Pre-Clinical Assessment of SAR442257, a CD38/CD3xCD28 Trispecific T Cell Engager in Treatment of Relapsed/Refractory Multiple Myeloma.

Current treatment strategies for multiple myeloma (MM) are highly effective, but most patients develop relapsed/refractory disease (RRMM). The anti-CD38/CD3xCD28 trispecific antibody SAR442257 targets CD38 and CD28 on MM cells and co-stimulates CD3 and CD28 on T cells (TCs). We evaluated different key aspects such as MM cells and T cells avidity interaction, tumor killing, and biomarkers for drug potency in three distinct cohorts of RRMM patients. We found that a significantly higher proportion of RRMM patients (86%) exhibited aberrant co-expression of CD28 compared to newly diagnosed MM (NDMM) patients (19%). Furthermore, SAR442257 mediated significantly higher TC activation, resulting in enhanced MM killing compared to bispecific functional knockout controls for all relapse cohorts (Pearson's r = 0.7). Finally, patients refractory to anti-CD38 therapy had higher levels of TGF-ß (up to 20-fold) compared to other cohorts. This can limit the activity of SAR442257. Vactoserib, a TGF-ß inhibitor, was able to mitigate this effect and restore sensitivity to SAR442257 in these experiments. In conclusion, SAR442257 has high potential for enhancing TC cytotoxicity by co-targeting CD38 and CD28 on MM and CD3/CD28 on T cells.

TOP CAR with TMIGD2 as a safe and effective costimulatory domain in CAR cells treating human solid tumors.

Chimeric antigen receptor (CAR)-T cell therapy shows impressive efficacy treating hematologic malignancies but requires further optimization in solid tumors. Here, we developed a TMIGD2 optimized potent/persistent (TOP) CAR that incorporated the costimulatory domain of TMIGD2, a T and NK cell costimulator, and monoclonal antibodies targeting the IgV domain of B7-H3, an immune checkpoint expressed on solid tumors and tumor vasculature. Comparing second- and third-generation B7-H3 CARs containing TMIGD2, CD28, and/or 4-1BB costimulatory domains revealed superior antitumor responses in B7-H3.TMIGD2 and B7-H3.CD28.4-1BB CAR-T cells in vitro. Comparing these two constructs using in vivo orthotopic human cancer models demonstrated that B7-H3.TMIGD2 CAR-T cells had equivalent or superior antitumor activity, survival, expansion, and persistence. Mechanistically, B7-H3.TMIGD2 CAR-T cells maintained mitochondrial metabolism; produced less cytokines; and established fewer exhausted cells, more central memory cells, and a larger CD8/CD4 T cell ratio. These studies demonstrate that the TOP CAR with TMIGD2 costimulation offered distinct benefits from CD28.41BB costimulation and is effective against solid tumors.

Associations between immune cell phenotypes and lung cancer subtypes: insights from mendelian randomization analysis.

INTRODUCTION: Lung cancer is a common malignant tumor, and different types of immune cells may have different effects on the occurrence and development of lung cancer subtypes, including lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). However, the causal relationship between immune phenotype and lung cancer is still unclear. METHODS: This study utilized a comprehensive dataset containing 731 immune phenotypes from the European Bioinformatics Institute (EBI) to evaluate the potential causal relationship between immune phenotypes and LUSC and LUAD using the inverse variance weighted (IVW) method in Mendelian randomization (MR). Sensitivity analyses, including MR-Egger intercept, Cochran Q test, and others, were conducted for the robustness of the results. The study results were further validated through meta-analysis using data from the Transdisciplinary Research Into Cancer of the Lung (TRICL) data. Additionally, confounding factors were excluded to ensure the robustness of the findings. RESULTS: Among the final selection of 729 immune cell phenotypes, three immune phenotypes exhibited statistically significant effects with LUSC. CD28 expression on resting CD4 regulatory T cells (OR 1.0980, 95% CI: 1.0627-1.1344, p < 0.0001) and CD45RA + CD28- CD8 + T cell %T cell (OR 1.0011, 95% CI: 1.0007; 1.0015, p < 0.0001) were associated with increased susceptibility to LUSC. Conversely, CCR2 expression on monocytes (OR 0.9399, 95% CI: 0.9177-0.9625, p < 0.0001) was correlated with a decreased risk of LUSC. However, no significant causal relationships were established between any immune cell phenotypes and LUAD. CONCLUSION: This study demonstrates that specific immune cell types are associated with the risk of LUSC but not with LUAD. While these findings are derived solely from European populations, they still provide clues for a deeper understanding of the immunological mechanisms underlying lung cancer and may offer new directions for future therapeutic strategies and preventive measures.

Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vß21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

Research and Analysis of Molecules such as CD28, CD45RA, CD45RO, CD38, HLA-DR, and CD57 on T Cells in Multiple Myeloma.

BACKGROUND: For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC). METHODS: For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells. RESULTS: We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group. CONCLUSIONS: Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

CD8+ T cells are necessary for improved sepsis survival induced by CD28 agonism in immunologically experienced mice.

Introduction: A hallmark of T cell dysregulation during sepsis is the downregulation of costimulatory molecules. CD28 is one of T cell costimulatory molecules significantly altered on memory T cells during sepsis. We recently showed that treatment with a αCD28 agonist in septic immunologically experienced mice led to improved survival. Therefore, here we aimed to identify the cell subset(s) necessary for the survival benefit observed in the context of CD28 agonism, and to further investigate the mechanism by which CD28 agonism improves sepsis survival in immunologically experienced mice. Methods: Mice received specific pathogen inoculation to generate memory T cell populations similar in frequency to that of adult humans. Once these infections were cleared and the T cell response had transitioned to the memory phase, animals were rendered septic via cecal ligation and puncture in the presence or absence of an agonistic anti-CD28 mAb. Results: Results demonstrated that CD8+ T cells, and not bulk CD4+ T cells or CD25+ regulatory T cells, were necessary for the survival benefit observed in CD28 agonist-treated septic immunologically experienced mice. Upon examination of these CD8+ T cells, we found that CD28 agonism in septic immunologically experienced mice was associated with an increase in Foxp3+ CD8+ T cells as compared to vehicle-treated controls. When CD8+ T cells were depleted in septic immunologically experienced mice in the setting of CD28 agonism, a significant increase in levels of inflammatory cytokines in the blood was observed. Discussion: Taken together, these results indicate that CD28 agonism in immunologically experienced mice effectively suppresses inflammation via a CD8+-dependent mechanism to decrease mortality during sepsis.

[Construction and Optimization of CD19 Chimeric Antigen Receptor T Cells Derived from C57BL/6J Mice].

OBJECTIVE: To explore the stimulation conditions, optimal culture time and infection time of C57BL/6J mice CD3+ T cells in vitro, so as to improve the infection efficiency of CD19 chimeric antigen receptor T cells (mCD19 CAR-T). METHODS: Purified C57BL/6J mice CD3+ T cells were cultured in anti-CD3/CD28 coated, anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated, respectively. The cells were stimulated in above three conditions for 12 h and 24 h, following with 24 h, 48 h and 72 h incubation and then the number of cell clones was recorded. C57BL/6J mice CD3+ T cells were stimulated for 12 h, 24 h, and 36 h under the above three conditions, then interleukin (IL)-2 (100 U/ml) was added. The number of cell clones was recorded under microscope at 24 h, 48 h, and 72 h of culture. After 24 h of stimulation, CD3+ T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells, and the percentage of GFP+ CAR-T cells was detected by flow cytometry. RESULTS: The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23% under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice. The number of clones of C57BL/6J mice CD3+ T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h. After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2, the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2, and the infection efficiency of CAR-T cells in this group reached 17.63%±4.17%. CONCLUSION: The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+ T cells are different from those of BABL/c mice. T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.

Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma.

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

Circulating Immune Landscape Profiling in Psoriasis Vulgaris and Psoriatic Arthritis by Mass Cytometry.

Background: Psoriasis, a systemic disorder mediated by the immune system, can appear on the skin, joints, or both. Individuals with cutaneous psoriasis (PsC) have an elevated risk of developing psoriatic arthritis (PsA) during their lifetime. Despite this known association, the cellular and molecular mechanisms underlying this progression remain unclear. Methods: We performed high-dimensional, in-depth immunophenotyping of peripheral blood mononuclear cells (PBMCs) in patients with PsA and psoriasis vulgaris (PsV) by mass cytometry. Blood samples were collected before and after therapy for a longitudinal study. Then three sets of comparisons were made here: active PsA vs. active PsV, untreated PsV vs. treated PsV, and untreated PsA vs. treated PsA. Results: Marked differences were observed in multiple lymphocyte subsets of PsA related to PsV, with expansion of CD4+ T cells, CD16- NK cells, and B cells. Notably, two critical markers, CD28 and CD127, specifically differentiated PsA from PsV. The expression levels of CD28 and CD127 on both Naïve T cells (TN) and central memory CD4+ T cells (TCM) were considerably higher in PsA than PsV. Meanwhile, after treatment, patients with PsV had higher levels of CD28hi CD127hi CD4+ TCM cells, CD28hi CD127hi CD4+ TN cells, and CD16- NK cells. Conclusion: In the circulation of PsA patients, the TN and CD4+ TCM are characterized with more abundant CD28 and CD127, which effectively distinguished PsA from PsV. This may indicate that individuals undergoing PsV could be stratified at high risk of developing PsA based on the circulating levels of CD28 and CD127 on specific cell subsets.

