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.

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.

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.

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.

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.

Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

The B7:CD28 family and friends: Unraveling coinhibitory interactions.

Immune responses must be tightly regulated to ensure both optimal protective immunity and tolerance. Costimulatory pathways within the B7:CD28 family provide essential signals for optimal T cell activation and clonal expansion. They provide crucial inhibitory signals that maintain immune homeostasis, control resolution of inflammation, regulate host defense, and promote tolerance to prevent autoimmunity. Tumors and chronic pathogens can exploit these pathways to evade eradication by the immune system. Advances in understanding B7:CD28 pathways have ushered in a new era of immunotherapy with effective drugs to treat cancer, autoimmune diseases, infectious diseases, and transplant rejection. Here, we discuss current understanding of the mechanisms underlying the coinhibitory functions of CTLA-4, PD-1, PD-L1:B7-1 and PD-L2:RGMb interactions and less studied B7 family members, including HHLA2, VISTA, BTNL2, and BTN3A1, as well as their overlapping and unique roles in regulating immune responses, and the therapeutic potential of these insights.

CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Disruption of SUV39H1-Mediated H3K9 Methylation Sustains CAR T-cell Function.

Suboptimal functional persistence limits the efficacy of adoptive T-cell therapies. CD28-based chimeric antigen receptors (CAR) impart potent effector function to T cells but with a limited lifespan. We show here that the genetic disruption of SUV39H1, which encodes a histone-3, lysine-9 methyl-transferase, enhances the early expansion, long-term persistence, and overall antitumor efficacy of human CAR T cells in leukemia and prostate cancer models. Persisting SUV39H1-edited CAR T cells demonstrate improved expansion and tumor rejection upon multiple rechallenges. Transcriptional and genome accessibility profiling of repeatedly challenged CAR T cells shows improved expression and accessibility of memory transcription factors in SUV39H1-edited CAR T cells. SUV39H1 editing also reduces expression of inhibitory receptors and limits exhaustion in CAR T cells that have undergone multiple rechallenges. Our findings thus demonstrate the potential of epigenetic programming of CAR T cells to balance their function and persistence for improved adoptive cell therapies. SIGNIFICANCE: T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.

Exploiting the CD200-CD200R immune checkpoint axis in multiple myeloma to enhance CAR T-cell therapy.

ABSTRACT: Patients with multiple myeloma (MM) treated with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells usually relapse with BCMA+ disease, indicative of CAR T-cell suppression. CD200 is an immune checkpoint that is overexpressed on aberrant plasma cells (aPCs) in MM and is an independent negative prognostic factor for survival. However, CD200 is not present on MM cell lines, a potential limitation of current preclinical models. We engineered MM cell lines to express CD200 at levels equivalent to those found on aPCs in MM and show that these are sufficient to suppress clinical-stage CAR T-cells targeting BCMA or the Tn glycoform of mucin 1 (TnMUC1), costimulated by 4-1BB and CD2, respectively. To prevent CD200-mediated suppression of CAR T cells, we compared CRISPR-Cas9-mediated knockout of the CD200 receptor (CD200RKO), to coexpression of versions of the CD200 receptor that were nonsignaling, that is, dominant negative (CD200RDN), or that leveraged the CD200 signal to provide CD28 costimulation (CD200R-CD28 switch). We found that the CD200R-CD28 switch potently enhanced the polyfunctionality of CAR T cells, and improved cytotoxicity, proliferative capacity, CAR T-cell metabolism, and performance in a chronic antigen exposure assay. CD200RDN provided modest benefits, but surprisingly, the CD200RKO was detrimental to CAR T-cell activity, adversely affecting CAR T-cell metabolism. These patterns held up in murine xenograft models of plasmacytoma, and disseminated bone marrow predominant disease. Our findings underscore the importance of CD200-mediated immune suppression in CAR T-cell therapy of MM, and highlight a promising approach to enhance such therapies by leveraging CD200 expression on aPCs to provide costimulation via a CD200R-CD28 switch.

Eribulin promotes proliferation of CD8+ T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells.

