Interferon-γ Production Research Articles

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Faecalibaculum rodentium remodels retinoic acid signaling to govern eosinophil-dependent intestinal epithelial homeostasis

The intestinal epithelium plays critical roles in sensing and integrating dietary and microbial signals. How microbiota and intestinal epithelial cell (IEC) interactions regulate host physiology in the proximal small intestine, particularly the duodenum, is unclear. Using single-cell RNA sequencing of duodenal IECs under germ-free (GF) and different conventional microbiota compositions, we show that specific microbiota members alter epithelial homeostasis by increasing epithelial turnover rate, crypt proliferation, and major histocompatibility complex class II (MHCII) expression. Microbiome profiling identified Faecalibaculum rodentium as a key species involved in this regulation. F.rodentium decreases enterocyte expression of retinoic-acid-producing enzymes Adh1, Aldh1a1, and Rdh7, reducing retinoic acid signaling required to maintain certain intestinal eosinophil populations. Eosinophils suppress intraepithelial-lymphocyte-mediated production of interferon-γ that regulates epithelial cell function. Thus, we identify a retinoic acid-eosinophil-interferon-γ-dependent circuit by which the microbiota modulates duodenal epithelial homeostasis.

The Role of D2-like Dopaminergic Receptor in Dopamine-mediated Modulation of Th17-cells in Multiple Sclerosis.

Dopamine is one of the main mediators capable regulate the neuroimmune interaction and is involved in multiple sclerosis (MS) pathogenesis. The aim of this study was to clarify the role of dopamine and its receptors in modulation of Th17-cells in MS. 34 relapsing-remitting MS patients and 23 healthy subjects were examined. To assess the effect of dopamine on Th17-cells, CD4+ T-cells were cultured in the presence of dopamine and antagonist or agonist of D1- or D2-like dopaminergic receptors and stimulated with anti-CD3/CD28- microbeads. The levels of cytokines in supernatants were assessed by ELISA. Production of interleukin-17 (IL-17), interferon-γ (IFN-γ), granulocyte-colony stimulating factor (GM-CSF), and IL-21 by CD4+ T-cells as well as dopamine were comparable between the groups. Dopamine suppressed cytokine secretion by activated СD4+ T-cells in both groups. Blockade of D1-like dopaminergic receptor with a specific antagonist SCH23390 did not affect dopaminemediated cytokine suppression. In contrast, blockade of D2-like dopaminergic receptor by sulpiride decreased dopamine's inhibitory effect on IL-17 secretion in both groups and GM-CSF and IL-21 production in MS patients. Blockade of D1-like dopaminergic receptor directly inhibited IL-17, IFN- γ, GM-CSF in both groups and IL-21 production in healthy subjects, while blockade of D2-like dopaminergic receptor had no effect on cytokine secretion. Finally, activation of D2-like dopaminergic receptor with a specific agonist quinpirole decreased cytokine production in both groups. These data suggest an inhibitory role of dopamine on Th17-cells in MS, which could be mediated by the activation of the D2-like dopaminergic receptor.

Wasabi Component 6-(Methylsulfinyl)hexly Isothiocyanate and Derivatives Improve the Survival of Skin Allografts.

We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14+ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.

Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including β-hydroxybutyrate (BHB)1–6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

SARS-CoV-2 spike and nucleocapsid proteins fail to activate human dendritic cells or γδ T cells.

γδ T cells are thought to contribute to immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanisms by which they are activated by the virus are unknown. Using flow cytometry, we investigated if the two most abundant viral structural proteins, spike and nucleocapsid, can activate human γδ T cell subsets, directly or in the presence of dendritic cells (DC). Both proteins failed to induce interferon-γ production by Vδ1 or Vδ2 T cells within fresh mononuclear cells or lines of expanded γδ T cells generated from healthy donors, but the same proteins stimulated CD3+ cells from COVID-19 patients. The nucleocapsid protein stimulated interleukin-12 production by DC and downstream interferon-γ production by co-cultured Vδ1 and Vδ2 T cells, but protease digestion and use of an alternative nucleocapsid preparation indicated that this activity was due to contaminating non-protein material. Thus, SARS-CoV-2 spike and nucleocapsid proteins do not have stimulatory activity for DC or γδ T cells. We propose that γδ T cell activation in COVID-19 patients is mediated by immune recognition of viral RNA or other structural proteins by γδ T cells, or by other immune cells, such as DC, that produce γδ T cell-stimulatory ligands or cytokines.