The mechanisms of MicroRNA 21 in premature ovarian insufficiency mice with mesenchymal stem cells transplantation : The involved molecular and immunological mechanisms.

Umbilical cord-derived mesenchymal stem cell (UCMSC) transplantation has been deeply explored for premature ovarian insufficiency (POI) disease. However, the associated mechanism remains to be researched. To explore whether and how the microRNA 21 (miR-21) functions in POI mice with UCMSCs transplantation, the autoimmune-induced POI mice model was built up, transplanted with or without UCMSCs transfect with the LV-hsa-miR-21-5p/LV-hsa-miR-21-5p-inhibition, with the transfection efficiency analyzed by QRT-PCR. Mice hormone secretion and the anti-Zona pellucida antibody (AZPAb) levels were analyzed, the ovarian morphological changes and folliculogenesis were observed, and the ovarian apoptosis cells were detected to evaluate ovarian function. The expression and localization of the PTEN/Akt/FOXO3a signal pathway-related cytokines were analyzed in mice ovaries.Additionally, the spleen levels of CD8 + CD28-T cells were tested and qualified with its significant secretory factor, interleukin 10 (IL-10). We found that with the LV-hsa-miR-21-5p-inhibition-UCMSCs transplantation, the mice ovarian function can be hardly recovered than mice with LV-NC-UCMSCs transplantation, and the PTEN/Akt/FOXO3a signal pathway was activated. The expression levels of the CD8 + CD28-T cells were decreased, with the decreased levels of the IL-10 expression. In contrast, in mice with the LV-hsa-miR-21-5p-UCMSCs transplantation, the injured ovarian function can be reversed, and the PTEN/AKT/FOXO3a signal pathway was detected activated, with the increased levels of the CD8 + CD28-T cells, and the increased serum levels of IL-10. In conclusion, miR-21 improves the ovarian function recovery of POI mice with UCMSCs transplantation, and the mechanisms may be through suppressing the PTEN/AKT/FOXO3a signal pathway and up-regulating the circulating of the CD8 + CD28-T cells.

The causal effects of immune cells on pancreatic cancer: A 2­sample Mendelian randomization study.

Leveraging publicly available genetic datasets, we conducted a comprehensive 2-sample Mendelian randomization (MR) analysis to explore the causal links between 731 immunophenotypes and the risk of pancreatic cancer (PC). To ensure the robustness of our findings, extensive sensitivity analyses were performed, evaluating stability, heterogeneity, and potential horizontal pleiotropy. Our analysis pinpointed 24 immunophenotypes significantly associated with the risk of PC. Notably, phenotypes such as CD4+ CD8dim %leukocyte (OR = 0.852, 95% CI = 0.729-0.995, P = .0430) and HLA DR+ CD4+ AC (OR = 0.933, 95% CI = 0.883-0.986) in TBNK were inversely correlated with PC risk. Conversely, phenotypes like CD28 on CD45RA- CD4 non-Treg (OR = 1.155, 95% CI = 1.028-1.297, P = .016) and CD25 on activated Treg (OR = 1.180, 95% CI = 1.014-1.374, P = .032) in Treg cells, among others, exhibited a positive correlation. These insights offer a valuable genetic perspective that could guide future clinical research in this area.

NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.

Causal relationship between immune cells and telomere length: mendelian randomization analysis.

BACKGROUND: The causal relationship between immune cells and telomere length remains controversial. METHODS: Data on the immune cells were obtained from a previous study with 3,757 participants. Data on telomere length were obtained from the OpenGWAS database. Genome-Wide Association Study (GWAS) data were obtained and screened for eligible instrumental variables (IVs) using the TwoSampleMR package and the Phenoscanner database. To investigate the genetic causality between immune cells and telomere length, Mendelian randomization (MR) analysis and Bayesian weighted Mendelian randomization (BWMR) analysis were used. RESULTS: MR analysis showed that there is indeed a genetic causal relationship between immune cells and telomere length. A total of 16 immune cells were successfully validated. A positive correlation was found between telomere length and immune cells such as CD28 + CD45RA + CD8br %CD8br (OR = 1.002, 95%CI: 1.000-1.003). A negative correlation was found between telomere length and immune cells such as Transitional AC (OR = 0.991, 95%CI: 0.984-0.997) (P < 0.05). Reverse MR analysis similarly confirmed that telomere length can affect four types of immune cells, including CD25 on IgD + CD24- (OR = 1.291, 95%CI: 1.060-1.571), at the genetic level. CONCLUSION: There is indeed a mutual genetic causality between immune cells and telomere length, which will provide theoretical basis and support for more subsequent clinical studies.

