IFN-γ Production In Response Research Articles

Vitamin A-treated natural killer cells reduce interferon-gamma production and support regulatory T-cell differentiation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that contribute to immune responses against stressed, transformed, or infected cells. NK cell effector functions are regulated by microenvironmental factors, including cytokines, metabolites, and nutrients. Vitamin A is an essential micronutrient that plays an indispensable role in embryogenesis and development, but was also reported to regulate immune responses. However, the role of vitamin A in regulating NK cell functions remains poorly understood. Here, we show that the most prevalent vitamin A metabolite, all-trans retinoic acid (atRA), induces transcriptional and functional changes in NK cells leading to altered metabolism and reduced IFN-γ production in response to a wide range of stimuli. atRA-exposed NK cells display a reduced ability to support dendritic cell (DC) maturation and to eliminate immature DCs. Moreover, they support the polarization and proliferation of regulatory T cells. These results imply that in vitamin A-enriched environments, NK cells can acquire functions that might promote tolerogenic immunity and/or immunosuppression.

Enhancement of subunit vaccine delivery with zinc-carnosine coordination polymer through the addition of mannan

Vaccines represent a pivotal health advancement for preventing infection. However, because carrier systems with repeated administration can invoke carrier-targeted immune responses that diminish subsequent immune responses (e.g., PEG antibodies), there is a continual need to develop novel vaccine platforms. Zinc carnosine microparticles (ZnCar MPs), which are composed of a one-dimensional coordination polymer formed between carnosine and the metal ion zinc, have exhibited efficacy in inducing an immune response against influenza. However, ZnCar MPs’ limited suspendability hinders clinical application. In this study, we address this issue by mixing mannan, a polysaccharide derived from yeast, with ZnCar MPs. We show that the addition of mannan increases the suspendability of this promising vaccine formulation. Additionally, since mannan is an adjuvant, we illustrate that the addition of mannan increases the antibody response and T cell response when mixed with ZnCar MPs. Mice vaccinated with mannan + OVA/ZnCar MPs had elevated serum IgG and IgG1 levels in comparison to vaccination without mannan. Moreover, in the mannan + OVA/ZnCar MPs vaccinated group, mucosal washes demonstrated increased IgG, IgG1, and IgG2c titers, and antigen recall assays showed enhanced IFN-γ production in response to MHC-I and MHC-II immunodominant peptide restimulation, compared to the vaccination without mannan. These findings suggest that the use of mannan mixed with ZnCar MPs holds potential for subunit vaccination and its improved suspendability further promotes clinical translation.

Liver sinusoidal endothelial cells orchestrate NK cell recruitment and activation in acute inflammatory liver injury

Liver sinusoidal endothelial cells (LSECs) rapidly clear lipopolysaccharide (LPS) from the bloodstream and establish intimate contact with immune cells. However, their role in regulating liver inflammation remains poorly understood. We show that LSECs modify their chemokine expression profile driven by LPS or interferon-γ (IFN-γ), resulting in the production of the myeloid- or lymphoid-attracting chemokines CCL2 and CXCL10, respectively, which accumulate in the serum of LPS-challenged animals. Natural killer (NK) cell exposure to LSECs invitro primes NK cells for higher production of IFN-γ in response to interleukin-12 (IL-12) and IL-18. In livers of LPS-injected mice, NK cells are the major producers of this cytokine. In turn, LSECs require exposure to IFN-γ for CXCL10 expression, and endothelial-specific Cxcl10 gene deletion curtails NK cell accumulation in the inflamed livers. Thus, LSECs respond to both LPS and immune-derived signals and fuel a positive feedback loop of immune cell attraction and activation in the inflamed liver tissue.

AFM13 enhances the anti‐tumor activity of AB‐101 towards CD30+ tumors, conferring tumor growth control in vivo