PURPOSE: Chemotherapeutic agents exert immunomodulatory effects on triple-negative breast cancer (TNBC) cells and immune cells. Eribulin favorably affects the immunological status of patients with breast cancer. However, the effects of eribulin on the immune cells remain unexplored. The aim of this study was to investigate the effects of eribulin on immune cells. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors and mouse splenocytes were stimulated with anti-CD3 and anti-CD28 antibodies. The effects of eribulin and paclitaxel on cell proliferation and differentiation status were analyzed using flow cytometry. RNA sequencing was performed to assess alterations in gene expression in CD8+ T cells following eribulin and paclitaxel treatment. Using TNBC cell lines (MDA-MB-231, Hs578T, and MDA-MB-157), the anti-tumor activity of CD3/CD28-stimulated T cells combined with eribulin or paclitaxel was evaluated. RESULTS: Eribulin did not affect CD3/CD28-stimulated PBMCs proliferation. However, eribulin significantly decreased the CD4/CD8 ratio in T cells, indicating that eribulin facilitates CD8+ T cell proliferation. Furthermore, eribulin significantly increased the frequency of less differentiated CD45RA+, CCR7+, and TCF1+ subsets of CD8+ T cells. RNA sequencing revealed that eribulin enhanced the expression of gene sets related to cell proliferation and immune responses. Moreover, eribulin augmented the anti-tumor effects of CD3/CD28-stimulated T cells against TNBC cells. These results were not observed in experiments using paclitaxel. CONCLUSIONS: Eribulin promoted CD8+ T cell proliferation, repressed effector T cell differentiation, and harnessed T cell-mediated anti-tumor effects. These mechanisms may be one of the cues that eribulin can improve the immunological status of tumor-bearing hosts.

Differential Runx3, Eomes, and T-bet expression subdivides MS-associated CD4+ T cells with brain-homing capacity.

Multiple sclerosis (MS) is a common and devastating chronic inflammatory disease of the CNS. CD4+ T cells are assumed to be the first to cross the blood-central nervous system (CNS) barrier and trigger local inflammation. Here, we explored how pathogenicity-associated effector programs define CD4+ T cell subsets with brain-homing ability in MS. Runx3- and Eomes-, but not T-bet-expressing CD4+ memory cells were diminished in the blood of MS patients. This decline reversed following natalizumab treatment and was supported by a Runx3+ Eomes+ T-bet- enrichment in cerebrospinal fluid samples of treatment-naïve MS patients. This transcription factor profile was associated with high granzyme K (GZMK) and CCR5 levels and was most prominent in Th17.1 cells (CCR6+ CXCR3+ CCR4-/dim ). Previously published CD28- CD4 T cells were characterized by a Runx3+ Eomes- T-bet+ phenotype that coincided with intermediate CCR5 and a higher granzyme B (GZMB) and perforin expression, indicating the presence of two separate subsets. Under steady-state conditions, granzyme Khigh Th17.1 cells spontaneously passed the blood-brain barrier in vitro. This was only found for other subsets including CD28- cells when using inflamed barriers. Altogether, CD4+ T cells contain small fractions with separate pathogenic features, of which Th17.1 seems to breach the blood-brain barrier as a possible early event in MS.

Full T-cell activation and function in teleosts require collaboration of first and co-stimulatory signals.

Mammalian T-cell responses require synergism between the first signal and co-stimulatory signal. However, whether and how dual signaling regulates the T-cell response in early vertebrates remains unknown. In the present study, we discovered that the Nile tilapia ( Oreochromis niloticus) encodes key components of the LAT signalosome, namely, LAT, ITK, GRB2, VAV1, SLP-76, GADS, and PLC-γ1. These components are evolutionarily conserved, and CD3ε mAb-induced T-cell activation markedly increased their expression. Additionally, at least ITK, GRB2, and VAV1 were found to interact with LAT for signalosome formation. Downstream of the first signal, the NF-κB, MAPK/ERK, and PI3K-AKT pathways were activated upon CD3ε mAb stimulation. Furthermore, treatment of lymphocytes with CD28 mAbs triggered the AKT-mTORC1 pathway downstream of the co-stimulatory signal. Combined CD3ε and CD28 mAb stimulation enhanced ERK1/2 and S6 phosphorylation and elevated NFAT1, c-Fos, IL-2, CD122, and CD44 expression, thereby signifying T-cell activation. Moreover, rather than relying on the first or co-stimulatory signal alone, both signals were required for T-cell proliferation. Full T-cell activation was accompanied by marked apoptosis and cytotoxic responses. These findings suggest that tilapia relies on dual signaling to maintain an optimal T-cell response, providing a novel perspective for understanding the evolution of the adaptive immune system.

CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo. METHODS: Four different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models. RESULTS: CD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice. CONCLUSIONS: CD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.

The Expression of LDL-R in CD8+ T Cells Serves as an Early Assessment Parameter for the Production of TCR-T Cells.

