Phenotype Of Subsets Research Articles

DOP125 CD11b−CD27− NK subsets account for NK-cell dysfunction in patients with inflammatory bowel disease

Abstract Background Natural killer (NK) cells play a crucial role in the dynamic interaction between innate and adaptive immune responses but their function is dysregulated in patients with inflammatory bowel disease (IBD)[1-4]. The mechanism that contributes to NK-cell dysregulation in IBD requires further elucidation. Methods Using flow cytometry staining, we precisely characterized the frequency, phenotype and function of NK subsets distinguished by CD27 and CD11b in 61 treatment-naive patients with active IBD (including 34 with Crohn’ s disease (CD) and 27 with ulcerative colitis (UC)), as compared to 30 healthy volunteers. Results We observed a significantly increased proportion of the peripheral CD11b-CD27- (double-negative, or DN) NK subsets in IBD patients. Notably, these relatively expanded DN NK subsets exhibited an inactive and immature phenotype. Functional analysis, conducted by stimulating and assessing the expression of CD107a and interferon-gamma (IFN-γ), revealed that the DN NK subsets had poor cytotoxic capacity and a deficient potential to produce IFN-γ. In addition, the percentages of DN NK subsets expressing CD107a and IFN-γ were positively correlated with NK degranulation and cytokine production, respectively. Interestingly, we also found that the frequency of DN NK subsets seemed to correlate with the plasma levels of IL-17A. High levels of IL-17A constrain the proliferation and maturation of NK cells in vitro. In some IBD patients who achieved clinical remission after receiving biological agent therapy, the percentage of DN NK subsets decreased accordingly. Conclusion The increased proportion of DN NK subsets may account for NK-cell dysfunction in IBD patients, potentially associated with elevated plasma levels of IL-17A. These alterations in NK cell subsets may contribute to defective killing and thus the secondary infections and increased risk of cancer observed in IBD patients.

EXamining the feasibility of exerCisE to manage symptoms of Lupus (EXCEL): a protocol for a randomised controlled pilot study

IntroductionSLE is a chronic autoimmune disease that results in sustained hyperactivation of innate and adaptive immune cells and widespread inflammatory damage. Regular exercise reduces SLE symptoms including fatigue and joint...

Liver damage and immune responses.

Chronic liver disease (CLD) has massive systemic repercussions including major impacts on the body's immune system. Abnormalities in phenotype, function and numbers of various immune cell subsets have been established by a large number of clinical and pre-clinical studies. The loss of essential immune functions renders CLD-patients exceptionally susceptible to bacterial and viral infections and also impairs the efficacy of vaccination. Consequently, infections represent a major clinical issue causing significant morbidity and mortality in these patients. Mechanistically, the immune dysfunction associated with CLD results from the increased translocation of bacteria and bacterial cues from the intestine. These trigger a signaling axis around the cytokines IFNI and IL-10 in hepatic myeloid cells, which aside from impairing the function of the myeloid cells themselves, also has notable negative impacts on the functionality of other immune cells. T cells in CLD-patients and -models are especially affected by this signaling axis and display a variety of quantitative and qualitative defects. Due to the high clinical relevance, understanding the mechanisms underlaying CED-associated immune dysfunction is of critical importance to discover and develop new therapeutic targets.

Comprehensive evaluation of immune dysregulation in secondary hemophagocytic lymphohistiocytosis