Introduction: Bispecific antibodies targeting CD16A and a tumor antigen have the potential to specifically redirect the cytotoxic potential of NK cells towards cancer cells through antibody-dependent cellular cytotoxicity (ADCC). It has been demonstrated that the bispecific CD16AxCD30 innate cell engager AFM13 significantly enhances the cytotoxic activity of CD16A+ NK cells towards CD30+ tumor cells. Combination of AFM13 with adoptive NK cell transfer achieved unprecedented clinical response rates in patients with relapsed/refractory (R/R) Hodgkin lymphoma (ORR 94.2% for patients treated with RP2D), while maintaining a favorable toxicity profile. AB-101 is a non-engineered, allogeneic, off-the-shelf, cryopreserved cord blood-derived NK cell product, currently being tested in a Phase 1/2 clinical trial as monotherapy and in combination with rituximab in patients with R/R B cell NHL. AB-101 is optimized for ADCC through pre-selection for the KIR-B haplotype and the natural high-affinity variant of CD16A (158V/V). In the present study, we investigated whether the combination of AFM13 with AB-101 leads to enhanced anti-tumor activity in vitro and in vivo. Methods: Cellular cytotoxic activity was assessed in 4-h calcein-release cytotoxicity assays. Cell viability, degranulation/CD107a expression and IFN-γ production were measured by flow cytometry. Tumor growth control in vivo was determined in a hematologic (Karpas-299 CD30+ T-cell lymphoma) human IL-15 NOG mouse xenograft model by whole body bioluminescence imaging. Results: We demonstrate robust, homogenous binding of AFM13 to AB-101 as assessed by flow cytometry when AB-101 cells were combined with AFM13 after thawing. Overall, the binding pattern of AFM13 was congruent to the binding pattern of high CD16A expression on AB-101, suggesting saturated loading of CD16A molecules by AFM13 on the surface of AB-101. Combination with AFM13 enhanced the cytotoxic activity of AB-101 towards CD30+ Karpas-299 tumor cells. AFM13-induced cytotoxic activity was associated with an increased functional activation status of AB-101, reflected by increased degranulation/CD107a expression and IFN-γ production in response to Karpas-299 tumor cells. Of note, viability of AB-101 cells was maintained upon exposure to AFM13 in the absence or presence of CD30+ tumor cells. Importantly, adoptive transfer of AB-101 co-administered with AFM13 conferred tumor growth control in vivo in a human IL-15 NOG mouse xenograft model. Conclusion: This study demonstrates synergistical anti-tumor activity in vivo for the combination of AFM13 and AB-101. Building on our clinical data with fresh cord blood-derived stimulated/expanded NK cells combined with AFM13 (NCT04074746), co-administration of cryopreserved AB-101 with AFM13 offers a promising highly scalable off-the-shelf immunotherapeutic treatment for patients with CD30+ malignancies. The research was funded by Affimed GmbH and Artiva Biotherapeutics Keywords: Cellular therapies, Combination Therapies, Immunotherapy Conflicts of interests pertinent to the abstract. J. Pahl Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH T. Haneke Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH L. Guerrettaz Employment or leadership position: Artiva Biotherapeutics S. Somanchi Employment or leadership position: Artiva Biotherapeutics H. Raymon Employment or leadership position: Artiva Biotherapeutics S. Pinto Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH P. Flynn Employment or leadership position: Artiva Biotherapeutics J. Koch Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH

Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis.

Inborn errors of IFN-γ immunity can underlie tuberculosis (TB). We report three patients from two kindreds without EBV viremia or disease but with severe TB and inherited complete ITK deficiency, a condition associated with severe EBV disease that renders immunological studies challenging. They have CD4+ αβ T lymphocytopenia with a concomitant expansion of CD4-CD8- double-negative (DN) αβ and Vδ2- γδ T lymphocytes, both displaying a unique CD38+CD45RA+T-bet+EOMES- phenotype. Itk-deficient mice recapitulated an expansion of the γδ T and DN αβ T lymphocyte populations in the thymus and spleen, respectively. Moreover, the patients' T lymphocytes secrete small amounts of IFN-γ in response to TCR crosslinking, mitogens, or forced synapse formation with autologous B lymphocytes. Finally, the patients' total lymphocytes secrete small amounts of IFN-γ, and CD4+, CD8+, DN αβ T, Vδ2+ γδ T, and MAIT cells display impaired IFN-γ production in response to BCG. Inherited ITK deficiency undermines the development and function of various IFN-γ-producing T cell subsets, thereby underlying TB.

Kidney-Protective Vagus Nerve Stimulation Modulates Splenic Immune Populations and Reduces IFNγ Production in Response to Toll-like Receptor Agonists

Kidney-Protective Vagus Nerve Stimulation Modulates Splenic Immune Populations and Reduces IFNγ Production in Response to Toll-like Receptor Agonists

Plasma metabolome predicts trained immunity responses after antituberculosis BCG vaccination.

