Unconventional T Cells Influence Clinical Outcome After Allogeneic Hematopoietic Cell Transplantation.

We evaluated the impact of early recovery of mucosal-associated invariant T cells (MAIT) and gamma-delta (γδ) T cells, especially Vδ2+ T cells, on the clinical outcomes of 76 patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT). MAIT cells were identified at day 20-30 post-transplant using flow cytometry and defined as CD3+ TCRVα7.2+CD161+. Two subsets of Vδ2+ T cells were analyzed according to the expression of CD26. The cytotoxicity profile of MAIT and Vδ2+ T cells was analyzed according to the intracellular expression of perforin and granzyme B, and intracellular IFN-γ was evaluated after in vitro activation. CD26+Vδ2+ T cells displayed higher intracellular levels of IFN-γ, whereas CD26- Vδ2+ T were found to be more cytotoxic. Moreover, MAIT cell frequency was correlated with the frequency of Vδ2+ T cells with a better correlation observed with Vδ2+CD26+ than with the Vδ2+CD26- T cell subset. By using the composite endpoint graft-versus-host disease (GvHD)-free, relapse-free survival (GRFS) as the primary endpoint, we found that patients with a higher MAIT cell frequency at day 20-30 after allo-HCT had a significantly increased GRFS and a better overall survival (OS) and disease-free survival (DFS). Moreover, patients with a low CD69 expression by MAIT cells had an increased cumulative incidence of grade 2-4 acute GvHD (aGvHD). These results suggest that MAIT cell reconstitution may provide mitigating effects early after allo-HCT depending on their activation markers and functional status. Patients with a high frequency of Vδ2+CD26+ T cells had a significantly higher GRFS, OS and DFS, but there was no impact on cumulative incidence of grade 2-4 aGVHD, non-relapse mortality and relapse. These results revealed that the impact of Vδ2+ T cells on the success of allo-HCT may vary according to the frequency of the CD26+ subset.

An innovative single-cell approach for phenotyping and functional genotyping of CAR NK cells.

BACKGROUND: To accelerate the translation of novel immunotherapeutic treatment approaches, the development of analytic methods to assess their efficacy at early in vitro stages is necessary. Using a droplet-based microfluidic platform, we have established a method for multiparameter quantifiable phenotypic and genomic observations of immunotherapies. Chimeric antigen receptor (CAR) natural killer (NK) cells are of increased interest in the current immunotherapy landscape and thus provide an optimal model for evaluating our novel methodology. METHODS: For this approach, NK cells transduced with a CD19 CAR were compared with non-transduced NK cells in their ability to kill a lymphoma cell line. Using our microfluidic platform, we were able to quantify the increase in cytotoxicity and synaptic contact formation of CAR NK cells over non-transduced NK cells. We then optimized our droplet sorter and successfully used it to separate NK cells based on target cell killing to perform transcriptomic analyses. RESULTS: Our data revealed expected improvement in cytotoxicity with the CD19 CAR but more importantly, provided unique insights into the factors involved in the cytotoxic mechanisms of CAR NK cells. This demonstrates a novel, improved system for accelerating the pre-clinical screening of future immunotherapy treatments. CONCLUSIONS: This study provides a new potential approach for enhanced early screening of immunotherapies to improve their development, with a highly relevant cell model to demonstrate. Additionally, our validation studies provided some potential insights into transcriptomic determinants influencing CAR NK cytotoxicity.

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice.

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Background: Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. Methods: We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. Results: We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. Conclusions: NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

Cell-mediated cytotoxicity within CSF and brain parenchyma in spinal muscular atrophy unaltered by nusinersen treatment.

5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity's role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.

Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma.

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αß T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, having innate antitumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allocompatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Rα-IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared with unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together, the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control.

Interleukin-2 is required for NKp30-dependent NK cell cytotoxicity by preferentially regulating NKp30 expression.

