IFN-γ Secretion In Response Research Articles

Neoantigen-augmented iPSC cancer vaccine combined with radiotherapy promotes antitumor immunity in poorly immunogenic cancers

Although irradiated induced-pluripotent stem cells (iPSCs) as a prophylactic cancer vaccine elicit an antitumor immune response, the therapeutic efficacy of iPSC-based cancer vaccines is not promising due to their insufficient antigenicity and the immunosuppressive tumor microenvironment. Here, we found that neoantigen-engineered iPSC cancer vaccines can trigger neoantigen-specific T cell responses to eradicate cancer cells and increase the therapeutic efficacy of RT in poorly immunogenic colorectal cancer (CRC) and triple-negative breast cancer (TNBC). We generated neoantigen-augmented iPSCs (NA-iPSCs) by engineering AAV2 vector carrying murine neoantigens and evaluated their therapeutic efficacy in combination with radiotherapy. After administration of NA-iPSC cancer vaccine and radiotherapy, we found that ~60% of tumor-bearing mice achieved a complete response in microsatellite-stable CRC model. Furthermore, splenocytes from mice treated with NA-iPSC plus RT produced high levels of IFNγ secretion in response to neoantigens and had a greater cytotoxicity to cancer cells, suggesting that the NA-iPSC vaccine combined with radiotherapy elicited a superior neoantigen-specific T-cell response to eradicate cancer cells. The superior therapeutic efficacy of NA-iPSCs engineered by mouse TNBC neoantigens was also observed in the syngeneic immunocompetent TNBC mouse model. We found that the risk of spontaneous lung and liver metastasis was dramatically decreased by NA-iPSCs plus RT in the TNBC animal model. Altogether, these results indicated that autologous iPSC cancer vaccines engineered by neoantigens can elicit a high neoantigen-specific T-cell response, promote tumor regression, and reduce the risk of distant metastasis in combination with local radiotherapy.

Transcriptomic signatures reveal a shift towards an anti-inflammatory gene expression profile but also the induction of type I and type II interferon signaling networks through aryl hydrocarbon receptor activation in murine macrophages.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a broad range of target genes involved in the xenobiotic response, cell cycle control and circadian rhythm. AhR is constitutively expressed in macrophages (Mϕ), acting as key regulator of cytokine production. While proinflammatory cytokines, i.e., IL-1β, IL-6, IL-12, are suppressed through AhR activation, anti-inflammatory IL-10 is induced. However, the underlying mechanisms of those effects and the importance of the specific ligand structure are not yet completely understood. Therefore, we have compared the global gene expression pattern in activated murine bone marrow-derived macrophages (BMMs) subsequently to exposure with either benzo[a]pyrene (BaP) or indole-3-carbinol (I3C), representing high-affinity vs. low-affinity AhR ligands, respectively, by means of mRNA sequencing. AhR dependency of observed effects was proved using BMMs from AhR-knockout (Ahr-/-) mice. In total, more than 1,000 differentially expressed genes (DEGs) could be mapped, covering a plethora of AhR-modulated effects on basal cellular processes, i.e., transcription and translation, but also immune functions, i.e., antigen presentation, cytokine production, and phagocytosis. Among DEGs were genes that are already known to be regulated by AhR, i.e., Irf1, Ido2, and Cd84. However, we identified DEGs not yet described to be AhR-regulated in Mϕ so far, i.e., Slpi, Il12rb1, and Il21r. All six genes likely contribute to shifting the Mϕ phenotype from proinflammatory to anti-inflammatory. The majority of DEGs induced through BaP were not affected through I3C exposure, probably due to higher AhR affinity of BaP in comparison to I3C. Mapping of known aryl hydrocarbon response element (AHRE) sequence motifs in identified DEGs revealed more than 200 genes not possessing any AHRE, and therefore being not eligible for canonical regulation. Bioinformatic approaches modeled a central role of type I and type II interferons in the regulation of those genes. Additionally, RT-qPCR and ELISA confirmed a AhR-dependent expressional induction and AhR-dependent secretion of IFN-γ in response to BaP exposure, suggesting an auto- or paracrine activation pathway of Mϕ.

Mycobacterial-specific secretion of cytokines and chemokines in healthcare workers with apparent resistance to infection with Mycobacterium tuberculosis.