The antituberculosis vaccine Bacillus Calmette–Guérin (BCG) induces nonspecific protection against heterologous infections, at least partly through induction of innate immune memory (trained immunity). The amplitude of the response to BCG is variable, but the factors that influence this response are poorly understood. Metabolites, either released by cells or absorbed from the gut, are known to influence immune responses, but whether they impact BCG responses is not known. We vaccinated 325 healthy individuals with BCG, and collected blood before, 2 weeks and 3 months after vaccination, to assess the influence of circulating metabolites on the immune responses induced by BCG. Circulating metabolite concentrations after BCG vaccination were found to have a more pronounced impact on trained immunity responses, such as the increase in IL-1β and TNF-α production upon Staphylococcus aureus stimulation, than on specific adaptive immune memory, assessed as IFN-γ production in response to Mycobacterium tuberculosis. Circulating metabolites at baseline were able to predict trained immunity responses at 3 months after vaccination and enrichment analysis based on the metabolites positively associated with trained immunity revealed enrichment of the tricarboxylic acid (TCA) cycle and glutamine metabolism, both of which were previously found to be important for trained immunity. Several new metabolic pathways that influence trained immunity were identified, among which taurine metabolism associated with BCG-induced trained immunity, a finding validated in functional experiments. In conclusion, circulating metabolites are important factors influencing BCG-induced trained immunity in humans. Modulation of metabolic pathways may be a novel strategy to improve vaccine and trained immunity responses.

Trained Immunity of IL-12-, IL-15-, and IL-18-Induced CD3+CD56+ NKT-Like Cells.

CD3+CD56+ natural killer T (NKT)-like cells have an immune function of T cells and NK cells, which play an important role in antitumor and antiviral immune responses. This study aims to establish a CD3+CD56+ NKT-like cell model by simulating the memory NK effect induced by cytokines IL-12, IL-15, and IL-18 (IL-12/15/18) and explore the formation mechanism. Our study found that the IL-12/15/18 preactivated CD3+CD56+ NKT-like cells exhibited enhanced IFN-γ production in response to restimulation with IL-12/15/18 for 6h on day 7. The intrinsic potential of these trained cells was significantly improved, showing an increase in IFN-γ, TNF-α, and cell proliferation potential. The IFN-γ release, granzyme B level, and proliferation ability significantly increased when stimulated by NK-cell-sensitive K562 tumor cells. Among these cytokines, the combination of IL-12/15/18 was particularly effective. After the preactivation of IL-12/15/18, some cell surface proteins related to function and differentiation, such as CD11b, CD62 L, NKp46, NKG2A, and CD127, showed an evident and consistent change trend. The CDK4/6 inhibitor can effectively weaken this effect, and the expression of cyclin D1, Rb protein phosphorylation, and E2F-1 decreased significantly. Our work revealed that cytokine IL-12/15/18 can induce CD3+CD56+ NKT-like cells to obtain enhanced training immunity, which was a memory-like phenomenon.

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.

Abstract 1333: Dual blockade of the EP2 and EP4 PGE2 receptors with TPST-1495 is an optimal approach for drugging the prostaglandin pathway