THEMIS enhances the magnitude of normal and neuroinflammatory type 1 immune responses by promoting TCR-independent signals.

Signals that determine the differentiation of naïve CD4+ T helper (TH) cells into specific effector cell subsets are primarily stimulated by cytokines, but additional signals are required to adjust the magnitude of TH cell responses and set the balance between effective immunity and immunological tolerance. By inducing the post-thymic deletion of the T cell lineage signaling protein THEMIS, we showed that THEMIS promoted the development of optimal type 1 immune responses to foreign antigens but stimulated signals that favored encephalitogenic responses to self-neuroantigens. THEMIS was required to stimulate the expression of the gene encoding the transcriptional regulator T-BET and the production of the cytokine interferon-γ (IFN-γ), and it enhanced the ability of encephalitogenic CD4+ T cells to migrate into the central nervous system. Consistently, analysis of THEMIS expression in polarized CD4+ T cells showed that THEMIS was selectively increased in abundance in TH1 cells. The stimulation of predifferentiated effector CD4+ T cells with antigen-presenting cells revealed a stimulatory function for THEMIS on type 1 cytokine responses, similar to those observed ex vivo after immunization. In contrast, THEMIS exerted opposing effects on naïve CD4+ T cells in vitro by inhibiting the T cell receptor (TCR)-mediated signals that lead to TH1 cell responses. These data suggest that THEMIS exerts TCR-independent functions in effector T cells, which increase the magnitude of normal and pathogenic TH1 cell-mediated responses.

Breakthrough SARS-CoV-2 infections, morbidity, and seroreactivity following initial COVID-19 vaccination series and additional dose in patients with SLE in New York City.

Breakthrough SARS-CoV-2 infections, morbidity, and seroreactivity following initial COVID-19 vaccination series and additional dose in patients with SLE in New York City.

Enhancing Mesenchymal Stromal Cell Potency: Inflammatory Licensing via Mechanotransduction.

Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE2) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach.

Pharmacological Role of Capparis decidua (Forssk.) Edgew in Preventing Cyclophosphamide-induced Myelosuppression and Modulating Innate and Adaptive Immune Response.

BackgroundCapparis decidua (Forssk.) Edgew is reported to be practiced in the traditional system of medicine for the management of various immunological pathologies.PurposeThe current study was designed to evaluate the modulatory effects of C decidua on different immune responses.Research DesignC decidua was extracted in 70% methanol and the crude extract (Cd.Cr) was analyzed by FTIR and GCMS. In vivo models were employed to assess the actions of Cd.Cr on cyclophosphamide-induced myelosuppression, innate and adaptive immune responses.ResultsGCMS and FTIR analysis indicated the presence of flavonoids, phenols, terpenoids and lipids. Cd.Cr evoked a significant and dose-dependent increase in percent neutrophil adhesion (15.97 ± .81, 27.47 ± .79 and 38.35 ± 1.08) and the phagocytic index (3.1 ± .04, 3.96 ± .06 and 5.28 ± .13) at the doses of 30, 100 and 300 mg/kg. Cd.Cr also potentiated haemagglutinating antibody titre, immunoglobulins and cytokines (interferon-γ and interleukin-2) production for 4 weeks, after exposure to sheep erythrocytes, and delayed type hypersensitivity reaction significantly (P < .05). The restoration of hematological profile and antioxidant enzyme activities, by Cd.Cr, indicated the prevention of cyclophosphamide-induced myelosuppression and oxidative stress.ConclusionsThe findings of this study suggest that C decidua holds immunomodulatory activity by thus possesses therapeutic potential for the management of immunological diseases.

