Distinct biological characteristics of mesenchymal stem cells separated from different components of human placenta.

Mesenchymal stem cells (MSCs) have tremendous potential in cell therapy and regenerative medicine. The placenta-derived MSCs (PMSCs) are becoming favorable sources as they are ethically preferable and rich in MSCs. Although several subgroups of PMSCs have been identified from human term placenta, optimal sources for specific clinical applications remain to be elucidated. This study aimed to isolate MSCs from various components of the placenta, and compare their biological characteristics, including morphology, proliferation, immunophenotype, differentiation potential, growth factor and cytokine secretion, and immunomodulatory properties. Finally, four distinct groups of PMSCs were isolated from the placenta: amniotic membrane-derived MSCs (AM-MSCs), chorionic membrane-derived MSCs (CM-MSCs), chorionic plate-derived MSCs (CP-MSCs), and chorionic villi-derived MSCs (CV-MSCs). The results showed that CV-MSCs had good proliferation ability, and were easier to induce osteogenic and chondrogenic differentiation; CP-MSCs exhibited the strongest inhibitory effect on the proliferation of activated T cells, secreted high levels of EGF and IL-6, and could well differentiate into osteoblasts, adipocytes, and chondroblasts; AM-MSCs showed good growth dynamics in the early generations, were able to grow at high density, and tended to induce differentiation into osteogenic and neural lineages. These findings may provide novel evidence for the selection of seed cells in clinical application.

Preclinical immunogenicity risk assessment of biotherapeutics using CD4 T cell assays.

T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.

An In Vitro Human Skin Test for Predicting Skin Sensitization and Adverse Immune Reactions to Biologics.

Biologics, including monoclonal antibodies (mAb), have proved to be effective and successful therapeutic agents, particularly in the treatment of cancer and immune-inflammatory conditions, as well as allergies and infections. However, their use carries an inherent risk of an immune-mediated adverse drug reaction. In this study, we describe the use of a novel pre-clinical human in vitro skin explant test for predicting skin sensitization and adverse immune reactions. The skin explant test was used to investigate the effects of therapeutic antibodies, which are known to cause a limited reaction in a small number of patients or more severe reactions. MATERIAL AND METHODS: Immune responses were determined by T cell proliferation and multiplex cytokine analysis, as well as histopathological analysis of skin damage (grades I-IV in increasing severity), predicting a negative (grade I) or positive (grade ≥ II) response for an adverse skin sensitization effect. RESULTS: T cell proliferation responses were significantly increased in the positive group (p < 0.004). Multiplex cytokine analysis showed significantly increased levels of IFNγ, TNFα, IL-10, IL-12, IL-13, IL-1ß, and IL-4 in the positive response group compared with the negative response group (p < 0.0001, p < 0.0001, p < 0.002, p < 0.01, p < 0.04, p < 0.006, and p < 0.004, respectively). CONCLUSIONS: Overall, the skin explant test correctly predicted the clinical outcome of 13 out of 16 therapeutic monoclonal antibodies with a correlation coefficient of 0.770 (p = 0.0001). This assay therefore provides a valuable pre-clinical test for predicting adverse immune reactions, including T cell proliferation and cytokine release, both associated with skin sensitization to monoclonal antibodies.

Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG1 antibodies.

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

Discovery of a T cell proliferation-associated regulator signature correlates with prognosis risk and immunotherapy response in bladder cancer.