Abstract Prostaglandin E2 (PGE2) is a bioactive lipid produced by tumor cells that drives disease progression through stimulating tumor proliferation, enhancing angiogenesis and suppressing immune function in the TME1, 2. PGE2 is also a mediator of adaptive resistance to immune checkpoint inhibitor therapy via the upregulation of cyclooxygenase-2 (COX-2). While the role of PGE2 signaling in cancer is clear, how best to inhibit PGE2 for cancer treatment remains under investigation. Inhibition of COX-1 and/or COX-2 has shown promising results in observational studies and meta-analyses, but inconsistent results in prospective studies. PGE2 signals through four receptors, EP1-4, that are variably expressed on tumor and immune cells and have distinct biological activities. The EP2 and EP4 receptors signal through cAMP and drive pro-tumor activities, while the EP1 and EP3 receptors signal through calcium flux and IP3 and drive immune activation and inflammation. While COX-2 and single EP inhibitors continue to be developed, the nature of PGE2 signaling supports our rationale to inhibit PGE2 by dual antagonism of the pro-tumor EP2/EP4 receptors, while sparing the pro-immune EP1/EP3 receptors. To our knowledge, TPST-1495 is the first clinical-stage dual inhibitor of both the EP2 and EP4 receptors. In mouse and human whole blood assays, dual blockade of EP2 and EP4 receptors with TPST-1495 reversed PGE2-mediated suppression of LPS-induced TNF-α, while single receptor antagonists were unable to block suppression at higher PGE2 concentrations. Similarly, in murine and human T cells in vitro, TPST-1495 inhibited PGE2-mediated suppression, resulting in a significant increase of IFN-γ production in response to stimulation with cognate peptide antigen. In vivo, TPST-1495 monotherapy significantly reduced tumor outgrowth in CT26 tumor-bearing mice and correlated with increased tumor infiltration by NK cells, CD8+ T cells, AH1-specific CD8+ T cells, and other anti-tumor myeloid and adaptive immune cell populations. The relative contribution of increased immune infiltration may be simultaneously dependent on the immunogenicity of the tumor model and on the direct antitumor effect of TPST-1495, because we also observed significant tumor regression in metastatic burden in the LS174T xenograft model in NSG mice as well as CT26 tumors in RAG2-/- animals, both of which are deficient in immune cell development. To that end, TPST- 1495 monotherapy significantly decreased the tumor burden compared to COX2 inhibition and EP2 or EP4 single antagonism in the Adenomatous Polyposis (APCmin/+) model, a model that is hypo-responsive to PD-1 monotherapy. TPST-1495 is currently being evaluated in an ongoing Phase 1 first-in-human study (NCT04344795) to characterize PK, PD, safety, and to identify a recommended phase 2 dose for expansion cohorts in key indications and biomarker-selected patients. Citation Format: Brian Francica, Justine Lopez, Anja Holtz, Dave Freund, Dingzhi Wang, Amanda Enstrom, Raymond Dubois, Francielle Kipper, Dipak Panigrahy, Chan Whiting, Sam Whiting, Thomas W. Dubensky. Dual blockade of the EP2 and EP4 PGE2 receptors with TPST-1495 is an optimal approach for drugging the prostaglandin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1333.

Vagus nerve stimulation leads to contraction of the spleen and dampened IFNγ production in response to TLR stimulation

Abstract Vagus nerve stimulation (VNS) generates an anti-inflammatory state and limits the pathology of inflammatory diseases, such as sepsis and acute kidney injury. Interestingly, splenectomy prior to stimulation results in a loss of this protection. Thus, the spleen is a central organ for converting VNS signals into protective anti-inflammation. To better understand the interplay between VNS and the spleen and gain insight into the mechanisms of anti-inflammation, we performed a broad query of the spleen post-VNS. Mice received either electrical stimulation of the left-cervical vagus nerve or sham surgery. Spleens were harvested and assessed for mass, immune cell populations, and splenocyte in vitro cytokine production in response to TLR4, 3, and 9 agonists (LPS, poly I:C, and CpG, respectively). We discovered that by 48 hours post-VNS, spleen mass was reduced and the number of CD45+ cells was significantly decreased compared to sham mice. Analysis of CD45+ cells revealed that many individual immune cell populations were diminished. B cells, T cells, NK cells, dendritic cells, and basophils were all significantly reduced and eosinophils and monocytes showed strong trends towards reduction, nearing significance. In response to TLR agonists, our Luminex analysis revealed a strong impact on TLR4 stimulation, with 12 of the 32 measured cytokines showing a significant reduction in supernatants from VNS samples. TLR3 and TLR9 stimulation also exhibited significant reductions in a subset of the measured cytokines, but only IFNγ exhibited strong signs of reduced production in all TLR stimulation conditions. Thus, VNS has profound impacts in the spleen and some of the observed protection could be due to tighter control of immune cell IFNγ secretion. Supported by grants from the NIH (R01 DK123248)

The ingenol-based protein kinase C agonist GSK445A is a potent inducer of HIV and SIV RNA transcription

Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 μg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.

Defective Interferon-Gamma Production Is Common in Chronic Pulmonary Aspergillosis.