ABSTRACT Host immune dysfunction plays a crucial role in the onset, progression, and outcome of hemophagocytic lymphohistiocytosis (HLH). This study aimed to comprehensively evaluate the peripheral immune profiles in patients with newly diagnosed secondary hemophagocytic lymphohistiocytosis (sHLH), and explore their predictive value for patient prognosis. A total of 77 patients with sHLH were enrolled in this study, with 31 of them experiencing mortality. Flow cytometry was used to assess the percentages, absolute numbers, and phenotypes of lymphocyte subsets. Simultaneously, cytokine levels and routine laboratory indicators were also collected. In sHLH patients, lymphocyte subset absolute numbers were significantly impaired, accompanied by T cell hyperactivation, B cell hyperactivation, and increased plasmablast proliferation. Prognostic analysis revealed that lower CD8+ T cell percentages, elevated APTT, IL-6, IL-10 levels, and increased CD4+CD28null T cell proportions were associated with poor patient outcomes. The study demonstrates dysregulation in the counts and phenotypes of lymphocyte subsets in sHLH patients. Several key factors, including IL-6, IL-10, APTT, and various T cell percentages, have potential as prognostic markers and therapeutic targets in sHLH.

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.

Application of γδ T cells with different memory phenotypes in clinical diagnosis of active pulmonary tuberculosis

ObjectiveTo investigate the application and clinical significance of different memory phenotypes of γδ T-cell subsets in the clinical diagnosis of pulmonary tuberculosis. MethodsIn total, 42 patients with tuberculosis (TB) according to the diagnostic criteria for tuberculosis (WS288-2017) who were treated at the Infectious Diseases Hospital affiliated with Soochow University from February 2023 to July 2023 were enrolled. Additionally, 16 patients with latent TB infection (LTBI) and 20 healthy controls (HCs) were included. Flow cytometry was used to measure the expression levels of γδ T cells, Vδ1 T-cell subsets/Vδ2 T-cell subsets, naive (CD45RA + CD27+) cells, central memory (CD45RA-CD27+) cells, effector memory (CD45RA-CD27−) cells, and terminally differentiated (CD45RA + CD27−) cells in the peripheral blood. The expression levels at different stages of TB infection were compared. ResultsThere were no significant differences in peripheral blood γδ T cells or Vδ1 T cells among the HC, LTBI and TB groups. The proportion of CD45RA-CD27+Vδ2 T cells in TB patients was significantly lower than that in HCs and LTBI patients, but the proportion of CD45RA + CD27-Vδ2 T cells was greater. ROC curve analysis revealed that CD45RA-CD27+Vδ2 T cells (AUC), CD45RA + CD27-Vδ2 T cells (AUC), and the combination of both (AUC) were effective in differentiating TB patients from LTBI patients. ConclusionThe proportions of CD45RA-CD27+Vδ2 T cells and CD45RA + CD27-Vδ2 T cells are potential biomarkers for the diagnosis of active TB infection and are helpful for distinguishing active TB infection from LTBI.

The Functional and Prognostic Impact of TIGIT Expression on Bone Marrow NK Cells in Core Binding Factor-Acute Myeloid Leukemia Patients at Diagnosis.

Background: The effect of the expression of the newly identified immune checkpoint, T cell immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) on NK cells in core binding factor-acute myeloid leukemia (CBF-AML) remains to be investigated. Methods: Fresh bone marrow samples from a total of 39 newly diagnosed CBF-AML patients and 25 healthy donors (HDs) were collected for testing the phenotype and function state of total NK, CD56bright, and CD56dim NK cell subsets after in vitro stimulation. Results: The frequencies of TIGIT+ cells in total NK, CD56bright, and CD56dim NK cell subsets had no significant difference between patients and HDs. TNF-α and INF-γ levels were uniformly lower in TIGIT+ cells than the corresponding TIGIT- cells in all HDs, whereas those for TIGIT+ to TIGIT- cells in patients were highly heterogenous; TIGIT expression was not related to PFP and GZMB expression in HDs, whereas it was related to higher intracellular PFP and GZMB levels in patients. Patients' TIGIT+ NK cells displayed lower K562 cell-killing activity than their TIGIT- NK cells. In addition, high frequencies of TIGIT+ cells in total NK and CD56dim NK cells were associated with poor RFS. Conclusions: TIGIT expression affected the diagnostic bone marrow-sited NK cell function and had prognostic significance in CBF-AML patients.