BACKGROUND/AIM: The quantity and the phenotypes of desired T cell receptor engineered T (TCR-T) cells in the final cell product determine their in vivo anti-tumor efficacy. Optimization of key steps in the TCR-T cell production process, such as T cell activation, has been shown to improve cell quality. MATERIALS AND METHODS: Using a modified TCR (mTCR) derived from mice transducing PBMCs, we assessed the proportions of low-density lipoprotein receptor (LDL-R) and mTCR expressing cells under the various activation conditions of CD3/CD28-Dynabeads or OKT3 via flow cytometry. RESULTS: We demonstrate that the proportion of T cells expressing LDL-R post activation is positively correlated with the percentage of mTCR+CD8+ T cells with their less differentiated subtypes in the final product. In addition, we show that shifting the CD3/CD28-Dynabeads activation duration from a typical 48 h to 24 h can significantly increase the production of the desired mTCR+CD8+ T cells. Importantly, the percentages of TCR-T cells with less-differentiated phenotypes, namely mTCR central memory T cells (TCM), were found to be preserved with markedly higher efficiency when T cell activation was optimized. CONCLUSION: Our findings suggest that the proportion of LDL-R+ T cells may serve as an early assessment parameter for evaluating TCR-T cell quality, possibly facilitating the functional and economical improvement of current adoptive therapy.

Expression of Tim-3/Galectin-9 pathway and CD8+T cells and related factors in patients with cystic echinococcosis.

OBJECTIVE: One of the primary reasons for the successful patriotization of Echinococcus multilocularis in patients is its ability to induce host immune tolerance. This study examined the expression of the immunosuppressive Tim-3/Galectin-9 pathway, CD8+T cells, and related factors in AE patients. The aim was to analyze the relationship between the Tim-3/Galectin-9 pathway and CD8+T cells in this disease and further understand the mechanism of immune tolerance induced by cystic echinococcosis. METHODS: Using flow cytometry, we evaluated the expression of CTL, CD8+CD28-T cells, CD8+CD28 + IFN-γ + T cells, CD8+CD28+perforin + T cells, CD8+CD28+granzyme B + T cells, CD8+CD28-IL-10 + T cells, CD8+CD28-TGF-ß+T cells, and Tim-3 expression on CD8+T cells in the peripheral blood of control (n = 30) and AE patients (n = 33). qRT-PCR was used to measure CD107a and Tim-3/Galectin-9 mRNA levels in PBMCs from the control and AE groups. Immunohistochemistry was employed to detect IL-10, TGF-ß, and Tim-3/Galectin-9 expressions in the infected livers of AE patients. RESULTS: AE patients exhibited a significant decrease in peripheral blood CTL ratio (P < 0.001) and an increase in CD8+CD28+IFN-γ+T cell ratio (P < 0.001). No significant changes were observed in the ratios of CD8+CD28+perforin + T cells (P = 0.720) and CD8+CD28+granzyme B + T cells (P = 0.051). The proportions of CD8+CD28-T cells (P < 0.001), CD8+CD28-IL-10 + T cells (P < 0.001), and CD8+CD28-TGF-ß+T cells (P < 0.001) were notably higher than in the control group. The expression of Tim-3 on CTL and CD8+CD28-T cells in AE patients was significantly upregulated (P < 0.001, P < 0.001). AE patients displayed a substantial decrease in peripheral blood PBMC CD107a mRNA levels (P < 0.001) and significant elevations in Tim-3/Galectin-9 mRNA levels (P < 0.001, P < 0.001). A negative correlation was observed between CD107a mRNA levels and both Tim-3 (r^2 = 0.411, P < 0.001) and Galectin-9 (r2 = 0.180, P = 0.019) mRNA levels. Expressions of IL-10 (P < 0.001), TGF-ß (P < 0.001), and Tim-3/Galectin-9 (P < 0.001, P < 0.001) in AE patient-infected livers were significantly higher than in uninfected regions. IL-10 and TGF-ß expressions showed a positive correlation with Tim-3/Galectin-9. CONCLUSION: This study suggests that the high expression of Tim-3 on CD8+T cell surfaces in AE patients might promote an increase in CD8+CD28-T cells and related factors, while suppressing CTL and related factor expressions. This potentially induces the onset of immune tolerance, which is unfavorable for the clearance of Echinococcus multilocularis in patients, leading to the exacerbation of persistent infections.