BACKGROUND: The efficacy of immunotherapy is heavily influenced by T cell activity. This study aimed to examine how T cell proliferation regulators can predict the prognosis and response to immunotherapy in patients with bladder cancer (BCa). METHODS: T cell proliferation-related subtypes were determined by employing the non-negative matrix factorization (NMF) algorithm that analyzed the expression patterns of T cell proliferation regulators. Subtypes were assessed for variations in prognosis, immune infiltration, and functional behaviors. Subsequently, a risk model related to T cell proliferation was created through Cox and Lasso regression analyses in the TCGA cohort and then confirmed in two GEO cohorts and an immunotherapy cohort. RESULTS: BCa patients were categorized into two subtypes (C1 and C2) according to the expression profiles of 31 T cell proliferation-related genes (TRGs) with distinct prognoses and immune landscapes. The C2 subtype had a shorter overall survival (OS), with higher levels of M2 macrophage infiltration, and the activation of cancer-related pathways than the C1 subtype. Following this, thirteen prognosis-related genes that were involved in T cell proliferation were utilized to create the prognostic signature. The model's predictive accuracy was confirmed by analyzing both internal and external datasets. Individuals in the high-risk category experienced a poorer prognosis, increased immunosuppressive factors in the tumor microenvironment, and diminished responses to immunotherapy. Additionally, the immunotherapeutic prediction efficacy of the model was further confirmed by an immunotherapy cohort (anti-PD-L1 in the IMvigor210 cohort). CONCLUSIONS: Our study characterized two subtypes linked to T cell proliferation in BCa patients with distinct prognoses and tumor microenvironment (TME) patterns, providing new insights into the heterogeneity of T cell proliferation in BCa and its connection to the immune landscape. The signature has prospective clinical implications for predicting outcomes and may help physicians to select prospective responders who prioritize current immunotherapy.

A Human Skin Explant Test as a Novel In Vitro Assay for the Detection of Skin Sensitization to Aggregated Monoclonal Antibodies.

Introduction: Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. Material and Methods: Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Results: Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II-III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. Conclusions: The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation.

Microfluidic-based preparation of artificial antigen-presenting gel droplets for integrated and minimalistic adoptive cell therapy strategies.

Adoptive T-cell transfer for cancer therapy is limited by the inefficiency ofin vitroT-cell expansion and the ability ofin vivoT-cells to infiltrate tumors. The construction of multifunctional artificial antigen-presenting cells is a promising but challenging approach to achieve this goal. In this study, a multifunctional artificial antigen-presenting gel droplet (AAPGD) was designed. Its surface provides regulated T-cell receptor (TCR) stimulation and co-stimulation signals and is capable of slow release of mitogenic cytokines and collagen mimetic peptide. The highly uniform AAPGD are generated by a facile method based on standard droplet microfluidic devices. The results of the study indicate that, T-cell proliferatedin vitroutilizing AAPGD have a fast rate and high activity. AAPGD increased the proportion ofin vitroproliferating T cells low differentiation and specificity. The starting number of AAPGDs and the quality ratio of TCR-stimulated and co-stimulated signals on the surface have a large impact on the rapid proliferation of low-differentiated T cellsin vitro. During reinfusion therapy, AAPGD also enhanced T-cell infiltration into the tumor site. In experiments using AAPGD for adoptive T cell therapy in melanoma mice, tumor growth was inhibited, eliciting a potent cytotoxic T-lymphocyte immune response and improving mouse survival. In conclusion, AAPGD promotes rapid low-differentiation proliferation of T cellsin vitroand enhances T cell infiltration of tumorsin vivo. It simplifies the preparation steps of adoptive cell therapy, improves the therapeutic effect, and provides a new pathway for overdosing T cells to treat solid tumors.

Immunophenotyping of canine T cell activation and proliferation by combined protein and RNA flow cytometry.

The limited availability of canine-reactive monoclonal antibodies restricts the analyses of immune cell subsets and their functions by flow cytometry. The PrimeFlow™ RNA Assay may serve as a potential solution to close this gap. Here we report a blood immunophenotyping method utilizing combined protein- and RNA-based flow cytometry to characterize canine T cell activation and proliferation within individual cells. In this assay, CD69 expression was detected by an RNA probe and CD25 and Ki67 were detected by antibodies. Canine peripheral blood mononuclear cells (PBMCs) were stimulated with three agents with different modes of action, anti-CD3/CD28 antibodies, phytohemagglutinin, or phorbol myristate acetate /ionomycin. Robust T cell activation (CD25+ and/or CD69+) and proliferation (Ki67+) were detected. Both CD69 and CD25 appear to be robust and sensitive T cell activation markers with early induction and low background expression. Upon stimulation, T cell proliferation occurred later than T cell activation and was associated with CD25 expression. This canine T cell activation and proliferation immunophenotyping method was evaluated in 5 independent experiments using PBMCs from 10 different beagle dogs with satisfactory assay performance. This method can greatly facilitate the evaluation of immune disease pathogenesis and immunotoxicity risk assessment in nonclinical drug development in canine.