Immune defects in chronic pulmonary aspergillosis (CPA) are poorly characterized. We compared peripheral blood cytokine profiles in patients with CPA versus healthy controls and explored the relationship with disease severity. Interferon-gamma (IFNγ), interleukin (IL)-17, tumor necrosis factor-α, IL-6, IL-12, and IL-10 were measured after in vitro stimulation of whole blood with lipopolysaccharide (LPS), phytohemagglutinin, β-glucan, zymosan (ZYM), IL-12 or IL-18, and combinations. Clinical parameters and mortality were correlated with cytokine production. Cytokine profiles were evaluated in 133 patients (57.1% male, mean age 61 years). In comparison to controls, patients with CPA had significantly reduced production of IFNγ in response to stimulation with β-glucan + IL-12 (312 vs 988 pg/mL), LPS + IL-12 (252 vs 1033 pg/mL), ZYM + IL-12 (996 vs 2347 pg/mL), and IL-18 + IL-12 (7193 vs 12330 pg/mL). Age >60 (hazard ratio [HR], 1.71; 95% confidence interval [CI], 1.00-2.91; P = .05) and chronic obstructive pulmonary disease (HR, 1.69; 95% CI, 1.03-2.78; P = .039) were associated with worse survival, whereas high IFNγ production in response to beta-glucan + IL-12 stimulation (HR, 0.48; 95% CI, .25-0.92; P = .026) was associated with reduced mortality. Patients with CPA show impaired IFNγ production in peripheral blood in response to stimuli. Defective IFNγ production ability correlates with worse outcomes. Immunotherapy with IFNγ could be beneficial for patients showing impaired IFNγ production in CPA.

Immune Recovery Following Autologous Hematopoietic Stem Cell Transplantation in HIV-Related Lymphoma Patients on the BMT CTN 0803/AMC 071 Trial.

We report a first in-depth comparison of immune reconstitution in patients with HIV-related lymphoma following autologous hematopoietic cell transplant (AHCT) recipients (n=37, lymphoma, BEAM conditioning), HIV(-) AHCT recipients (n=30, myeloma, melphalan conditioning) at 56, 180, and 365 days post-AHCT, and 71 healthy control subjects. Principal component analysis showed that immune cell composition in HIV(+) and HIV(-) AHCT recipients clustered away from healthy controls and from each other at each time point, but approached healthy controls over time. Unsupervised feature importance score analysis identified activated T cells, cytotoxic memory and effector T cells [higher in HIV(+)], and naïve and memory T helper cells [lower HIV(+)] as a having a significant impact on differences between HIV(+) AHCT recipient and healthy control lymphocyte composition (p<0.0033). HIV(+) AHCT recipients also demonstrated lower median absolute numbers of activated B cells and lower NK cell sub-populations, compared to healthy controls (p<0.0033) and HIV(-) AHCT recipients (p<0.006). HIV(+) patient T cells showed robust IFNγ production in response to HIV and EBV recall antigens. Overall, HIV(+) AHCT recipients, but not HIV(-) AHCT recipients, exhibited reconstitution of pro-inflammatory immune profiling that was consistent with that seen in patients with chronic HIV infection treated with antiretroviral regimens. Our results further support the use of AHCT in HIV(+) individuals with relapsed/refractory lymphoma.

NK cells eliminate Epstein-Barr virus bound to B cells through a specific antibody-mediated uptake.

Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.

Cytomegalovirus mediates expansion of IL-15-responsive innate-memory cells with SIV killing function.

Interindividual immune variability is driven predominantly by environmental factors, including exposure to chronic infectious agents such as cytomegalovirus (CMV). We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells-and marked expansion of innate-memory CD8+ T cells. These cells express high levels of NKG2A/C and the IL-2 and IL-15 receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C+CD122+CD8+ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I-deficient K562 targets and prompt IFN-γ production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1-vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C+CD8+ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMV-vectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innate-memory expansion could be achieved by other vaccine platforms expressing IL-15.

Allotransplantation Is Associated With Exacerbation of CD8 T-Cell Senescence: The Particular Place of the Innate CD8 T-Cell Component.