The generation of stem cell-like memory cells early after BNT162b2 vaccination is associated with durability of memory CD8+ Tcell responses.

COVID-19 vaccines elicit humoral and cellular immune responses. Durable maintenance of vaccine-induced immunity is required for long-term protection of the host. Here, we examine activation and differentiation of vaccine-induced CD8+ T cells using MHC class I (MHC-I) multimers and correlations between early differentiation and the durability of CD8+ T cell responses among healthcare workers immunized with two doses of BNT162b2. The frequency of MHC-I multimer+ cells is robustly increased by BNT162b2 but decreases 6 months post-second vaccination to 2.4%–65.6% (23.0% on average) of the peak. MHC-I multimer+ cells dominantly exhibit phenotypes of activated effector cells 1–2 weeks post-second vaccination and gradually acquire phenotypes of long-term memory cells, including stem cell-like memory T (TSCM) cells. Importantly, the frequency of TSCM cells 1–2 weeks post-second vaccination significantly correlates with the 6-month durability of CD8+ T cells, indicating that early generation of TSCM cells determines the longevity of vaccine-induced memory CD8+ T cell responses.

Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation.

Visceral leishmaniasis (VL) is a neglected tropical disease that causes substantial morbidity and mortality and is a growing health problem in Ethiopia, where this study took place. Most individuals infected with Leishmania donovani parasites will stay asymptomatic, but some develop VL that, if left untreated, is almost always fatal. This stage of the disease is associated with a profound immunosuppression, characterised by impaired production of Interferonγ (IFNγ), a cytokine that plays a key role in the control of Leishmania parasites, and high expression levels of an inhibitory receptor, programmed cell death 1 (PD1) on CD4+ T cells. Here, we tested the contribution of the interaction between the immune checkpoint PD1 and its ligand PDL-1 on the impaired production of IFNγ in VL patients. Our results show that in the blood of VL patients, not only CD4+, but also CD8+ T cells express high levels of PD1 at the time of VL diagnosis. Next, we identified PDL-1 expression on different monocyte subsets and neutrophils and show that PDL-1 levels were significantly increased in VL patients. PD1/PDL-1 inhibition resulted in significantly increased production of IFNγ, suggesting that therapy using immune checkpoint inhibitors might improve disease control in these patients.

P1435: PRECLINICAL EVALUATION OF ZILOVERTAMAB-BASED ANTI-ROR1 CHIMERIC ANTIGEN RECEPTORS IN NK AND T CELLS

Background: The receptor tyrosine kinase-like orphan receptor ROR1 is a promising target for immunotherapies of many cancers, including mantle cell lymphoma, diffuse large B-cell lymphoma, B-cell acute lymphocytic leukemia and chronic lymphocytic leukemia. Normally only expressed during embryonic development and only minimally, if at all, in adult normal tissues, ROR1 is overexpressed on malignant cells. Zilovertamab is a monoclonal antibody binding ROR1 that is currently in Phase 1/2 clinical trials. The antibody blocks survival signals mediated by Wnt5a via the ROR1 receptor. A different approach is the use of the antigen binding domain of the antibody in chimeric antigen receptors (CARs) expressed in NK or T cells to induce cellular cytotoxicity against ROR1-expressing cancer cells. Aims: This study aimed to evaluate different CAR designs in NK cells and T cells for cytotoxicity and cytokine production in vitro and control of cancer growth in a murine in vivo model. Methods: Two CARs using different linkers between the single chain antibody fragment (scFv) and the transmembrane domain were designed and cloned into lentiviral vectors. The two constructs were expressed in the cell line NK92 and the cells’ response against ROR1-positive and negative cancer cell lines was measured by degranulation and interferon-γ production. The construct that showed a stronger activation was tested in an in vivo model. Immune deficient NSG mice were injected with ROR1-positive JEKO-1 cells transgenic for luciferase. The cancer cell burden was monitored by intravital imaging. When cancer cell growth was established, the mice received either 1, 3, or 9 x 106 human T cells expressing the CAR or untransduced T cells. Results: NK92 cells expressing either of the two CARs showed enhanced degranulation and IFN-γ production in response to the ROR1-expressing cancer cell lines RPMI 8226 and JEKO-1. NK cells expressing the CAR containing the shorter linker between the scFv and the transmembrane domain exhibited higher activation levels. Primary human T cells expressing this CAR construct demonstrated degranulation and cytokine production during co-incubation with the ROR1-expressing JEKO-1 cell line but not the ROR1-negative Jurkat cell line. Injection of anti-ROR1 human CAR-T cells into NSG mice with established cancer growth greatly reduced the cell burden of JEKO-1 while untransduced T cells were unable to control cancer cell growth. Summary/Conclusion: The zilovertamab-based CAR tested in this study enhances the NK cell response towards ROR1-expressing cancer cells and enables T cells to clear fast-growing cancer cells in an in vivo model. These findings support the development of anti-ROR1 cell therapies for the potential treatment of ROR1-positive hematological malignancies.