Currently, diagnosis of latent TB infection (LTBI) is based on the secretion of IFN-γ in response to Mycobacterium tuberculosis (Mtb) antigens, the absence of which is regarded as no infection. Some individuals appear to resist Mtb infection despite sustained exposure (resisters). In this study, we aimed to assess cytokines, chemokines and antibodies that may be associated with resistance to Mtb infection. We hypothesized that there may be an alternative immune response to Mtb exposure in the absence of IFN-γ in resisters. We enrolled HIV-uninfected healthcare workers who had worked in high TB-exposure environments for 5 years or longer. We screened them for LTBI using the tuberculin skin test and the QuantiFERON-TB Gold Plus assay. We performed multiplex Luminex to measure concentrations of T cell-associated cytokines and chemokines as well as total antibodies in plasma collected from unstimulated fresh whole blood and supernatants from QuantiFERON-TB Gold Plus tubes following incubation of whole blood for 16-24 hours with ESAT6/CFP10 peptides. Samples from 78 individuals were analyzed: 33 resisters (TST<10mm; IGRA<0.35 IU/mL), 33 with LTBI (TST≥10mm and IGRA≥0.35 IU/mL) and 12 discordant (TST=0mm; IGRA≥1.0 IU/mL). There were no differences in concentrations of cytokines and chemokines in plasma between the different groups. Resisters had significantly lower concentrations of IFN-γ, IL-2, TNF-α, MIP-1α, MIP-1β, ITAC, IL-13 and GM-CSF in supernatants compared with LTBI group. There were no significant differences in the concentrations in supernatants of IL-10, IL-1β, IL-17A, IL-21, IL-23, MIP-3α, IL-4, IL-5, IL-6, IL-7, IL-8, Fractalkine and IL-12p70 between the groups. We observed that resisters had similar concentrations of total antibodies (IgG1, IgG2, IgG3, IgG4, IgA, and IgM) in plasma and supernatants compared to the LTBI and discordant groups. Resistance to Mtb infection despite sustained exposure is associated with lower Mtb-specific secretion of Th1-associated cytokines and chemokines. However, resisters showed secreted concentrations after Mtb stimulation of total antibodies and cytokines/chemokines associated with innate and Th17 immune responses similar to those with Mtb infection. This suggests an ability to mount non-IFN-γ immune responses to Mtb in apparent resisters.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Higher SARS-CoV-2 Spike Binding Antibody Levels and Neutralization Capacity 6 Months after Heterologous Vaccination with AZD1222 and BNT162b2.

Within a year after the emergence of SARS-CoV-2, several vaccines had been developed, clinically evaluated, proven to be efficacious in preventing symptomatic disease, and licensed for global use. The remaining questions about the vaccines concern the duration of protection offered by vaccination and its efficacy against variants of concern. Therefore, we set out to analyze the humoral and cellular immune responses 6 months into homologous and heterologous prime-boost vaccinations. We recruited 190 health care workers and measured their anti-spike IgG levels, their neutralizing capacities against the Wuhan-Hu-1 strain and the Delta variant using a surrogate viral neutralization test, and their IFNγ-responses towards SARS-CoV-2-derived spike peptides. We here show that IFNγ secretion in response to peptide stimulation was significantly enhanced in all three vaccination groups and comparable in magnitude. In contrast, the heterologous prime-boost regimen using AZD1222 and BNT162b2 yielded the highest anti-spike IgG levels, which were 3–4.5 times more than the levels resulting from homologous AZD1222 and BNT162b2 vaccination, respectively. Likewise, the neutralizing capacity against both the wild type as well as the Delta receptor binding domains was significantly higher following the heterologous prime-boost regimen. In conclusion, our results suggest that mixing different SARS-CoV-2 vaccines might lead to more efficacious and longer-lasting humoral protection against breakthrough infections.

Effect of different fatty acids on the proliferation and cytokine production of dairy cow peripheral blood mononuclear cells