Immunosenescence is a physiological process that is associated with changes in the immune system, particularly among CD8 T-cells. Recent studies have hypothesized that senescent CD8 T-cells are produced with chronologic age by chronic stimulation, leading to the acquisition of hallmarks of innate-like T-cells. While conventional CD8 T-cells are quite well characterized, CD8 T-cells sharing features of NK cells and memory CD8 T-cells, are a newly described immune cell population. They can be distinguished from conventional CD8 T-cells by their combined expression of panKIR/NKG2A and Eomesodermin (E), a unique phenotype closely associated with IFN-γ production in response to innate stimulation. Here, we first provided new evidence in favor of the innate character of panKIR/NKG2A(+) E(+) CD8 T-cells in normal subjects, documenting their position at an intermediate level in the innateness gradient in terms of both innate IFN-γ production and diminished mitochondrial mass. We also revealed that CD8 E(+) panKIR/NKG2A(+) T-cells, hereafter referred to as Innate E(+) CD8 T-cells, exhibit increased senescent (CD27(-) CD28(-)) phenotype, compared to their conventional memory counterparts. Surprisingly, this phenomenon was not dependent on age. Given that inflammation related to chronic viral infection is known to induce NK-like marker expression and a senescence phenotype among CD8 T-cells, we hypothesized that innate E(+) CD8 T-cells will be preferentially associated with exacerbated cellular senescence in response to chronic alloantigen exposure or CMV infection. Accordingly, in a pilot cohort of stable kidney allotransplant recipients, we observed an increased frequency of the Innate E(+) CD8 T-cell subset, together with an exacerbated senescent phenotype. Importantly, this phenotype cannot be explained by age alone, in clear contrast to their conventional memory counterparts. The senescent phenotype in CD8 T-cells was further increased in cytomegalovirus (CMV) positive serology transplant recipients, suggesting that transplantation and CMV, rather than aging by itself, may promote an exacerbated senescent phenotype of innate CD8 T-cells. In conclusion, we proposed that kidney transplantation, via the setting of inflammatory stimuli of alloantigen exposure and CMV infection, may exogenously age the CD8 T-cell compartment, especially its innate component. The physiopathological consequences of this change in the immune system remain to be elucidated.

Potential Role of CXCL10 in Monitoring Response to Treatment in Leprosy Patients.

The treatment of multibacillary cases of leprosy with multidrug therapy (MDT) comprises 12 doses of a combination of rifampicin, dapsone and clofazimine. Previous studies have described the immunological phenotypic pattern in skin lesions in multibacillary patients. Here, we evaluated the effect of MDT on skin cell phenotype and on the Mycobacterium leprae-specific immune response. An analysis of skin cell phenotype demonstrated a significant decrease in MRS1 (SR-A), CXCL10 (IP-10) and IFNG (IFN-γ) gene and protein expression after MDT release. Patients were randomized according to whether they experienced a reduction in bacillary load after MDT. A reduction in CXCL10 (IP-10) in sera was associated with the absence of a reduction in the bacillary load at release. Although IFN-γ production in response to M. leprae was not affected by MDT, CXCL10 (IP-10) levels in response to M. leprae increased in cells from patients who experienced a reduction in bacillary load after treatment. Together, our results suggest that CXCL10 (IP-10) may be a good marker for monitoring treatment efficacy in multibacillary patients.

Immune Checkpoint Blockade Enhances Mutated Calreticulin-Induced T Cell Immunity in Myeloproliferative Neoplasms