MicroRNA-142 regulates gut associated lymphoid tissues and group 3 innate lymphoid cells

The transcriptomic signatures that shape responses of innate lymphoid cells (ILCs) have been well characterised, however post-transcriptional mechanisms which regulate their development and activity remain poorly understood. We demonstrate that ILC groups of the intestinal lamina propria express mature forms of microRNA-142 (miR-142), an evolutionarily conserved microRNA family with several non-redundant regulatory roles within the immune system. Germline Mir142 deletion alters intestinal ILC compositions, resulting in the absence of T-bet+ populations and significant defects in the cellularity and phenotypes of ILC3 subsets including CCR6+ LTi-like ILC3s. These effects were associated with decreased pathology in an innate-immune cell driven model of colitis. Furthermore, Mir142−/− mice demonstrate defective development of gut-associated lymphoid tissues, including a complete absence of mature Peyer’s patches. Conditional deletion of Mir142 in ILC3s (RorcΔMir142) supported cell-intrinsic roles for these microRNAs in establishing or maintaining cellularity and functions of LTi-like ILC3s in intestinal associated tissues. RNAseq analysis revealed several target genes and biological pathways potentially regulated by miR-142 microRNAs in these cells. Finally, lack of Mir142 in ILC3 led to elevated IL-17A production. These data broaden our understanding of immune system roles of miR-142 microRNAs, identifying these molecules as critical post-transcriptional regulators of ILC3s and intestinal mucosal immunity.

In-Depth Analysis of the Peripheral Immune Profile of HER2+ Breast Cancer Patients on Neoadjuvant Treatment with Chemotherapy Plus Trastuzumab Plus Pertuzumab.

Currently, therapy for early-stage human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) is based on the combination of trastuzumab and pertuzumab plus chemotherapy in a neoadjuvant regimen. The INMUNOHER study aimed to detect immunological markers in peripheral blood and their association with treatment response. Sixty-two HER2+ BC patients were recruited. Pre-treatment samples were obtained before the start of treatment, while post-treatment samples were obtained after completing therapy and before surgery and were analyzed by flow cytometry. The pathologic complete response (pCR) rate achieved was 82.3%. The expression of the NKp30, PD-1, and TIM-3 receptors was reduced in the Natural Killer (NK)-CD56dim subset of patients who did not achieve pCR. Following therapy, many changes were found in leukocytes, including alterations in T cell lymphocyte proportions. Also, the percentage of NK cells decreased, and several phenotypic changes were observed in this population. After treatment, IFN-γ production by NK cells against HER2+-cells with or without trastuzumab was significantly reduced. HER2-targeted therapy plus chemotherapy demonstrated high efficacy in most patients, reducing the statistical power for finding immunological markers. However, NK subset phenotypes correlated better with response groups, and numerous changes in the percentage of leukocytes and T and NK cells, as well as changes in the functionality of NK cells, were observed in most patients after treatment, encouraging further research into these immune populations.

Gut Microbiota Defines Functional Direction of Colonic Regulatory T Cells with Unique TCR Repertoires.

Intestinal microbiota and selected strains of commensal bacteria influence regulatory T (Treg) cell functionality in the colon. Nevertheless, whether and how microbiota changes the transcriptome profile and TCR specificities of colonic Tregs remain to be precisely defined. In this study, we have employed single-cell RNA sequencing and comparatively analyzed colonic Tregs from specific pathogen-free and germ-free (GF) mice. We found that microbiota shifts the activation trajectory of colonic Tregs toward a distinct phenotypic subset enriched in specific pathogen-free but not in GF mice. Moreover, microbiota induced the expansion of specific Treg clonotypes with shared transcriptional specificities. The microbiota-induced subset of colonic Tregs, identified as PD-1- CXCR3+ Tregs, displayed enhanced suppressive capabilities compared with colonic Tregs derived from GF mice, enhanced production of IL-10, and were the primary regulators of enteric inflammation in dextran sodium sulfate-induced colitis. These findings identify a hitherto unknown gut microbiota and immune cell interaction module that could contribute to the development of a therapeutic modality for intestinal inflammatory diseases.