P799: SPATIAL ANALYSIS IDENTIFIES A SPECTRUM OF IMMUNE DYSREGULATION IN ACQUIRED BONE MARROW FAILURE CONDITIONS

Background: Poor Graft Function (PGF), manifested by multilineage cytopenias with complete donor chimerism post allogeneic stem cell transplantation, and acquired Aplastic Anaemia (AA) are acquired bone marrow failure syndromes. Both are thought to result from T cell activation resulting in excessive interferon-γ (IFNγ) production, which in turn suppresses haematopoiesis. In AA, this can promote clonal stem cell selection and potentially progression to myeloid malignancy. Despite similarities in the clinical presentation and proposed mechanism, no studies to date have compared the immunobiology of the bone marrow (BM) microenvironment in these conditions. Aims: To examine the immune microenvironment of the BM in archival AA and PGF BM trephines and identify common immunopathologies. Methods: Archival BM trephines were sourced from patients with PGF (n=20), AA at Diagnosis (AA_DX) (n=15), AA at Progression (AA_PROG) to MDS/AML (n=15) and normal controls (NC) (n=20). 6 x 300µm regions identified by dual CD3/CD45 immunofluorescent staining were analysed per BM trephine using NanoString GeoMX™ digital spatial profiling for the expression of 57 proteins with an immunology focused panel. Data was analysed using a Limma-Voom bioinformatics pipeline. Results: Significant dysregulation was identified across multiple proteins. Overall, 22 proteins had significantly different expression in AA_DX samples (13 up, 9 down) compared to NC with AA_PROG and PGF having 8 (4 up, 4 down) and 14 (7 up, 7 down) respectively. As would be expected given the hypocellular BM, expression of CD45 was significantly reduced compared to NC across AA_DX (adj P = 5.413e-10), AA_PROG (adj P = 8.299e-8) and PGF (adj P = 8.634e-9) groups. When AA_DX patients were stratified into those who progressed (n=6) vs those who did not (n=9), or when AA_DX vs AA_PROG was compared in patients with matched samples (n=6) there were no significantly differentially expressed proteins identified. This suggests that while AA provides an immune microenvironment that is permissive to MDS/AML pathogenesis, it may not directly impact on clonal evolution. Total monocytes were unchanged by CD11c expression. However, AA and PGF samples exhibited upregulation of CD163 compared to NC (AA_DX adj P = 4.5e-6; AA_PROG adj P = 1.2e-5; PGF adj P = 1.58e-8) suggesting an increase in monocyte/macrophage lineage cells. Expression of CD66b was also downregulated in AA_DX (adj P = 8.459e-24), AA_PROG (adj P = 1.193e-10) and PGF (adj P = 2.244e-14) suggesting a reduction in non-classical monocytes, neutrophils or granulocytic MDSCs. Importantly, VISTA was downregulated across AA_DX (adj P = 6.84e-27), AA_PROG (adj P = 1.1e-5) and PGF (adj P = 1.58e-8) and STING was downregulated in AA_DX compared to NC (adj P = 6.139e-13) with a trend towards reduced expression in PGF. Decreased expression of these key immunoregulatory proteins may have wide ranging effects on BM resident macrophages, dendritic cells, MDSCs and T cells leading to reduced immunoregulation and increased T cell activation/IFNγ production, ultimately resulting in stem cell depletion. Data on cell specific expression is currently being obtained to refine this model. Summary/Conclusion: Spatial analysis demonstrated that patients with AA and PGF exhibit similar patterns of protein expression likely resulting in a BM immune microenvironment of decreased immunoregulation. AA_DX samples exhibited a greater degree of dysregulation of multiple markers compared to PGF suggesting that these diseases represent a spectrum of immune dysregulation.