▪Somatic mutations in the calreticulin (CALR) gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. Each CALR mutation leads to a shift in the reading frame resulting in the formation of an altered protein with an identical 36-amino acid sequence in the C-terminus. We hypothesized that the recurrence and uniformity of the novel sequence would provide the mutated CALR an attractive candidate as a MPN-specific tumor antigen that might elicit anti-tumor immune responses. Therefore, we investigated the immunogenicity of the altered CALR by employing in silico peptide binding prediction algorithms to interrogate the binding affinities of mutated CALR peptides to human leukocyte antigens (HLA). These analyses indicated that the mutated epitopes can bind to class I and II HLA alleles with high affinity supporting the hypothesis that mutant CALR could be immunogenic in vivo . We then tested the immunogenicity of the overlapping peptides spanning the mutated region in vitro by utilizing intracellular staining (ICS) and Enzyme-Linked ImmunoSPOT (ELISPOT) assays. Naïve T cells derived from the peripheral blood mononuclear cells (PBMNCs) isolated from healthy donors displayed effector functions after priming with the mutated peptides but not with the corresponding wild type peptides . Incubation withthe mutated CALR peptides induced T cells proliferation, upregulation of CD137 (4-1BB) and production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Moreover, as suggested by in silico peptide-HLA binding predictions, both CD8+ and CD4+ T cell precursors displayed increased IFN-γ production in response to stimulation with mutated CALR peptides. There was an overall 4±3-fold increase in IFN-γ production by CD4+ T cells and a 2.5±0.5-fold increase by CD8+ T cells after stimulation with mutated peptides as compared to control self-antigens (n = 14 healthy donors out of which 11 showed CD4+ and 5 showed CD8+ T cell responses). These results demonstrate that mutated CALR elicits T cell responses in vitro supporting its potential immunogenic functions in vivo .We next investigated the presence of mutant CALR-specific T cell immunity in MPN patients with CALR mutations. PBMNCs from a total of 18 CALR-mutated MPN patients with ET (7), MF (5) or MF arising from ET (6) were evaluated. MPN T cells were then stimulated with the pooled mutant CALR peptides and T cell responses were assessed by ELISPOT. Out of 18 patients, increased IFN-γ production upon stimulation was observed in only 2 patients (one with ET and one with ET-MF). The less than robust MPN T cell responses suggested that immune suppressive mechanisms might be operating. To this end, we evaluated the expression of cell surface inhibitory receptors, programmed cell death (PD)-1 and the cytotoxic T-lymphocyte-associated antigen (CTLA)-4, by T cells from MPN patients. Both CD8+ and CD4+ T cells from MPN patients exhibited increased levels of PD-1 and CTLA-4 when compared to the healthy controls (HC). For instance, 9.3±8.2% of MPN patients CD4+ T cells expressed PD-1 (n = 12) as compared to 2.5±1.7% of HC CD4+ T cells (n=10) (p value = 0.0482). Similarly, there were twice as many PD-1 expressing CD8+ T cells in MPN patients as compared to HC. We next assessed MPN T cell responses against mutated peptides in the presence of neutralizing PD-1 or CTLA-4 antibodies. PD-1 inhibition restored T cell immunity in cultures derived from 3 MPN patients and CTLA-4 inhibition restored response in one patient (n=16). Of note, following the PD-1 or CTLA-4 inhibition, the number of IFN-γ producing cells and the amount of IFN-γ produced on a per-cell-basis increased upon stimulation with mutant-CALR peptides as compared to control peptides. These results indicate that the limited MPN T cell responses to CALR mutated peptides were due to T cell exhaustion caused, at least in part, by activation of the PD-1/CTLA-4 pathway.In summary, our studies establish mutated CALR as a MPN-specific tumor antigen thereby providing a rationale for the development of immunotherapies targeting mutated CALR in patients. Moreover, our data indicate that immune checkpoint signaling might be responsible for T cell exhaustion in some MPN patients thereby supporting the use of checkpoint blockade inhibitors for treatment of these patients. DisclosuresMascarenhas:Incyte: Other: Clinical Trial Steering Committee , Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding; Merck: Research Funding; Novartis: Other: DSMB member , Research Funding; Promedior: Research Funding. Bhardwaj:Merck & Co., Inc.: Membership on an entity's Board of Directors or advisory committees; Dendreon Corporation: Membership on an entity's Board of Directors or advisory committees; Dynavax Technologies Corporation: Consultancy; Crucell: Patents & Royalties; Neostem, Inc.: Patents & Royalties.

Children With Asthma Have Impaired Innate Immunity and Increased Numbers of Type 2 Innate Lymphoid Cells Compared With Healthy Controls

BackgroundAsthma is the most frequent cause of hospitalisation among children; however, little is known regarding the effects of asthma on immune responses in children.ObjectiveThe present study aimed to evaluate cytokine responses of peripheral blood mononuclear cells (PBMCs), PBMC composition and lung function in children with and without asthma.MethodsUsing a case-control design, we compared 48 children with asthma aged 3-11 years with 14 age-matched healthy controls. PBMC composition and cytokine production including interferon (IFN)-γ, interleukin (IL)-1β, IL-5 and lL-6 following stimulation with rhinovirus-1B (RV1B), house dust mite (HDM) and lipopolysaccharide (LPS) were measured. Lung function was assessed using impulse oscillometry and nitrogen multiple breath washout.ResultsThe frequency of group 2 innate lymphoid cells were significantly higher in asthmatics and PBMCs from asthmatics had deficient IFN-γ production in response to both RV1B and LPS compared with controls (P<0.01). RV1B-induced IL-1β response and HDM-stimulated IL-5 production was higher in asthmatics than controls (P<0.05). In contrast, IL-1β and IL-6 were significantly reduced in response to HDM and LPS in asthmatics compared to controls (P<0.05). Children with asthma also had reduced pulmonary function, indicated by lower respiratory reactance as well as higher area of-reactance and lung clearance index values compared with controls (P<0.05).ConclusionOur study indicates that children with asthma have a reduced lung function in concert with impaired immune responses and altered immune cell subsets. Improving our understanding of immune responses to viral and bacterial infection in childhood asthma can help to tailor management of the disease.