Anti-HLA serologic response to CD38-targeting desensitization therapy is challenged by peripheral memory B cells in highly sensitized kidney transplant candidates

High human leukocyte antigen (HLA) sensitization limits access to compatible transplantation. New CD38-targeting agents have been shown to reduce anti-HLA antibodies, although with important interpatient variability. Thus, pretreatment identification of responder and nonresponder (NR) patients is needed for treatment decision-making. We analyzed 26 highly sensitized (HS) patients from 2 desensitization trials using anti-CD38 monoclonal antibodies. Hierarchical clustering identified 3 serologic responder groups: high responders, low responders, and NR. Spectral flow cytometry and functional HLA-specific memory B cell (mBC) assessment were first conducted on peripheral blood mononuclear cells and bone marrow samples from 16 patients treated with isatuximab (NCT04294459). Isatuximab effectively depleted bone marrow plasma cells, peripheral CD38-expressing plasmablasts, plasma cells, transitional B cells, and class-switch mBCs, ultimately reducing frequencies of HLA-specific immunoglobulin G (IgG)-producing mBCs. Multidimensional spectral flow cytometry with partial least squares discriminant analysis revealed that pretreatment abundance of specific circulating mBC phenotypes, especially CD38neg class-switch mBCs, accurately distinguished between high serologic responders and low responders or NR (AUC 0.958, 0.860-1.000, P = .009), who also displayed significantly lower frequencies of HLA-specific IgG-producing mBCs (P < .0001). This phenotypical mBC signature predicting response to therapy was validated in an external HS patient cohort (n = 10) receiving daratumumab (NCT04204980). This study identifies critical circulating mBC subset phenotypes that distinguish HS patients with successful serologic responses to CD38-targeting desensitization therapies, potentially guiding treatment decision-making.

IL-10-modulated dendritic cells from birch pollen- and hazelnut-allergic patients facilitate Treg-mediated allergen-specific and cross-reactive tolerance.

Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) invitro and invivo. CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor-specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. After IL-10 DC priming and allergen-specific restimulation (Bet or Cor), non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder Th2 cell responses invitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). Invivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms, and immune responses. Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.

The expression of Siglec-10 on naive B cells is involved in the pathology of systemic lupus erythematosus.

Previous studies have reported the expansion of CD19+Siglec-10+ B cells in systemic lupus erythematosus (SLE) patients. However, the composition of this cell population, phenotype and characteristics are still unknown. We examined this memory B-cell subset's composition and phenotype and determined the SYK and AKT phosphorylation levels by flow cytometry. Additionally, we explored the relationship between Siglec-10 expression on B-cell subsets and clinical manifestations. Our results indicated elevated levels of Siglec-10 on naive B cells in active SLE patients. Compared with healthy controls (HCs) and inactive SLE patients, the Siglec-10+ B cells in active SLE patients exhibited elevated CD40 and CD21low levels. The levels of Siglec-10 on naive B cells were positively correlated with the proportion of CD21low double negative (DN) B cells and the SLEDAI-2K score. The results indicate that the upregulation of Siglec-10+/naive B cells may function as a feedback mechanism to regulate B cell hyperreactivity. Monitoring the proportion of Siglec-10+/naive B cells may contribute to the evaluation of disease progression in SLE.

#1339 The characteristic of memory B-cell subsets in patients with IgA-nephropathy