Natural killer (NK) cells are key effectors in cancer immunosurveillance, eliminating a broad spectrum of cancer cells without major histocompatibility complex (MHC) specificity and graft-versus-host diseases (GvHD) risk. The use of allogeneic NK cell therapies from healthy donors has demonstrated favorable clinical efficacies in treating diverse cancers, particularly hematologic malignancies, but it requires cytokines such as IL-2 to primarily support NK cell persistence and expansion. However, the role of IL-2 in the regulation of activating receptors and the function of NK cells expanded for clinical trials is poorly understood and needs clarification for the full engagement of NK cells in cancer immunotherapy. Here, we demonstrated that IL-2 deprivation significantly impaired the cytotoxicity of primary expanded NK cells by preferentially downregulating NKp30 but not NKp46 despite their common adaptor requirement for expression and function. Using NK92 and IL-2-producing NK92MI cells, we observed that NKp30-mediated cytotoxicity against myeloid leukemia cells such as K562 and THP-1 cells expressing B7-H6, a ligand for NKp30, was severely impaired by IL-2 deprivation. Furthermore, IL-2 deficiency-mediated NK cell dysfunction was overcome by the ectopic overexpression of an immunostimulatory NKp30 isoform such as NKp30a or NKp30b. In particular, NKp30a overexpression in NK92 cells improved the clearance of THP-1 cells in vivo without IL-2 supplementation. Collectively, our results highlight the distinct role of IL-2 in the regulation of NKp30 compared to that of NKp46 and suggest NKp30 upregulation, as shown here by ectopic overexpression, as a viable modality to harness NK cells in cancer immunotherapy, possibly in combination with IL-2 immunocytokines.

AML/T cell interactomics uncover correlates of patient outcomes and the key role of ICAM1 in T cell killing of AML.

T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human-engineered cytotoxic CD4+ T cells. Single-cell RNA-seq of primary AML samples and CD4+ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4+ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro. Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1, a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing by primary ex vivo-isolated CD8+ T cells in vitro, and engineered CD4+ T cells in vitro and in vivo. While AML heterogeneity implies that multiple factors may determine their sensitivity to T cell killing, these data show that ICAM1 acts as an immune trigger, allowing T cell killing, and could play a role in AML patient survival in vivo.

Pre-existing cell populations with cytotoxic activity against SARS-CoV-2 in people with HIV and normal CD4/CD8 ratio previously unexposed to the virus.

Introduction: HIV-1 infection may produce a detrimental effect on the immune response. Early start of antiretroviral therapy (ART) is recommended to preserve the integrity of the immune system. In fact, people with HIV (PWH) and normal CD4/CD8 ratio appear not to be more susceptible to severe forms of COVID-19 than the general population and they usually present a good seroconversion rate in response to vaccination against SARS-CoV-2. However, few studies have fully characterized the development of cytotoxic immune populations in response to COVID-19 vaccination in these individuals. Methods: In this study, we recruited PWH with median time of HIV-1 infection of 6 years, median CD4/CD8 ratio of 1.0, good adherence to ART, persistently undetectable viral load, and negative serology against SARS-CoV-2, who then received the complete vaccination schedule against COVID-19. Blood samples were taken before vaccination against COVID-19 and one month after receiving the complete vaccination schedule. Results: PWH produced high levels of IgG against SARS-CoV-2 in response to vaccination that were comparable to healthy donors, with a significantly higher neutralization capacity. Interestingly, the cytotoxic activity of PBMCs from PWH against SARS-CoV-2-infected cells was higher than healthy donors before receiving the vaccination schedule, pointing out the pre-existence of activated cell populations with likely unspecific antiviral activity. The characterization of these cytotoxic cell populations revealed high levels of Tgd cells with degranulation capacity against SARS-CoV-2-infected cells. In response to vaccination, the degranulation capacity of CD8+ T cells also increased in PWH but not in healthy donors. Discussion: The full vaccination schedule against COVID-19 did not modify the ability to respond against HIV-1-infected cells in PWH and these individuals did not show more susceptibility to breakthrough infection with SARS-CoV-2 than healthy donors after 12 months of follow-up. These results revealed the development of protective cell populations with broad-spectrum antiviral activity in PWH with normal CD4/CD8 ratio and confirmed the importance of early ART and treatment adherence to avoid immune dysfunctions.

Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

Self-sufficient primary natural killer cells engineered to express T cell receptors and interleukin-15 exhibit improved effector function and persistence.

Background: NK cells can be genetically engineered to express a transgenic T-cell receptor (TCR). This approach offers an alternative strategy to target heterogenous tumors, as NK:TCR cells can eradicate both tumor cells with high expression of HLA class I and antigen of interest or HLA class I negative tumors. Expansion and survival of NK cells relies on the presence of IL-15. Therefore, autonomous production of IL-15 by NK:TCR cells might improve functional persistence of NK cells. Here we present an optimized NK:TCR product harnessed with a construct encoding for soluble IL-15 (NK:TCR/IL-15), to support their proliferation, persistence and cytotoxic capabilities. Methods: Expression of tumor-specific TCRs in peripheral blood derived NK-cells was achieved following retroviral transduction. NK:TCR/IL-15 cells were compared with NK:TCR cells for autonomous cytokine production, proliferation and survival. NK:BOB1-TCR/IL-15 cells, expressing a HLA-B*07:02-restricted TCR against BOB1, a B-cell lineage specific transcription factor highly expressed in all B-cell malignancies, were compared with control NK:BOB1-TCR and NK:CMV-TCR/IL-15 cells for effector function against TCR antigen positive malignant B-cell lines in vitro and in vivo. Results: Viral incorporation of the interleukin-15 gene into engineered NK:TCR cells was feasible and high expression of the TCR was maintained, resulting in pure NK:TCR/IL-15 cell products generated from peripheral blood of multiple donors. Self-sufficient secretion of IL-15 by NK:TCR cells enables engineered NK cells to proliferate in vitro without addition of extra cytokines. NK:TCR/IL-15 demonstrated a marked enhancement of TCR-mediated cytotoxicity as well as enhanced NK-mediated cytotoxicity resulting in improved persistence and performance of NK:BOB1-TCR/IL-15 cells in an orthotopic multiple myeloma mouse model. However, in contrast to prolonged anti-tumor reactivity by NK:BOB1-TCR/IL-15, we observed in one of the experiments an accumulation of NK:BOB1-TCR/IL-15 cells in several organs of treated mice, leading to unexpected death 30 days post-NK infusion. Conclusion: This study showed that NK:TCR/IL-15 cells secrete low levels of IL-15 and can proliferate in an environment lacking cytokines. Repeated in vitro and in vivo experiments confirmed the effectiveness and target specificity of our product, in which addition of IL-15 supports TCR- and NK-mediated cytotoxicity.

Transient TCR-based T cell therapy in a patient with advanced treatment-resistant MSI-high colorectal cancer.

We previously demonstrated the antitumor effectiveness of transiently T cell receptor (TCR)-redirected T cells recognizing a frameshift mutation in transforming growth factor beta receptor 2. We here describe a clinical protocol using mRNA TCR-modified T cells to treat a patient with progressive, treatment-resistant metastatic microsatellite instability-high (MSI-H) colorectal cancer. Following 12 escalating doses of autologous T cells electroporated with in-vitro-transcribed Radium-1 TCR mRNA, we assessed T cell cytotoxicity, phenotype, and cytokine production. Tumor markers and growth on computed tomography scans were evaluated and immune cell tumor infiltrate at diagnosis assessed. At diagnosis, tumor-infiltrating CD8+ T cells had minimal expression of exhaustion markers, except for PD-1. Injected Radium-1 T cells were mainly naive and effector memory T cells with low expression of exhaustion markers, except for TIGIT. We confirmed cytotoxicity of transfected Radium-1 T cells against target cells and found key cytokines involved in tumor metastasis, growth, and angiogenesis to fluctuate during treatment. The treatment was well tolerated, and despite his advanced cancer, the patient obtained a stable disease with 6 months survival post-treatment. We conclude that treatment of metastatic MSI-H colorectal cancer with autologous T cells electroporated with Radium-1 TCR mRNA is feasible, safe, and well tolerated and that it warrants further investigation in a phase 1/2 study.

Magnetic CAR T cell purification using an anti-G4S linker antibody.