During the transition period, dairy cows often experience negative energy balance, which induces metabolic and immunological disturbances. Our previous work has shown a relationship between the inhibition of immune functions and increased blood nonesterified fatty acid (NEFA) levels. In this study, we evaluated the effect of 11 fatty acids (palmitoleic, myristic, palmitic, stearic, oleic, linoleic, docosahexaenoic, conjugated linoleic, lauric, eicosapentaenoic, and linolenic acids) as well as a mix that represented the NEFA profile observed during the transition period at different concentrations (0, 50, 100, and 250 µM) on proliferation and cytokines secretion of lymphocytes. To assess lymphoproliferation, peripheral blood mononuclear cell (PBMC) from 5 healthy cows (166-189 d in milk) were isolated, stimulated with the mitogenic lectin concanavalin A (ConA), incubated for 72 h with or without fatty acids, and subjected to flow cytometry analysis. Our results showed that all fatty acids, except lauric acid, significantly reduced proliferation of PBMC in a dose-dependent manner. The most detrimental effect was observed with conjugated linoleic and stearic acids, where proliferation of PBMC was already inhibited at the lowest dose (50 µM). For cytokine secretion, we found that levels of IL-4 in culture supernatant of ConA-stimulated PBMC were reduced after a 24-h exposure to the lowest dose (50 µM) of oleic and palmitoleic acids. A dose of 100 µM of eicosapentaenoic acid, NEFA mixture, and myristic acid was necessary to observe a reduction in IL-4 levels. The PBMC also showed a decrease in the secretion of IFN-γ in response to lauric, linolenic, palmitoleic, and stearic acids at 50 µM and myristic acid at 100 µM. Overall, polyunsaturated fatty acids were more potent inhibitors of cytokine secretions than saturated fatty acids. In addition, we detected an inverse relationship between the melting points of fatty acids and their ability to inhibit IL-4 and IFN-γ secretions, as evidenced by greater inhibition with low-melting point fatty acids. Overall, our study confirmed that NEFA have a negative effect on some lymphocyte functions, and that their inhibitory effect on cytokine secretions increases with the degree of unsaturation.

The Effect of Delivery Systems on the Induction of T Helper 1 Cell Response to an ESAT6-Like Protein Rv3619c and Identification of Its Immunodominant Peptides

Objective: This study determined the effects of chemical adjuvants, incomplete Freund’s adjuvant (IFA) and aluminum hydroxide (Alum), mycobacteria, and a DNA plasmid as delivery systems on the induction of protective Th1 (interferon-gamma (IFN-γ)) and nonprotective Th2 (IL-5) and Treg (IL-10) cytokine responses to Rv3619c and its peptides. Rv3619c is an immunodominant Mycobacterium tuberculosis-specific antigen and belongs to the early-secreted antigenic target of 6 kDa-family of proteins. Delivery systems are needed to deliver such antigens in animal models and induce protective immune responses. Methods: The rv3619c gene was amplified from the genomic DNA of M. tuberculosis and cloned into appropriate vectors for expression in Escherichia coli, Mycobacterium smegmatis, and eukaryotic cells. Spleen cells from mice immunized with rv3619c using different delivery systems were stimulated in vitro with synthetic peptides (P1 to P6) of Rv3619c, and secreted cytokines were estimated by ELISA. Results: The recombinant M. smegmatis and DNA plasmid induced the secretion of the protective cytokine IFN-γ in response to peptide-pool of Rv3619c and all the individual peptides, whereas rv3619c/IFA induced the secretion of IFN-γ in response to the peptide pool, and the peptides P5 and P6. However, the secretions of the nonprotective cytokines IL-5 and IL-10 were induced to none of the peptides with the delivery systems used. Conclusion: Rv3619c is a major antigen of M. tuberculosis with multiple immunogenic epitopes; however, immune responses to individual epitopes can vary based on delivery systems used.

Systematic analysis of CD39, CD103, CD137, and PD-1 as biomarkers for naturally occurring tumor antigen-specific TILs.

The detection of tumor-specific T cells in solid tumors is integral to interrogate endogenous antitumor responses and to advance downstream therapeutic applications. Multiple biomarkers are reported to identify endogenous tumor-specific tumor-infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39; however, a direct comparison of these molecules has yet to be performed. We evaluated these biomarkers in primary human ovarian tumor samples using single-cell mass cytometry to compare their relative phenotypic profiles, and examined their response to autologous tumor cells ex vivo. PD-1+ , CD103+ , and CD39+ TILs all contain a CD137+ cell subset, while CD137+ TILs highly co-express the aforementioned markers. CD137+ TILs exhibit the highest expression of cytotoxic effector molecules compared to PD-1+ , CD103+ , or CD39+ TILs. Removal of CD137+ cells from PD-1+ , CD103+ , or CD39+ TILs diminish their IFN-γsecretion in response to autologous tumor cell stimulation, while CD137+ TILs maintain high HLA-dependent IFN-γ secretion. CD137+ TILs exhibited an exhausted phenotype but with CD28 co-expression, suggesting possible receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1+ , CD103+ , and CD39+ TILs are mainly derived from a subset of CD137-expressing TILs, implicating CD137 as a more selective biomarker for naturally occurring tumor-specific TILs.