Coordinated Priming of NKG2D Pathway by IL-15 Enhanced Functional Properties of Cytotoxic CD4+CD28- T Cells Expanded in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder, and numerous aberrations of T cell responses have been reported and were implicated in its pathophysiology. Recently, CD4-positive T cells with cytotoxic potential were shown to be involved in autoimmune disease progression and tissue damage. However, the effector functions of this cell type and their potential molecular mechanisms in SLE patients remain to be elucidated. In this study, we find that cytotoxic CD4+CD28- T cells are expanded in SLE patients with flow cytometry analysis, and the percentage of CD4+CD28- T cells positively correlates with the Systemic Lupus International Collaborating Clinics/ACR Damage Index (SDI). Furthermore, our study suggests that interleukin-15 (IL-15) promotes the expansion, proliferation, and cytotoxic function of CD4+CD28- T cells in SLE patients through activation of the Janus kinase3-STAT5 pathway. Further study indicates that IL-15 not only mediates the upregulation of NKG2D, but also cooperates with the NKG2D pathway to regulate the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Together, our study demonstrated that proinflammatory and cytolytic CD4+CD28- T cells expand in SLE patients. The pathogenic potential of these CD4+CD28- T cells is driven by the coupling of the IL-15/IL-15R signaling pathway and the NKG2D/DAP10 signaling pathway, which may open new avenues for therapeutic intervention to prevent SLE progression.

Complexities in comparing the impact of costimulatory domains on approved CD19 CAR functionality.

Chimeric antigen receptors (CARs) are engineered to target T cells specifically to tumor cells, resulting in the engineered T cell killing the tumor cell. This technology has been developed to target a range of cancers, with the most notable successes in the treatment of B-cell malignancies where four approved therapies, all targeting CD19, are on the market. These four products differ in the costimulation domains, with axicabtagene ciloleucel (Yescarta) and brexucabtagene autoleucel (Tecartus) both utilizing the CD28 costimulation domain whilst tisagenlecleucel (Kymriah) and lisocabtagene maraleucel (Breyanzi) both utilizing the 4-1BB costimulation domain. There are clearly defined differences in how the CD28 and 4-1BB domains signal, yet it is difficult to ascertain which domain affords a superior mechanism of action given many other differences between these products, including overall CAR architecture and manufacturing methods. Additionally, while in vitro and preclinical in vivo studies have compared CARs with different costimulation domains, it remains a challenge to extrapolate differences observed in this biology across different experimental systems to the overall product performance. While there has been extensive preclinical and clinical work looking at CARs with a variety of targeting domains and architectures, this review will focus on the differences between the four marketed anti-CD19 CAR-Ts, with an additional focus on the impact of hinge and transmembrane domain on CAR activity and interaction with the target cell as well as other proteins on the surface of the T-cell.

The ß2-adrenergic receptor agonist terbutaline upregulates T helper-17 cells in a protein kinase A-dependent manner.

BACKGROUND: T helper 17 (Th17) cells produce IL-17A cytokine and can exacerbate autoimmune diseases and asthma. The ß2 adrenergic receptor is a g protein-coupled receptor that induces cAMP second messenger pathways. We tested the hypothesis that terbutaline, a ß2-adrenergic receptor-specific agonist, promotes IL-17 secretion by memory Th17 cells in a cAMP and PKA-dependent manner. METHODS: Venous peripheral blood mononuclear cells (PBMC) from healthy human participants were activated with anti-CD3 and anti-CD28 antibodies. Secreted IL-17A was measured by enzyme linked immunosorbent assay, intracellular IL-17A, and RORγ were measured using flow cytometry, and RORC by qPCR. Memory CD3+CD4+CD45RA-CD45RO+ T cells were obtained by immunomagnetic negative selection and activated with tri-antibody complex CD3/CD28/CD2. Secreted IL-17A, intracellular IL-17A, RORC were measured, and phosphorylated-serine133-CREB was measured by western blotting memory Th cells. RESULTS: Terbutaline increased IL-17A (p < 0.001), IL-17A+ cells (p < 0.05), and RORC in activated PBMC and memory Th cells. The PKA inhibitors H89 (p < 0.001) and Rp-cAMP (p < 0.01) abrogated the effects of terbutaline on IL-17A secretion in PBMC and memory T cells. Rolipram increased IL-17A (p < 0.01) to a similar extent as terbutaline. P-Ser133-CREB was increased by terbutaline (p < 0.05) in memory T cells. CONCLUSION: Terbutaline augments memory Th17 cells in lymphocytes from healthy participants. This could exacerbate autoimmune diseases or asthma, in cases where Th17 cells are considered to be pro-inflammatory.