Abstract Background and Aims Impaired B-cell development has been postulated to play an important role in the maintaining of autoimmune inflammation what makes memory B-cells a perspective target for a new therapeutic strategy in IgA-nephropathy (IgAN). However, to date there are few and controversial data about the precise role of circulating memory B-cell subsets in clinical progression of IgAN. The aim was to estimate the immunological profile of memory B-cell subpopulations in the peripheral blood of IgAN patients. Method The peripheral blood was obtained from 20 IgAN patients (aged of 32.0 (27.0-36.0) y.o., male/female ratio as 13/7) and 15 donors (aged of 38.0 (30.0-46.0) y.o., male/female ratio as 10/5). The diagnosis was confirmed by morphological examination of the biopsy material. The phenotype of B-cells subsets was determined by a panel of CD38-PE/CD19-ECD/CD27-PC5/IgD-APC antibodies; γδT-lymphocytes—by a panel of TCRγδ-FITC/TCRαβ-PE/HLA-DR-ECD/TCRVδ1-PC7/CD3-APC-A750/TCRVδ2-PB/ CD45-KRO antibodies using flow cytometry method (Cytoflex, Beckman Coulter, USA). The concentration of B-cell activating factor (BAFF) was measured using enzyme-linked immunosorbent assay. Statistical data processing was done in Statistica 10.0 program. Results Based on cell surface markers expression the following B-cells subsets were identified in the peripheral blood: naïve B-cells (CD19+CD27−IgD+), pre-switched B-cells (CD19+CD27+IgD+), switched B-cells (CD19+CD27+IgD−), plasmablasts (CD19+CD27+CD38+) and long-lived plasma cells (CD27hiCD38hi) (Fig. 1). The altered balance in B-cells maturation characterizing by a significant lower percent of pre-switched B-cells and plasmablasts (p &amp;lt; 0.05) as well as a higher trend (&amp;gt;55%) in the naive B-cells (p &amp;lt; 0.01) was established in IgAN patients as compared to healthy donors reflecting the exhaustion of peripheral blood plasmablasts due to constant generation of antibody-producing plasma cells. Moreover, the lower pre-switched B-cells percentage was detected in the blood, the more BAFF production was observed in serum of IgAN patients (R = 0.50; p &amp;lt; 0.05) and the higher proteinuria level was detected (R = 0.47; p &amp;lt; 0.05). The increased total number of γδТ-cells up to 9.42 (6.25-12.44)% in IgAN patients as compared to donors (p = 0.03) was correlated with total memory CD19+CD27+B-cells numbers (R = 0.54; p &amp;lt; 0.05) as well as pre-switched B-cells (R = 0.45; p &amp;lt; 0.05), plasmablasts (R = 0.57; p &amp;lt; 0.01) and long-lived plasma cells (R = 0.51; p &amp;lt; 0.05). A detailed subsets analysis revealed an elevated level of tissue-resident mucosa-associated cells (Vδ1+ and Vδ3+T-lymphocytes) as well as enhanced HLA-DR expression on them (p &amp;lt; 0.05) in combination with decreased normally circulating Vδ2+T-cells numbers (p &amp;lt; 0.01) as compared to healthy donors what characterized Vδ1+ and Vδ3+T-cells subsets as professional antigen presenting cells and indicated their possible role in the priming of antigen specific B-cells. Conclusion Suggesting that most peripheral B-lymphocytes are naïve, the obtained data may reflect a γδT-cells role in migration of pre-switched B-cells from the circulation to mucosa sites for possible further maturation and class-switching what allows us to consider these two parameters as potential useful biomarkers of IgAN disease prognosis in clinical practice.

Characterization of innate lymphoid cell subsets infiltrating melanoma and epithelial ovarian tumors

ABSTRACT The innate lymphoid cell (ILC) family is composed of heterogeneous innate effector and helper immune cells that preferentially reside in tissues where they promote tissue homeostasis. In cancer, they have been implicated in driving both pro- and anti-tumor responses. This apparent dichotomy highlights the need to better understand differences in the ILC composition and phenotype within different tumor types that could drive seemingly opposite anti-tumor responses. Here, we characterized the frequency and phenotype of various ILC subsets in melanoma metastases and primary epithelial ovarian tumors. We observed high PD-1 expression on ILC subsets isolated from epithelial ovarian tumor samples, while ILC populations in melanoma samples express higher levels of LAG-3. In addition, we found that the frequency of cytotoxic ILCs and NKp46+ILC3 in tumors positively correlates with monocytic cells and conventional type 2 dendritic cells, revealing potentially new interconnected immune cell subsets in the tumor microenvironment. Consequently, these observations may have direct relevance to tumor microenvironment composition and how ILC subset may influence anti-tumor immunity.