Immunogenicity of Pfizer-BioNTech COVID-19 vaccine in patients with inborn errors of immunity

BackgroundIn mid-December 2020, Israel started a nationwide mass vaccination campaign against coronavirus disease 2019 (COVID-19). In the first few weeks, medical personnel, elderly citizens, and patients with chronic diseases were prioritized. As such, patients with primary and secondary immunodeficiencies were encouraged to receive the vaccine. Although the efficacy of RNA-based COVID-19 vaccines has been demonstrated in the general population, little is known about their efficacy and safety in patients with inborn errors of immunity (IEI).ObjectiveOur aim was to evaluate the humoral and cellular immune response to COVID-19 vaccine in a cohort of patients with IEI.MethodsA total of 26 adult patients were enrolled, and plasma and peripheral blood mononuclear cells were collected from them 2 weeks following the second dose of Pfizer-BioNTech COVID-19 vaccine. Humoral response was evaluated by testing anti–SARS-CoV-2 spike (S) receptor-binding domain and antinucleocapsid antibody titers and evaluating neutralizing ability by inhibition of receptor-binding domain–angiotensin-converting enzyme 2 binding. Cellular immune response was evaluated by using ELISpot, estimating IL-2 and IFN-γ secretion in response to pooled SARS-CoV-2 S- or M-peptides.ResultsOur cohort included 18 patients with a predominantly antibody deficiency, 2 with combined immunodeficiency, 3 with immune dysregulation, and 3 with other genetically defined diagnoses. Twenty-two of them were receiving immunoglobulin replacement therapy. Of the 26 patients, 18 developed specific antibody response, and 19 showed S-peptide–specific T-cell response. None of the patients reported significant adverse events.ConclusionVaccinating patients with IEI is safe, and most patients were able to develop vaccine-specific antibody response, S-protein–specific cellular response, or both.

Simulated microgravity disarms human NK-cells and inhibits anti-tumor cytotoxicity in vitro

Long-duration spaceflight impairs natural killer (NK) cell function, which could compromise immune surveillance in exploration class mission crew. To determine if microgravity can impair NK-cell function, we established a rotary cell culture system to expose human peripheral blood mononuclear cells to simulated microgravity (SMG) in vitro. We found that 12 h of SMG suppressed NK-cell cytotoxic activity (NKCA) by ~50% against K562, U266 and 721.221 tumor target cells when returned to the 1G environment. Mass cytometry was used to identify 37 individual markers associated with NK-cell activation, maturation and cytotoxicity, revealing that SMG causes reductions in NK-cell degranulation and effector cytokine production. Extended flow cytometry confirmed that SMG lowered NK cell perforin and granzyme b expression by 25% and 17% respectively, but did not affect the surface expression of various activating (NKG2D, NKp30) and inhibitory (NKG2A, KLRG1) receptors or the ability of NK-cells to conjugate with target cells. Flow cytometry further revealed that SMG impaired NK-cell degranulation (reduced CD107a+ expression) and suppressed TNFα and IFNγ secretion in response to stimulation with K562 target cells. These findings indicate that SMG ‘disarms’ human NK-cells of cytolytic granules and impairs NKCA against a range of tumor target cells in vitro. Exposure to microgravity could be a factor that contributes to impaired NK-cell function during long duration space travel.

974. PD-1 Immune Checkpoint Blockade Improves Survival and Promotes Fungal Clearance in an Immunosuppressed Murine Invasive Pulmonary Aspergillosis (IPA) Model