Myeloid caspase-8 restricts RIPK3-dependent proinflammatory IL-1β production and CD4 T cell activation in autoimmune demyelination

Caspase-8 functions at the crossroad of programmed cell death and inflammation. Here, using genetic approaches and the experimental autoimmune encephalomyelitis model of inflammatory demyelination, we identified a negative regulatory pathway for caspase-8 in infiltrated macrophages whereby it functions to restrain interleukin (IL)-1β-driven autoimmune inflammation. Caspase-8 is partially activated in macrophages/microglia in active lesions of multiple sclerosis. Selective ablation of Casp8 in myeloid cells, but not microglia, exacerbated autoimmune demyelination. Heightened IL-1β production by caspase-8-deficient macrophages underlies exacerbated activation of encephalitogenic T cells and production of GM-CSF and interferon-γ. Mechanistically, IL-1β overproduction by primed caspase-8-deficient macrophages was mediated by RIPK1/RIPK3 through the engagement of NLRP3 inflammasome and was independent of cell death. When instructed by autoreactive CD4 T cells in the presence of antigen, caspase-8-deficient macrophages, but not their wild-typecounterparts, released significant amount of IL-1β that in turn acted through IL-1R to amplify T cell activation. Moreover, the worsened experimental autoimmune encephalomyelitis progression in myeloid Casp8 mutant mice was completely reversed when Ripk3 was simultaneously deleted. Together, these data reveal a functional link between T cell-driven autoimmunity and inflammatory IL-1β that is negatively regulated by caspase-8, and suggest that dysregulation of the pathway may contribute to inflammatory autoimmune diseases, such as multiple sclerosis.

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

Immune-Enhancing Effects of Co-treatment With Kalopanax pictus Nakai Bark and Nelumbo nucifera Gaertner Leaf Extract in a Cyclophosphamide-Induced Immunosuppressed Rat Model.

ObjectiveImmune system disorders can result in various pathological conditions, such as infections and cancer. Identifying therapies that enhance the immune response might be crucial for immunocompromised individuals. Therefore, we assessed the immune-enhancing effect of co-treatment with Kalopanax pictus Nakai Bark and Nelumbo nucifera Gaertner leaf extract (KPNN) in a cyclophosphamide (Cy)-induced immunosuppressed rat model.Materials and MethodsFor in vitro studies, macrophages and splenocytes were treated with various KPNN doses in the presence or absence of Cy. Macrophage viability, nitric oxide production, splenocyte viability, cytokine production and natural killer (NK) cell activity were analyzed. For in vivo studies, analysis of weekly body weight, dietary intake, tissue weight, immune-related blood cell count, cytokine levels, and spleen biopsy was performed in a Cy-induced immunocompromised animal model.ResultsKPNN significantly increased phospho-NF-κB and phospho-ERK protein levels and cell viability in macrophages. KPNN significantly increased the NK cell activity in splenocytes compared to that in the control. Cy treatment decreased tumor necrosis factor (TNF)-α, interleukin (IL)-6, and interferon-γ production. In the Cy-induced immunosuppression rat model, KPNN-treated rats had significantly higher body weights and tissue weights than the Cy-treated rats. Additionally, KPNN treatment restored the immune-related factors, such as total leukocyte, lymphocyte, and intermediate cell contents, to their normal levels in the blood. The blood cytokines (TNF-α and IL-6) were increased, and spleen tissue damage was significantly alleviated.ConclusionsCollectively, KPNN exerts an immune-enhancing effect suggesting their potential as an immunostimulatory agent or functional food.