Abstract 1055: Atxn2 Inhibition Enhances The Contractile Phenotype Of Vascular Smooth Muscle Cells

Introduction: Phenotype switching of vascular smooth muscle cells (VSMC) between their synthetic and differentiated contractile state is a critical player in controlling the stability of atherosclerotic plaque. Hundreds of genes associated with coronary artery disease (CAD) have been identified through Genome-Wide Association Studies (GWAS). However, the involvement of most identified genes in the pathogenesis of atherosclerosis remains unknown. ATXN2 was identified as a CAD GWAS candidate gene with unclear biological role in atherosclerosis. ATXN2 regulates the endocytic internalization of the EGF receptor, which is essential in the VSMC phenotype switching. Therefore, we aimed to investigate whether ATXN2 is involved in the VSMC phenotype switching. Methods and Results: Using immunofluorescence, we demonstrated that ATXN2 siRNA knockdown resulted in a 5-fold increase in the ratio of the contractile phenotype of human aortic VSMC that expressed a high alpha-smooth muscle cell actin content compared to controls. In addition, we found that ATXN2 inhibition reduced the VSMC proliferation and migration as assessed by the EdU cell proliferation assay and the wound healing migration assay, respectively. Next, we confirmed that ATXN2 siRNA knockdown increased the mRNA expression of contractile markers: calponin, alpha-smooth muscle cell actin, and smooth muscle protein 22 alpha using quantitative PCR. We found that the enhancing effect of VSMC contractile phenotype by ATXN2 siRNA was less pronounced in the absence of TGF-beta. Indeed, the enhancing effect of ATXN2 inhibition on the VSMC contractile phenotype was abolished by SMAD4 gene silencing, suggesting the involvement of TGF-beta signaling. Furthermore, we validated the expression signature of the ATXN2 gene in different phenotypic subsets of VSMC from human atherosclerotic plaque samples using single-cell RNA sequencing analysis. We detected that ATXN2 expression was negatively correlated with the contractile VSMC subset. In conclusion, our findings suggests ATXN2, CAD GWAS candidate gene, as a novel potential therapeutic target to enhance the VSMC contractile phenotype for stabilizing atherosclerotic plaque in coronary artery disease patients.

Abstract LT02: Regulation of Exhausted CD8+ T cell Differentiation by IKZF Transcription Factors

Abstract CD8+ T cells are indispensable for pathogen and tumour clearance, although can induce immunopathology if left unrestricted. T cell exhaustion, progressively programmed in the context of persistent antigen exposure, ensures partial control while mitigating immunopathological risk. However, exhaustion is characterised by curtailed proliferative capacity, cytokine production and cytotoxic functions, which tumours and chronic pathogens exploit to persist. As such, transiently disrupting exhaustion has emerged as a therapeutic strategy for treating cancer. Exhausted cells transition through multiple differentiation states, from stem-like progenitors that mediate response to checkpoint blockade, to terminal effector or exhausted cells. Unfortunately, only a fraction of patients respond to checkpoint blockade long-term, in part due to loss of the therapy responsive progenitor population. Therefore, understanding the molecular pathways moderating the exhausted states, hindering the effector populations, and promoting terminal exhaustion is essential for augmenting cancer immunotherapy. We identify IKZF transcription factors, Ikaros (IKZF1), Helios (IKZF2) and Aiolos (IKZF3), as key mediators of T cell exhaustion using gene knock-out mice and a CRISPR-mediated genetic knock-out system in both chronic infection and tumour models. We elucidate the molecular pathways involved in regulation of T cell exhaustion by IKZF transcription factor using an array of RNA sequencing, ATAC sequencing and flow cytometric validation technologies. IKZF1 and IKZF3 are critical regulators of exhausted subset expansion, retention, phenotype and cytokine function. Strikingly, dual IKZF1 and IKZF3 ablation boosts the response of anti-tumour and anti-viral exhausted T cells, potentially via the formation of atypical, exhausted T cell populations and the enrichment of functional exhausted subsets. Excitingly, dual ablation vastly improves survival and tumour control in a solid tumour, HER2-specific CAR T cell model. Cumulatively, our data highlight a novel role for IKZF transcription factors in biasing exhausted T cell differentiation and provides potentially important clinical implications, directing future cancer immunotherapies targeting T cell exhaustion. Citation Format: Sinead M. Reading, Isabelle Munoz, Maria N. de Menezes, Nicole Y. L. Saw, Krutika Ambani, Antonio Ahn, Simone Nussing, Sara Roth, Shienny Sampurno, Kelly M. Ramsbottom, Joseph A. Trapani, Kim L. Good-Jacobson, Ricky W. Johnstone, Shom Goel, Paul A. Beavis, Ian A. Parish. Regulation of Exhausted CD8+ T cell Differentiation by IKZF Transcription Factors [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT02.

