.05). APA, represented by IgG to cardiolipin, were detected in 47 (47.5%) pregnant women MG and 4 (8.0%) women pregnant CG (p&lt;0.05). Conclusions. Іn pregnant women with МОS, there is an immune maladaptation that progressively increases in the dynamics of gestation and is manifested by excessive activation of natural killers (CD56+), an increase in the immunoregulatory coefficient (CD4+/CD8+), an increase in the percentage of B-lymphocytes (CD19+), dysimmunoglobulinemia, which is accompanied by high indicators of the general hemolytic activity of the classical pathway of complement activation (CH50), the presence of APA. The research was conducted according to principles of Declaration of Helsinki. Protocol of research was proved by local ethical committee, mentioned in institution’s work. A informed sonsennt was collected in order to carry out the research. The author is stating no conflict of interests is declared.

Abstract 6553: Targeting macrophages and regulatory T cells improves response to chemotherapy in high-grade serous ovarian cancer

Abstract Despite the initial response to chemotherapy, most high-grade serous ovarian cancer (HGSOC) patients will suffer disease relapse within two years of diagnosis with no currently successful immunotherapy options.We performed single-cell RNA sequencing (scRNAseq) on omental metastasis from HGSOC patients. Analysis of 64,097 immune cells showed that neoadjuvant chemotherapy (NACT) induced immunosuppressive mechanisms that counteracted its potentially positive effects. We showed that while NACT enhanced phagocytosis and antigen presentation, this was counterbalanced by upregulation of Stabilin1 (STAB1) in eight out of eleven macrophage subpopulations we identified. The NACT associated increase in macrophage STAB1 expression was validated in a separate cohort of eighty HGSOC patients at the protein level. STAB1 is a macrophage scavenger receptor that transports phagocytosed targets to undergo significant lysosomal degradation reducing the efficiency of antigen presentation. In vitro anti-stabilin1 antibody (anti-stab1 ab) -treated macrophages had significantly reduced antigen degradation and higher CD11C expression than isotype-treated ones. Furthermore, T cells co-cultured with anti-stab1 ab -treated macrophages had increased T cell proliferation and killing.We identified thirteen CD4 subpopulations by scRNAseq, five of which were Tregs. We showed that NACT significantly upregulated and activated Tregs. Tregs displayed a shared developmental trajectory with naïve and effector T cells suggesting that those are induced rather than natural Tregs. This was supported by TCR clonal sharing between Tregs and other effector cells. In vitro T cells treated with FOXP3 anti-sense oligonucleotide (ASO), AZD8701, had an anti-tumor cytokine profile and enhanced tumor cell killing. Analysis of 69,781 scRNAseq cells from control and chemotherapy-treated HGSOC syngeneic mouse model showed similar responses. In two HGSOC syngeneic mouse models (HGS2 and 30200), combinations of chemotherapy with anti-stab1 ab and/or Foxp3-ASO significantly increased median survival of mice with established peritoneal disease compared to chemotherapy alone. Bulk RNAseq of tumors treated with anti-stab1 ab were enriched with CXCL9 positive macrophages, while Foxp3-ASO treatment showed significant increase in T helper cell infiltration and activation. Potentially cured long-term survivors (300 days+) were resistant to tumor rechallenge.In a third HGSOC mouse model (60577), in which tumors relapsed after a good initial response to chemotherapy, combinations of chemotherapy with anti-stab1 ab and/or Foxp3-ASO significantly increased the post relapse survival compared to chemotherapy alone.We believe that modulating Tregs and STAB1 could improve response to chemotherapy and can have translational potential as each AZD8701 and a humanized anti-stab1 ab are in phase I clinical trials. Citation Format: Samar Elorbany, Chiara Berlato, Larissa Carnevalli, Simon Barry, Eleni Maniati, Jun Wang, Ranjit Manchanda, Julia Kzhyshkowska, Frances Balkwill. Targeting macrophages and regulatory T cells improves response to chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6553.