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.

Enhanced cytotoxic activity of natural killer cells from increased calcium influx induced by electrical stimulation.

Natural killer (NK) cells play a crucial role in immunosurveillance independent of antigen presentation, which is regulated by signal balance via activating and inhibitory receptors. The anti-tumor activity of NK cells is largely dependent on signaling from target recognition to cytolytic degranulation; however, the underlying mechanism remains unclear, and NK cell cytotoxicity is readily impaired by tumor cells. Understanding the activation mechanism is necessary to overcome the immune evasion mechanism, which remains an obstacle in immunotherapy. Because calcium ions are important activators of NK cells, we hypothesized that electrical stimulation could induce changes in intracellular Ca2+ levels, thereby improving the functional potential of NK cells. In this study, we designed an electrical stimulation system and observed a correlation between elevated Ca2+ flux induced by electrical stimulation and NK cell activation. Breast cancer MCF-7 cells co-cultured with electrically stimulated KHYG-1 cells showed a 1.27-fold (0.5 V/cm) and 1.55-fold (1.0 V/cm) higher cytotoxicity, respectively. Electrically stimulated KHYG-1 cells exhibited a minor increase in Ca2+ level (1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) higher), which also led to increased gene expression of granzyme B (GZMB) by 1.36-fold (0.5 V/cm) and 1.58-fold (1.0 V/cm) by activating Ca2+-dependent nuclear factor of activated T cell 1 (NFAT1). In addition, chelating Ca2+ influx with 5 µM BAPTA-AM suppressed the gene expression of Ca2+ signaling and lytic granule (granzyme B) proteins by neutralizing the effects of electrical stimulation. This study suggests a promising immunotherapeutic approach without genetic modifications and elucidates the correlation between cytolytic effector function and intracellular Ca2+ levels in electrically stimulated NK cells.

Dual T cell receptor/chimeric antigen receptor engineered NK-92 cells targeting the HPV16 E6 oncoprotein and the tumor-associated antigen L1CAM exhibit enhanced cytotoxicity and specificity against tumor cells.

The human papillomavirus type 16 (HPV16) causes a large fraction of genital and oropharyngeal carcinomas. To maintain the transformed state, the tumor cells must continuously synthesize the E6 and E7 viral oncoproteins, which makes them tumor-specific antigens. Indeed, specific T cell responses against them have been well documented and CD8+ T cells engineered to express T cell receptors (TCRs) that recognize epitopes of E6 or E7 have been tested in clinical studies with promising results, yet with limited clinical success. Using CD8+ T cells from peripheral blood of healthy donors, we have identified two novel TCRs reactive to an unexplored E618-26 epitope. These TCRs showed limited standalone cytotoxicity against E618-26-HLA-A*02:01-presenting tumor cells. However, a single-signaling domain chimeric antigen receptor (ssdCAR) targeting L1CAM, a cell adhesion protein frequently overexpressed in HPV16-induced cancer, prompted a synergistic effect that significantly enhanced the cytotoxic capacity of NK-92/CD3/CD8 cells armored with both TCR and ssdCAR when both receptors simultaneously engaged their respective targets, as shown by live microscopy of 2-D and 3-D co-cultures. Thus, virus-specific TCRs from the CD8+ T cell repertoire of healthy donors can be combined with a suitable ssdCAR to enhance the cytotoxic capacity of the effector cells and, indirectly, their specificity.

Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in tuberculosis patients.