Differential Impact of CD43 and CD28 on T-Cell Differentiation Depending on the Order of Engagement with the TCR.

The combination of signals from the T-cell receptor (TCR) and co-stimulatory molecules triggers transcriptional programs that lead to proliferation, cytokine secretion, and effector functions. We compared the impact of engaging the TCR with CD28 and/or CD43 at different time points relative to TCR engagement on T-cell function. TCR and CD43 simultaneous engagement resulted in higher CD69 and PD-1 expression levels than in TCR and CD28-stimulated cells, with a cytokine signature of mostly effector, inflammatory, and regulatory cytokines, while TCR and CD28-activated cells secreted all categories of cytokines, including stimulatory cytokines. Furthermore, the timing of CD43 engagement relative to TCR ligation, and to a lesser degree that of CD28, resulted in distinct patterns of expression of cytokines, chemokines, and growth factors. Complete cell activation was observed when CD28 or CD43 were engaged simultaneously with or before the TCR, but ligating the TCR before CD43 or CD28 failed to complete a cell activation program regarding cytokine secretion. As the order in which CD43 or CD28 and the TCR were engaged resulted in different combinations of cytokines that shape distinct T-cell immune programs, we analyzed their upstream sequences to assess whether the combinations of cytokines were associated with different sets of regulatory elements. We found that the order in which the TCR and CD28 or CD43 are engaged predicts the recruitment of specific sets of chromatin remodelers and TFSS, which ultimately regulate T-cell polarization and plasticity. Our data underscore that the combination of co-stimulatory molecules and the time when they are engaged relative to the TCR can change the cell differentiation program.

Higher Circulating Lymphocytes and the Incidence of Pre-eclampsia and Eclampsia.

Excessive immune activation contributes to the onset of early dysfunction of the maternal-fetal interface, and it is closely linked to the development of pre-eclampsia. However, the effect of specific immune cells on the risk of pre-eclampsia and eclampsia remains controversial. We investigated the causal relationship between immune cells and pre-eclampsia and eclampsia. For exposure, we extracted genetic variants associated with immune cell-related traits, and for outcomes, we used summary genetic data of pre-eclampsia/eclampsia. A two-sample Mendelian randomization (MR) analysis was then performed to assess the causal relationship. Robustness of the MR results was then evaluated through colocalization analysis. We found that genetically proxied circulating lymphocyte absolute count was causally associated with total eclampsia (odds ratio (OR) = 1.53, 95% confidence interval (CI) (1.31-1.79), p = 1.15E - 07) and pre-eclampsia (OR = 1.50, 95% CI (1.28-1.77), p = 9.18E - 07); T cell absolute count was causally associated with total eclampsia (OR = 1.49, 95% CI (1.28-1.73), p = 2.73E - 07) and pre-eclampsia (OR = 1.47, 95% CI (1.25-1.72), p = 1.76E - 06). And CD28- CD25+ CD8+ T cell absolute count was causally associated with total eclampsia (OR = 1.83, 95% CI (1.44-2.32), p = 7.11E - 07) and pre-eclampsia (OR = 1.77, 95% CI (1.38-2.26), p = 6.55E - 06). Colocalization analysis revealed that immune cell-related traits shared the same variant with pre-eclampsia/eclampsia. Our study suggested causal effects of genetic predisposition to high lymphocyte absolute count levels, T cell absolute count, and CD28- CD25+ CD8+ T cell absolute count on eclampsia, particularly pre-eclampsia risk, providing crucial new insights into the potential prevention target for eclampsia and pre-eclampsia.

Practical 10-Color T-Cell Panel for Phenotyping Diverse Populations Using Spectral Flow Cytometry: A Beginner's Guide.