Leucine-Rich Glioma-Inactivated 1 (LGI1) Protein Stimulates Proliferation and IL-10 Production in Peripheral Blood Mononuclear Cells of Patients with LGI1 Antibody-Mediated Autoimmune Encephalitis In Vitro.

Limbic encephalitis (LE) due to anti-leucine-rich glioma-inactivated 1 (LGI1) antibodies is an autoimmune disease characterized by distinct clinical features unique to LGI1 LE, such as faciobrachial dystonic seizures. However, it is unclear whether an additional disease-related LGI1 antigen-specific T cell response is involved in the pathogenesis of this disease. To address this question, we studied the effect of recombinant LGI1 on the proliferation and effector-specific cytokine production (IFN-γ, IL-5, IL-10, and IL-17) of peripheral blood mononuclear cells (PBMCs) from patients with LGI1 LE and healthy controls. We observed that recombinant LGI1 stimulated the proliferation of PBMCs from patients with LGI1 LE, but not from healthy controls. Cytokine measurement of cell culture supernatants from PBMCs incubated with recombinant LGI1 revealed a highly significant increase in IL-10 release in PBMCs from patients with LGI1 LE in comparison with healthy controls. These results suggest that LGI1-mediated stimulation of PBMCs from patients with LGI1 LE leads to the establishment of an IL-10-dominated immunosuppressive cytokine milieu, which may inhibit Th1 differentiation and support B cell proliferation, IgG production, and IgG subclass switching.

Accumulation of circulating myeloid-derived suppressor cell subsets: predicting poor clinical efficacy and prognosis through T cell suppression in non-Hodgkin's lymphoma.

Myeloid-derived suppressor cells (MDSCs) are implicated in the regulation of immune responses closely associated with poor clinical outcomes in cancer. However, the MDSC subtypes in non-Hodgkin's lymphoma (NHL) have not been systematically investigated. So, we investigated the percentage of MDSC subsets in 78 newly diagnosed NHL patients by flow cytometry. The results showed that all MDSC subsets increased in NHL patients compared with healthy donors. Notably, MDSCs, monocytic MDSCs, and CD14 + CD66b + MDSCs significantly increased in NHL patients compared with those with lymphadenitis donors. polymorphonuclear MDSCs (PMN-MDSCs), early-stage MDSCs (e-MDSCs), and the International Prognostic Index were independent risk factors for poor clinical efficacy and were involved in constructing the nomogram for predicting clinical efficacy. Progression-free survival (PFS) was significantly shorter in patients with high level of MDSC subsets, and PMN-MDSCs emerged as an independent prognostic factor for PFS. PMN-MDSCs, e-MDSCs, and the International Prognostic Index were involved in constructing the nomogram for predicting PFS. Patients with a higher percentage of MDSCs, PMN-MDSCs, e-MDSCs, and CD14 + CD66b + MDSCs experienced a shorter overall survival compared with those with lower percentages. In addition, research on mechanisms found that T cell function was suppressed and mediated by the expansion of MDSCs via involving arginase-1 and interleukin-10 in vitro and in vivo. In conclusion, our study demonstrates that the increased circulating MDSC subsets predict poor clinical efficacy and prognosis in NHL, potentially involving T cell suppression through MDSC subset expansion. These findings indicate the potential of MDSC subsets as comprehensive diagnostic, prognostic biomarkers, and therapeutic targets for NHL.

Characterizing Foxp3+ and Foxp3- T cells in the homeostatic state and after allo-activation: resting CD4+Foxp3+ Tregs have molecular characteristics of activated T cells.

Due to the intracellular expression of Foxp3 it is impossible to purify viable Foxp3+ cells on the basis of Foxp3 staining. Consequently CD4+Foxp3+ regulatory T cells (Tregs) in mice have mostly been characterized using CD4+CD25+ T cells or GFP-Foxp3 reporter T cells. However, these two populations cannot faithfully represent Tregs as the expression of CD25 and Foxp3 does not completely overlap and GFP+Foxp3+ reporter T cells have been reported to be functionally altered. The aim of this study was to characterize normal Tregs without separating Foxp3+ and Foxp3- cells for the expression of the main functional molecules and proliferation behaviors by flow cytometry and to examine their gene expression characteristics through differential gene expression. Our data showed that the expressions of Foxp3, CD25, CTLA-4 (both intracellular and cell surface) and PD-1 was mostly confined to CD4+ T cells and the expression of Foxp3 did not completely overlap with the expression of CD25, CTLA-4 or PD-1. Despite higher levels of expression of the T cell inhibitory molecules CTLA-4 and PD-1, Tregs maintained higher levels of Ki-67 expression in the homeostatic state and had greater proliferation in vivo after allo-activation than Tconv. Differential gene expression analysis revealed that resting Tregs exhibited immune activation markers characteristic of activated Tconv. This is consistent with the flow data that the T cell activation markers CD25, CTLA-4, PD-1, and Ki-67 were much more strongly expressed by Tregs than Tconv in the homeostatic state.