BackgroundCheckpoint blockade (CPB) has brought a revolution in modern oncology and may offer new strategies for antifungal immunotherapy. In vitro studies have demonstrated that blockade of the PD-1/PDL-1 interaction increased IFN-γ secretion in response to Aspergillus antigens, suggesting a potential role for anti-PD-1 therapy in promoting anti-Aspergillus immunity. We sought to evaluate the therapeutic efficacy of low-dose anti-PD-1 therapy in a murine IPA model.MethodsEight- to twelve-week-old female BALB/c mice were immunosuppressed with cyclophosphamide and cortisone acetate and infected intra-nasally with 5 × 104 of A. fumigatus Af293 conidia (panel A). Mice were then treated intraperitoneally with 4 doses of either 200 µL PBS (PBS control), 250 µg/kg BW IgG antibody (isotype control), or a monoclonal PD-1 antibody (anti-PD-1). Survival was monitored daily until day 8 post-infection. 24–28 mice per treatment were assessed in 3 independent experiments. Pulmonary fungal burden was determined by 18S qPCR either on day 8 post-infection or upon death. Additional mice were sacrificed on day 1 and 4 post-infection to assess serum concentrations of selected cytokines by ELISA.ResultsInfected mice receiving treatment with either PBS or the isotype antibody exhibited 8 day survival rates of 33% and 36%, respectively. In contrast, 68% of the mice in the PD-1 antibody treatment group survived (panel B). Accordingly, pulmonary fungal burden was significantly reduced in anti-PD-1 vs. isotype-treated infected mice (median spore equivalent: 0.39 vs. 2.06 × 109, P = 0.015). No signs of toxicity or early mortality were seen in anti-PD-1-treated mice, and no elevated serum levels of pro-inflammatory cytokines TNF-α and INF-γ were found in those mice (compared with isotype-treated infected mice).ConclusionWe found that anti-PD-1 immune checkpoint blockade has independent beneficial effects in untreated immunosuppressed mice with IPA. We are in the process of measuring pulmonary cytokines to deepen our understanding of protective anti-Aspergillus immunity conferred by low-dose CPB. In addition, future studies would address the combined application of CPB and conventional antifungal drugs that have immune-regulatory activity such as echinocandins. Disclosures All Authors: No reported Disclosures.

LFA-1 Ligation by High-Density ICAM-1 Is Sufficient To Activate IFN-γ Release by Innate T Lymphocytes.

By binding to its ligand ICAM-1, LFA-1 is known to mediate both adhesion and costimulatory signaling for T cell activation. The constitutively high LFA-1 cell surface expression of invariant NKT (iNKT) cells has been shown to be responsible for their distinctive tissue homing and residency within ICAM-rich endothelial vessels. However, the functional impact of LFA-1 on the activation of iNKT cells and other innate T lymphocyte subsets has remained largely unexplored. In particular, it is not clear whether LFA-1 contributes to innate-like pathways of T cell activation, such as IFN-γ secretion in response to IL-12. Using a recombinant ICAM-1-Fc fusion protein to stimulate human iNKT cells in the absence of APCs, we show that LFA-1 engagement enhances their IL-12-driven IFN-γ production. Surprisingly, exposure to high densities of ICAM-1 was also sufficient to activate iNKT cell cytokine secretion independently of IL-12 and associated JAK/STAT signaling. LFA-1 engagement induced elevated cytoplasmic Ca2+ and rapid ERK phosphorylation in iNKT cells, and the resulting IFN-γ secretion was dependent on both of these pathways. Analysis of freshly isolated human PBMC samples revealed that a fraction of lymphocytes that showed elevated LFA-1 cell surface expression produced IFN-γ in response to plate-bound ICAM-1-Fc. A majority of the responding cells were T cells, with the remainder NK cells. The responding T cells included iNKT cells, MAIT cells, and Vδ2+ γδ T cells. These results delineate a novel integrin-mediated pathway of IFN-γ secretion that is a shared feature of innate lymphocytes.

Identification of Tumoricidal TCRs from Tumor-Infiltrating Lymphocytes by Single-Cell Analysis.

T-cell receptor (TCR) gene therapy is a promising next-generation antitumor treatment. We previously developed a single-T-cell analysis protocol that allows the rapid capture of paired TCRα and β cDNAs. Here, we applied the protocol to analyze the TCR repertoire of tumor-infiltrating lymphocytes (TIL) of various cancer patients. We found clonally expanded populations of T cells that expressed the same clonotypic TCR in 50% to 70% of CD137+CD8+ TILs, indicating that they responded to certain antigens in the tumor environment. To assess the tumor reactivity of the TCRs derived from those clonally expanded TILs in detail, we then analyzed the CD137+CD8+ TILs from the tumor of B16F10 melanoma cells in six C57BL/6 mice and analyzed their TCR repertoire. We also found clonally expanded T cells in 60% to 90% of CD137+CD8+ TILs. When the tumor reactivity of dominant clonotypic TCRs in each mouse was analyzed, 9 of 13 TCRs induced the secretion of IFNγ in response to, and showed killing of, B16F10 cells in vitro, and 2 of them showed strong antitumor activity in vivo Concerning their antigen specificity, 7 of them reacted to p15E peptide of endogenous murine leukemia virus-derived envelope glycoprotein 70, and the rest reacted to tumor-associated antigens expressed on EL4 lymphoma as well as B16 melanoma cells. These results show that our strategy enables us to simply and rapidly obtain the tumor-specific TCR repertoire with high fidelity in an antigen- and MHC haplotype-independent manner from primary TILs. Cancer Immunol Res; 6(4); 378-88. ©2018 AACR.