Flow cytometry stands as the most employed high-throughput single-cell analysis technique, facilitating the profiling of remarkably diverse samples, such as blood, bone marrow and body fluids. In addition, it allows for the discrimination of diverse immune cell subsets, including infrequently encountered types like T regulatory cells and exhausted CD28Null T cells. However, analyzing rare immune cell subsets with conventional flow cytometry poses challenges stemming from factors like fluorophore overlap, compensation issues, and limited flexibility in fluorophore selection. Therefore, spectral flow cytometry offers advantages over traditional flow cytometry. It measures the full emission spectrum and then separates it to identify different fluorochromes. This enables the use of fluorochromes with significant overlap in a single test, allowing for the analysis of more protein markers. Following this, spectral technology employs precise calculations to separate individual fluorochromes, thereby enabling the detection and elimination of autofluorescent signals originating from cells within the entire emission spectrum. This capability is pivotal in achieving deep phenotyping of immune cells with the requisite sensitivity and resolution essential for monitoring the immune systems of patients with compromised immunity, such as cancer and autoimmune disorders. Additionally, it allows for the exploration of interactions between distinct immune subsets. In this context, we introduce an optimized protocol utilizing spectral flow cytometry for precise T-cell characterization and differentiation, encompassing the assessment of their activation states. Furthermore, this protocol extends its applicability to the identification of less common circulating T-cell populations, notably T-regulatory and CD28Null T cells, following autofluorescence correction within the spectrum. This protocol provides a set of steps and reagents for the surface and intracellular staining of human T cells using whole peripheral blood. The spectral-based design of this panel allows for its applicability to other spectral machines, providing a versatile and efficient tool for T-cell analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Achieving optimal staining through effective antibody titration Basic Protocol 2: Single-cell staining Basic Protocol 3: Comprehensive panel staining post-titration and spectral library integration.

Persistence of peripheral CD8 + CD28- T cells indicates a favourable outcome and tumour immunity in first-line HER2-positive metastatic breast cancer.

BACKGROUND: The contradictory role of CD8 + CD28- T cells in tumour immunity has been reported, while their biological and clinical significance in HER2-positive metastatic breast cancer (MBC) is still unknown. METHODS: HER2-positive MBC patients with no prior therapy in the metastatic setting were retrospectively recruited at two medical centres. Peripheral CD8 + CD28- T cells (pTCD8+CD28-) were detected at baseline and following therapeutic intervals. Progression-free survival (PFS) was compared according to pTCD8+CD28- levels. The molecular features of pTCD8+CD28- and its correlation with tumour immunity were also investigated. RESULTS: A total of 252 patients were enrolled, and the median follow-up time was 29.6 months. pTCD8+CD28- high at baseline has prolonged PFS compared to pTCD8+CD28- low (P = 0.001). Patients who maintained pTCD8+CD28- high had a longer PFS than those who kept pTCD8+CD28- low (P < 0.001). The enhanced pTCD8+CD28- level also indicates a longer PFS compared to pTCD8+CD28- low (P = 0.025). Here, pTCD8+CD28- was demonstrated as an antigen-experienced effector T cell. Higher IL-2 level (P = 0.034) and lower TGF-ß level (P = 0.016) in the serum and highly infiltrated CD8 + CD28- T cells (P = 0.037) were also connected to pTCD8+CD28- high. CONCLUSIONS: High pTCD8+CD28- level is associated with a favourable tumour immunity and a better PFS of HER2-targeting therapy in MBC patients.

Immune profiling analysis of double-negative T cells in patients with systemic sclerosis.

OBJECTIVE: To construct a molecular immune map of patients with systemic sclerosis (SSc) by mass flow cytometry, and compare the number and molecular expression of double-negative T (DNT) cell subsets between patients and healthy controls (HC). METHODS: Peripheral blood mononuclear cells (PBMCs) were extracted from the peripheral blood of 17 SSc patients and 9 HC. A 42-channel panel was set up to perform mass cytometry by time of flight (CyTOF) analysis for DNT subgroups. Flow cytometry was used to validate subpopulation functions. The clinical data of patients were collected for correlation analysis. RESULTS: Compared with HC, the number of total DNT cells decreased in SSc patients. Six DNT subsets were obtained from CyTOF analysis, in which the proportion of cluster1 increased, while the proportion of cluster3 decreased. Further analysis revealed that cluster1 was characterized by high expression of CD28 and CCR7, and cluster3 was characterized by high expression of CD28 and CCR5. After in vitro stimulation, cluster1 secreted more IL-4 and cluster3 secreted more IL-10 in SSc patients compared to HC. Clinical correlation analysis suggested that cluster1 may play a pathogenic role while cluster3 may play a protective role in SSc. ROC curve analysis further revealed that cluster3 may be a potential indicator for determining disease activity in SSc patients. CONCLUSION: We found a new CCR5+CD28+ DNT cell subset, which played a protective role in the pathogenesis of SSc. Key Points • The number of DNT cells decreased in SSc patients' peripheral blood. • DNT cells do not infiltrate in the skin but secrete cytokines to participate in the pathogenesis of SSc. • A CCR5+CD28+ DNT cell population may play a protective role in SSc.