Identification of novel T cell proliferation patterns, potential biomarkers and therapeutic drugs in colorectal cancer.

Background: T cells are crucial components of antitumor immunity. A list of genes associated with T cell proliferation was recently identified; however, the impact of T cell proliferation-related genes (TRGs) on the prognosis and therapeutic responses of patients with colorectal cancer (CRC) remains unclear. Methods: 33 TRG expression information and clinical information of patients with CRC gathered from multiple datasets were subjected to bioinformatic analysis. Consensus clustering was used to determine the molecular subtypes associated with T cell proliferation. Utilizing the Lasso-Cox regression, a predictive signature was created and verified in external cohorts. A tumor immune environment analysis was conducted, and potential biomarkers and therapeutic drugs were identified and confirmed via in vitro and in vivo studies. Results: CRC patients were separated into two TRG clusters, and differentially expressed genes (DEGs) were identified. Patient information was divided into three different gene clusters, and the determined molecular subtypes were linked to patient survival, immune cells, and immune functions. Prognosis-associated DEGs in the three gene clusters were used to evaluate the risk score, and a predictive signature was developed. The ability of the risk score to predict patient survival and treatment response has been successfully validated using multiple datasets. To discover more possible biomarkers for CRC, the weighted gene co-expression network analysis algorithm was utilized to screen key TRG variations between groups with high- and low-risk. CDK1, BATF, IL1RN, and ITM2A were screened out as key TRGs, and the expression of key TRGs was confirmed using real-time reverse transcription polymerase chain reaction. According to the key TRGs, 7,8-benzoflavone was identified as the most significant drug molecule, and MTT, colony formation, wound healing, transwell assays, and in vivo experiments indicated that 7,8-benzoflavone significantly suppressed the proliferation and migration of CRC cells. Conclusion: T cell proliferation-based molecular subtypes and predictive signatures can be utilized to anticipate patient results, immunological landscape, and treatment response in CRC. Novel biomarker candidates and potential therapeutic drugs for CRC were identified and verified using in vitro and in vivo tests.

Homeodomain-only protein suppresses proliferation and contributes to differentiation- and age-related reduced CD8+ T cell expansion.

T cell activation is a tightly controlled process involving both positive and negative regulators. The precise mechanisms governing the negative regulators in T cell proliferation remain incompletely understood. Here, we report that homeodomain-only protein (HOPX), a homeodomain-containing protein, and its most abundant isoform HOPXb, negatively regulate activation-induced proliferation of human T cells. We found that HOPX expression progressively increased from naïve (TN) to central memory (TCM) to effector memory (TEM) cells, with a notable upregulation following in vitro stimulation. Overexpression of HOPXb leads to a reduction in TN cell proliferation while HOPX knockdown promotes proliferation of TN and TEM cells. Furthermore, we demonstrated that HOPX binds to promoters and exerts repressive effects on the expression of MYC and NR4A1, two positive regulators known to promote T cell proliferation. Importantly, our findings suggest aging is associated with increased HOPX expression, and that knockdown of HOPX enhances the proliferation of CD8+ T cells in older adults. Our findings provide compelling evidence that HOPX serves as a negative regulator of T cell activation and plays a pivotal role in T cell differentiation and in age-related-reduction in T cell proliferation.

Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia.

Severe combined immunodeficiency (SCID) is characterized by a severe deficiency in T cell numbers. We analyzed data collected (n = 307) for PHA-based T cell proliferation from the PIDTC SCID protocol 6901, using either a radioactive or flow cytometry method. In comparing the two groups, a smaller number of the patients tested by flow cytometry had <10% of the lower limit of normal proliferation as compared to the radioactive method (p = 0.02). Further, in patients with CD3+ T cell counts between 51 and 300 cells/µL, there was a higher proliferative response with the PHA flow assay compared to the 3H-T assay (p < 0.0001), suggesting that the method of analysis influences the resolution and interpretation of PHA results. Importantly, we observed many SCID patients with profound T cell lymphopenia having normal T cell proliferation when assessed by flow cytometry. We recommend this test be considered only as supportive in the diagnosis of typical SCID.