BACKGROUND: Human T cells play an important role in immunity against tuberculosis (TB) infection. Activating receptor HLA-DR and inhibitory receptor KLRG1 are critical regulators of T cell function during viral infection and tumorigenesis, but they have been less studied in TB infection. METHODS: In this study, we explored the relationship between CD3+ T cell expression of HLA-DR and KLRG1 receptors and function against TB infection. Flow cytometry was conducted to assess the immunomodulatory effects of HLA-DR and KLRG1 receptors on CD3+ T cells in patients with different TB infection status. RESULTS: We found activating receptors HLA-DR, NKG2C, CD57 and NKP46, and inhibitory receptors KLRG1 and KIR on CD3+ T cells in different TB infection status showed different distribution patterns; the cytotoxic potential and cytokine secretion capacity of CD3+ T cells after Mtb-specific antigen stimulation were significantly enhanced in TB infection groups. Further studies revealed HLA-DR+ T and KLRG1+ T cells expressed higher activating and inhibitory receptors than the negative population. In addition, the expression of cytotoxic potential and cytokine secretion capacity of HLA-DR+ T and KLRG1+ T cells was significantly higher than that of HLA-DR- T and KLRG1- T cells. CONCLUSIONS: Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3+ T cells in TB patients, suggesting CD3+ T cells expressing HLA-DR and KLRG1 are important effector cell phenotypes involved in the host anti-TB infection. HLA-DR and KLRG1 expressed by CD3+ T cells may be potential predictive markers of TB disease progression and clinical immune assessment.

The quantity, function and anti-tumor effect of Mucosal associated invariant T cells in patients with bladder cancer.

BACKGROUND: Bladder cancer (BC), a prevalent malignancy in the urinary system, often poses challenges for effective treatment. Immunotherapy, harnessing the immune system, has exhibited promise in early-stage clinical trials. Mucosal associated invariant T (MAIT) cells, a subset of immune cells implicated in various diseases, including certain cancer, have yet to be explored in BC patients. We aimed to investigate the quantity, function, and anti-tumor effects of MAIT cells in BC patients. METHODS: A total of 75 newly diagnosed BC patients and 183 healthy volunteers were included. Blood samples were collected and analyzed to evaluate the quantity and function of MAIT cells. Surgical resection provided BC tissues for further analysis, and the clinical features of BC tumors were collected and their relationship with MAIT cells was explored. RESULTS: MAIT cells were identified in both healthy individuals and BC patients. The proportion of MAIT cells in the peripheral blood of BC patients did not significantly differ from that of healthy controls. However, the study revealed a correlation between the proportion of IFN-γ producing MAIT cells and tumor number and invasion in BC patients. Furthermore, MAIT cells exhibited cytotoxic effects on BC cells in vitro and in vivo. CONCLUSIONS: This study sheds light on the role of MAIT cells in BC. While the quantity of MAIT cells showed no significant change in BC patients, their functional attributes and association with tumor characteristics suggest their potential as an immunotherapy target in BC treatment.

The inhibitory effects of live and UV-killed Akkermansia muciniphila and its derivatives on cytotoxicity and inflammatory response induced by Clostridioides difficile RT001 in vitro

Clostridioides difficile infection (CDI) is the leading cause of healthcare-acquired infections worldwide. Probiotics are widely recommended to prevent CDI and its recurrences. Akkermansia muciniphila, as a therapeutic symbiont colonizing the intestinal mucosal layer, is considered to be a promising next-generation probiotic. In this work, we assessed the inhibitory effects of A. muciniphila MucT and its derivatives on cytotoxicity and inflammatory response induced by C. difficile RT001 in Caco-2 cells. The results obtained from SEM revealed that the morphology of UV-killed A. muciniphila remained unchanged after UV inactivation. TEM analysis showed that A. muciniphila–isolated extracellular vesicles (EVs) were spherical and ranged from 50 to 200 nm in size. Toxigenic supernatant (Tox-S) of C. difficile RT001 (500 μg/ml) significantly (P <0.01) reduced the cell viability of Caco-2 cells. Caco-2 cells treated with live (MOI 10), UV-killed (MOI 10), cell-free supernatant (CFS, 106 cfu/ml), and EVs (20 μg/ml) of A. muciniphila exhibited over 90% viability in comparison to untreated control. The neutralized CFS preparation using A. muciniphila and its derivatives could notably reduce the expression level of inflammatory markers. Additionally, A. muciniphila and its derivatives modulated the production of IL-1β, TNF-α, and IL-10 in Tox-S stimulated Caco-2 cells. We demonstrated that A. muciniphila and its derivatives can modulate changes in the gut barrier–related genes and inflammatory response caused by C. difficile Tox-S in Caco-2 cells. (AU)