CD40 is Required For Protective T Cell Responses Against Cryptosporidium

Abstract The apicomplexan intestinal parasite Cryptosporidium infects intestinal epithelial cells (IECs) and is a leading cause of life-threatening disease in infants, toddlers, and immunocompromised patients. No vaccine against Cryptosporidium exists, and current treatments are ineffective in immunocompromised individuals. A prominent immunodeficiency associated with susceptibility to Cryptosporidium is hyper-IgM syndrome, which is caused by defects in CD40 signaling. In a murine model of Cryptosporidiosis, mice deficient in CD154 (CD40L) are defective in parasite control and clearance, and complementation of CD40L restores protection. Therefore, characterizing CD40-mediated immune responses against Cryptosporidium may enhance progress toward effective therapy in at-risk individuals. CD40 engagement on dendritic cells (DCs) promotes IL-12 production that induces differentiation and proliferation of T helper type 1 (Th1) cells and their production of interferon-γ (IFN-γ), which is known to be essential for control and clearance of Cryptosporidium infection. Our findings suggest a defect in T cell responses in CD40−/− mice infected with Cryptosporidium. CD40 deletion on Xcr1+ DCs also resulted in increased susceptibility to Cryptosporidium along with a defect in CD8+ T cell expansion. Therefore, we conclude that CD8+ T cell responses against Cryptosporidium require CD40 on cDC1s. Supported by NIH grant U01AI163671

Cell surface–tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy

Immune-activating cytokines such as interleukin-12 (IL-12) hold strong potential for cancer immunotherapy but have been limited by high systemic toxicities. We describe here an approach to safely harness cytokine biology for adoptive cell therapy through uniform and dose-controlled tethering onto the surface of the adoptively transferred cells. Tumor-specific T cells tethered with IL-12 showed superior antitumor efficacy across multiple cell therapy models compared to conventional systemic IL-12 coadministration. Mechanistically, the IL-12–tethered T cells supported a strong safety profile by driving interferon-γ production and adoptively transferred T cell activity preferentially in the tumor. Immune profiling revealed that the tethered IL-12 reshaped the suppressive tumor immune microenvironment, including triggering a pronounced repolarization of monocytic myeloid-derived suppressor cells into activated, inflammatory effector cells that further supported antitumor activity. This tethering approach thus holds strong promise for harnessing and directing potent immunomodulatory cytokines for cell therapies while limiting systemic toxicities.

Antibody-mediated blockade of the IL23 receptor destabilizes intratumoral regulatory T cells and enhances immunotherapy

Regulatory T cells (Treg) can impede antitumor immunity and currently represent a major obstacle to effective cancer immunotherapy. Targeting tumor-infiltrating regulatory Treg while sparing systemic Treg represents an optimal approach to this problem. Here, we provide evidence that the interleukin 23 receptor (IL23R) expressed by tumor-infiltrating Treg promotes suppressive activity. Disruption of the IL23R results in increased responsiveness of destabilized Treg to the IL12 cytokine, the production of γ-interferon, and the recruitment of CD8 T cells that inhibit tumor growth. Since the Treg destabilization pathway that is initiated by IL23R blockade is distinct and independent from the destabilization pathway coupled to glucocorticoid-induced TNFR-related protein (GITR) activation, we examined the impact of the coordinate induction of the two destabilization pathways on antitumor immune responses. Combined GITR and IL23R antibody treatment of mice inoculated with MC38 tumors resulted in robust and synergistic antitumor responses. These findings indicate that the delineation of independent Treg destabilization pathways may allow improved approaches to the development of combination immunotherapy for cancers.