Survival and division fate programs are preserved but retuned during the naïve to memory CD8+ T-cell transition.

Memory T cells are generated from naïve precursors undergoing proliferation during the initial immune response. Both naïve and memory T cells are maintained in a resting, quiescent state and respond to activation with a controlled proliferative burst and differentiation into effector cells. This similarity in the maintenance and response dynamics points to the preservation of key cellular fate programs; however, whether memory T cells have acquired intrinsic changes in these programs that may contribute to the enhanced immune protection in a recall response is not fully understood. Here we used a quantitative model-based analysis of proliferation and survival kinetics of in vitro-stimulated murine naïve and memory CD8+ T cells in response to homeostatic and activating signals to establish intrinsic similarities or differences within these cell types. We show that resting memory T cells display heightened sensitivity to homeostatic cytokines, responding to interleukin (IL)-2 in addition to IL-7 and IL-15. The proliferative response to αCD3 was equal in size and kinetics, demonstrating that memory T cells undergo the same controlled division burst and automated return to quiescence as naïve T cells. However, perhaps surprisingly, we observed reduced expansion of αCD3-stimulated memory T cells in response to activating signals αCD28 and IL-2 compared with naïve T cells. Overall, we demonstrate that although sensitivities to cytokine and costimulatory signals have shifted, fate programs regulating the scale of the division burst are conserved in memory T cells.

11-deoxycortisol positively correlates with T cell immune traits in physiological conditions.

BACKGROUND: Endogenous steroid hormones have significant effects on inflammatory and immune processes, but the immunological activities of steroidogenesis precursors remain largely unexplored. METHODS: We conducted a systematic approach to examine the association between steroid hormones profile and immune traits in a cohort of 534 healthy volunteers. Serum concentrations of steroid hormones and their precursors (cortisol, progesterone, testosterone, androstenedione, 11-deoxycortisol and 17-OH progesterone) were determined by liquid chromatography-tandem mass spectrometry. Immune traits were evaluated by quantifying cellular composition of the circulating immune system and ex vivo cytokine responses elicited by major human pathogens and microbial ligands. An independent cohort of 321 individuals was used for validation, followed by in vitro validation experiments. FINDINGS: We observed a positive association between 11-deoxycortisol and lymphoid cellular subsets numbers and function (especially IL-17 response). The association with lymphoid cellularity was validated in an independent validation cohort. In vitro experiments showed that, as compared to androstenedione and 17-OH progesterone, 11-deoxycortisol promoted T cell proliferation and Candida-induced Th17 polarization at physiologically relevant concentrations. Functionally, 11-deoxycortisol-treated T cells displayed a more activated phenotype (PD-L1high CD25high CD62Llow CD127low) in response to CD3/CD28 co-stimulation, and downregulated expression of T-bet nuclear transcription factor. INTERPRETATION: Our findings suggest a positive association between 11-deoxycortisol and T-cell function under physiological conditions. Further investigation is needed to explore the potential mechanisms and clinical implications. FUNDING: Found in acknowledgements.

Nanoparticles (NPs)-mediated Siglec15 silencing and macrophage repolarization for enhanced cancer immunotherapy.

T cell infiltration and proliferation in tumor tissues are the main factors that significantly affect the therapeutic outcomes of cancer immunotherapy. Emerging evidence has shown that interferon-gamma (IFNγ) could enhance CXCL9 secretion from macrophages to recruit T cells, but Siglec15 expressed on TAMs can attenuate T cell proliferation. Therefore, targeted regulation of macrophage function could be a promising strategy to enhance cancer immunotherapy via concurrently promoting the infiltration and proliferation of T cells in tumor tissues. We herein developed reduction-responsive nanoparticles (NPs) made with poly (disulfide amide) (PDSA) and lipid-poly (ethylene glycol) (lipid-PEG) for systemic delivery of Siglec15 siRNA (siSiglec15) and IFNγ for enhanced cancer immunotherapy. After intravenous administration, these cargo-loaded could highly accumulate in the tumor tissues and be efficiently internalized by tumor-associated macrophages (TAMs). With the highly concentrated glutathione (GSH) in the cytoplasm to destroy the nanostructure, the loaded IFNγ and siSiglec15 could be rapidly released, which could respectively repolarize macrophage phenotype to enhance CXCL9 secretion for T cell infiltration and silence Siglec15 expression to promote T cell proliferation, leading to significant inhibition of hepatocellular carcinoma (HCC) growth when combining with the immune checkpoint inhibitor. The strategy developed herein could be used as an effective tool to enhance cancer immunotherapy.