FEATURES OF THE T-IMMUNE SYSTEM IN PATIENTS WITH GLOMERULONEPHRITISES WITH NEPHROTIC SYNDROME

The study of issues related to glomerulonephritises with nephrotic syndrome is one of the urgent problems of medicine due to their prevalence worldwide, mainly in the young age group. Medical workers distinguish primary (idiopathic) nephrotic syndrome, which occurs in 80–90% of cases, and secondary nephrotic syndrome, mainly associated with systemic autoimmune diseases, diabetes mellitus and neoplasms. Glomerulonephritises, manifested by nephrotic syndrome (membranous nephropathy, focal segmental glomerulosclerosis, nephropathy with minimal changes), are known to be autoimmune diseases. To date, the immunological mechanisms of the pathogenesis of glomerulonephritises with nephrotic syndrome associated with the T-system of adaptive immunity remain unexplored. The aim of the study was to study the role of the T–immune system in the pathogenesis of primary nephrotic syndrome based on the study of immunoregulatory, activated T-cell subsets in patients with this pathology. Material and methods. 136 patients with chronic glomerulonephritis with nephrotic syndrome were selected for the study. The assessment of the T-immune system included determination of the lymphocyte phenotype of immunoregulatory T-cell subsets (T-helper/inducers, cytotoxic T-lymphocytes), various subpopulations of activated T-cells (activated T-lymphocytes; activated T-lymphocytes expressing CD25–alpha chain of IL-2 receptor; activated cytotoxic T-lymphocytes expressing HLA-DR and CD38) and regulatory T-cells (Treg cells). Study results. In the patients of the examined cohort, an increase in the number of T-lymphocytes and T-helper cells, as well as activated T-lymphocytes expressing HLA-DR antigens, was found. At this, the content of cytotoxic T-cells and the number of activated T-cells expressing the IL-2 – CD25 receptor did not differ from similar indicators in healthy individuals. The levels of Treg cells and activated cytotoxic T-lymphocytes with the CD3+CD8brightCD38+ phenotype were reduced. The immunoregulatory index (T-helpers/cytotoxic T-lymphocytes) was increased, due to an increase in the number of T-helper cells against the background of an unchanged number of cytotoxic T-lymphocytes. Conclusions. The results of the study indicate that the main features of the T-system of the immune response in primary nephrotic syndrome are imbalance in the ratio of the content of immunoregulatory cells due to predominance of T-helper cells and a decrease in the number of Treg cells.

Abstract 1332: Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies

Abstract Invariant Natural Killer T (iNKT) cells are a unique subset of innate lymphocytes, constituting &amp;lt;1% of human T cells. iNKT cells express a semi-invariant TCR (iTCR) recognizing glycolipids presented by the monomorphic, MHC-like molecule CD1d. Previous studies indicate that due to distinctive TCR constitution and antigen recognition properties, iNKT cells do not induce acute graft-versus-host disease. Based on the CD4 and CD8 expression, mature human iNKT cells can be classified into CD4+CD8-, CD4-CD8- &amp; CD4-CD8+ subsets with overlapping and distinct functions. The functional profiling of CAR19-iNKT cell subsets serves the dual purpose of ensuring their safety and efficacy as a therapeutic intervention. Here we characterized the phenotypic and functional profile of CD4+ and CD4- CAR19-iNKT cells. iNKT cells were isolated with &amp;gt;99% purity from healthy donors' peripheral blood and were transduced with a CD19 CAR-encoding 3rd generation lentivirus. After expansion, the CAR19-iNKT cells were cryopreserved and later analyzed post-thawing. Differential expression of CD27 and PD1 was noted in CAR+CD4+ and CAR+CD4- iNKT cells compared to other markers. For functional profiling, CAR+CD4+ iNKT cells were positively selected, and 24h cytotoxicity assays were performed using several tumor cell lines including SEM, Ramos and C1R-CD1d (+/- α-GalCer). The CAR+CD4- iNKT cells exhibited superior cytotoxicity to multiple tumor cell lines compared to that of CAR+CD4+ iNKT cells from two donors. However, both subsets lysed α-GalCer pulsed C1R-CD1d cells in a comparable manner indicating the potential of these subsets to recognize antigen through the iTCR. Subsequently, we assessed the proliferation ability of the CAR+CD4+ and CAR+CD4- subsets by exposing the cells to three rounds of stimulation, 24h apart, using SEM (CD19+CD1d-) or K562 (CD19-CD1d-) tumor cells with or without IL-15. Proliferation was assessed seven days following the first stimulation. The CAR+CD4+ subset of CAR19-iNKT cells demonstrated faster proliferation than CAR+CD4- cells in the presence of IL-15 after repeated exposure to tumor cells. Ongoing RNA seq analysis of CAR+CD4+/CAR+CD4- cells will further elucidate differences between these subsets. The outcomes of these studies have shown encouraging results, indicating the potential benefit of having diverse subsets among CAR19-iNKT cells for treating CD19+ cancers. The therapeutic potential of CAR+CD4+ iNKT cell subset could be enhanced to match that of CAR+CD4- subset with the use of α-GalCer. In summary, inclusion of both CD4+ and CD4- iNKT cells is critical for the functionality of allogeneic CAR-iNKT cell therapy. Extensive characterization is crucial for clinical translation, but additional research, including preclinical and clinical trials, is essential to determine the safety and effectiveness of CAR19-iNKT cell therapy in real-world applications. Citation Format: Kanagaraju Ponnusamy, Simon Poon, Nicole van der Weerden, Robson Dossa, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies [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 1332.

Early elevated IFNα is a key mediator of HIV pathogenesis

BackgroundA complete understanding of the different steps of HIV replication and an effective drug combination have led to modern antiretroviral regimens that block HIV replication for decades, but these therapies are not curative and must be taken for life. “Elite controllers” (ECs) is a term for the 0.5% of HIV-infected persons requiring no antiretroviral therapy, whose status may point the way toward a functional HIV cure. Defining the mechanisms of this control may be key to understanding how to replicate this functional cure in others.MethodsIn ECs and untreated non-EC patients, we compared IFNα serum concentration, distribution of immune cell subsets, and frequency of cell markers associated with immune dysfunction. We also investigated the effect of an elevated dose of IFNα on distinct subsets within dendritic cells, natural killer cells, and CD4+ and CD8 + T cells.ResultsSerum IFNα was undetectable in ECs, but all immune cell subsets from untreated non-EC patients were structurally and functionally impaired. We also show that the altered phenotype and function of these cell subsets in non-EC patients can be recapitulated when cells are stimulated in vitro with high-dose IFNα.ConclusionsElevated IFNα is a key mediator of HIV pathogenesis.