Osteogenic Differentiation of Human Periodontal Ligament Stromal Cells Influences Their Immunosuppressive Potential toward Allogenic CD4+ T Cells.

The differentiation ability of human periodontal ligament mesenchymal stromal cells (hPDL-MSCs) in vivo is limited; therefore, some studies considered strategies involving their pre-differentiation in vitro. However, it is not known how the differentiation of hPDL-MSCs influences their immunomodulatory properties. This study investigated how osteogenic differentiation of hPDL-MSCs affects their ability to suppress CD4+ T-lymphocyte proliferation. hPDL-MSCs were cultured for 21 days in osteogenic differentiation or standard culture media. Allogeneic CD4+ T lymphocytes were co-cultured with undifferentiated and differentiated cells in the presence or absence of interferon (IFN)-γ, interleukin (IL)-1ß or tumor necrosis factor (TNF)-α, and their proliferation and apoptosis were measured. Additionally, the effects of these cytokines on the expression of immunomodulatory or pro-inflammatory factors were investigated. Our data show that osteogenic differentiation of hPDL-MSCs reduced their ability to suppress the proliferation of CD4+ T lymphocytes in the presence of IFN-γ and enhanced this ability in the presence of IL-1ß. These changes were accompanied by a slightly decreased proportion of apoptotic CD4+ in the presence of IFN-γ. The osteogenic differentiation was accompanied by decreases and increases in the activity of indoleamine-2,3-dioxygenase in the presence of IFN-γ and IL-1ß, respectively. The basal production of interleukin-8 by hPDL-MSCs was substantially increased upon osteogenic differentiation. In conclusion, this study suggests that pre-differentiation strategies in vitro may impact the immunomodulatory properties of hPDL-MSCs and subsequently affect their therapeutic effectiveness in vivo. These findings provide important insights for the development of MSC-based therapies.

Review of T cell proliferation regulatory factors in treatment and prognostic prediction for solid tumors.

T cell proliferation regulators (Tcprs), which are positive regulators that promote T cell function, have made great contributions to the development of therapies to improve T cell function. CAR (chimeric antigen receptor) -T cell therapy, a type of adoptive cell transfer therapy that targets tumor cells and enhances immune lethality, has led to significant progress in the treatment of hematologic tumors. However, the applications of CAR-T in solid tumor treatment remain limited. Therefore, in this review, we focus on the development of Tcprs for solid tumor therapy and prognostic prediction. We summarize potential strategies for targeting different Tcprs to enhance T cell proliferation and activation and inhibition of cancer progression, thereby improving the antitumor activity and persistence of CAR-T. In summary, we propose means of enhancing CAR-T cells by expressing different Tcprs, which may lead to the development of a new generation of cell therapies.

Mind the GAP: RASA2 and RASA3 GTPase-activating proteins as gatekeepers of T cell activation and adhesion.

Following stimulation, the T cell receptor (TCR) and its coreceptors integrate multiple intracellular signals to initiate T cell proliferation, migration, gene expression, and metabolism. Among these signaling molecules are the small GTPases RAS and RAP1, which induce MAPK pathways and cellular adhesion to activate downstream effector functions. Although many studies have helped to elucidate the signaling intermediates that mediate T cell activation, the molecules and pathways that keep naive T cells in check are less understood. Several recent studies provide evidence that RASA2 and RASA3, which are GAP1-family GTPase-activating proteins (GAPs) that inactivate RAS and RAP1, respectively, are crucial molecules that limit T cell activation and adhesion. In this review we describe recent data on the roles of RASA2 and RASA3 as gatekeepers of T cell activation and migration.