GRAIL and Otubain-1 are Related to T Cell Hyporesponsiveness during Trypanosoma cruzi Infection.

BackgroundTrypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens and is characterized by decreased IL-2 synthesis. In addition, the acquisition of the anergic phenotype is correlated with upregulation of “gene related to anergy in lymphocytes” (GRAIL) protein in CD4 T cells. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during T. cruzi infection.Methodology/Principal FindingsBalb/c mice were infected intraperitoneally with 500 blood-derived trypomastigotes of Tulahuen strain, and spleen cells from control non-infected or infected animals were obtained. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenic T cells was measured by real-time PCR, flow cytometry and Western blot. We found increased GRAIL expression at the early stages of infection, coinciding with the peak of parasitemia, with these findings correlating with impaired proliferation and poor IL-2 and IFN-γ secretion in response to plate-bound antibodies. In addition, we showed that the expression of GRAIL E3-ubiquitin ligase in CD4 T cells during the acute phase of infection was complemented by a high expression of inhibitory receptors such as PD-1 and CTLA-4. We demonstrated that GRAIL expression during infection was modulated by the mammalian target of the rapamycin (mTOR) pathway, since addition of IL-2 or CTLA-4 blockade in splenocytes from mice 21 days post infection led to a reduction in GRAIL expression. Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell proliferation.ConclusionsWe hypothesize that GRAIL expression induced by the parasite may be maintained by the increased expression of inhibitory molecules, which blocked mTOR activation and IL-2 secretion. Consequently, the GRAIL regulator Otubain-1 was not expressed and GRAIL maintained the brake on T cell proliferation. Our findings reveal a novel association between increased GRAIL expression and impaired CD4 T cell proliferation during Trypanosoma cruzi infection.

Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection.

ESAT-6-dependent cytosolic pattern recognition drives noncognate tuberculosis control in vivo.

IFN-γ is a critical mediator of host defense against Mycobacterium tuberculosis (Mtb) infection. Antigen-specific CD4+ T cells have long been regarded as the main producer of IFN-γ in tuberculosis (TB), and CD4+ T cell immunity is the main target of current TB vaccine candidates. However, given the recent failures of such a TB vaccine candidate in clinical trials, strategies to harness CD4-independent mechanisms of protection should be included in future vaccine design. Here, we have reported that noncognate IFN-γ production by Mtb antigen-independent memory CD8+ T cells and NK cells is protective during Mtb infection and evaluated the mechanistic regulation of IFN-γ production by these cells in vivo. Transfer of arenavirus- or protein-specific CD8+ T cells or NK cells reduced the mortality and morbidity rates of mice highly susceptible to TB in an IFN-γ-dependent manner. Secretion of IFN-γ by these cell populations required IL-18, sensing of mycobacterial viability, Mtb protein 6-kDa early secretory antigenic target-mediated (ESAT-6-mediated) cytosolic contact, and activation of NLR family pyrin domain-containing protein 3 (NLRP3) inflammasomes in CD11c+ cell subsets. Neutralization of IL-18 abrogated protection in susceptible recipient mice that had received noncognate cells. Moreover, improved Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine-induced protection was lost in the absence of ESAT-6-dependent cytosolic contact. Our findings provide a comprehensive mechanistic framework for antigen-independent IFN-γ secretion in response to Mtb with critical implications for future intervention strategies against TB.

Anti-Human T-Lymphocyte Immunoglobulin (ATG)-Induced T Regulatory Cells and Their Soluble Factors Suppress T Cell Proliferation: Potential Role in Allogeneic Stem Cell Transplantation

Polyclonal anti-human T-lymphocyte immunoglobulin(ATG) have been recently shown, in two randomized studies, to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells is suggested as one of the possible mechanisms involved. The aim of our current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance in patients receiving intravenous ATG (ATG-Fresenius ® S, Neovii Biotech) as part of their pre HSCT conditioning.Phenotypic characterization of regulatory cells markers revealedthattwo days culture of peripheral blood mononuclear cells (PBMCs) with ATG-F (30-120 µg/ml) resulted in significant increase in CD25 expression on CD4+ T cells. The percentages of cells expressing CTLA4, GITR, CD95 and FoxP3 was also significantly elevated on CD4+ cells compared to rabbit IgG-treated PBMCs. In addition, expression of CD127 and VLA-4 molecules was significantly decreased on CD4+CD25+ cells upon ATG-F treatment (p<0.01).Next, tolerance ability of ATG-F-induced cells was examined. Addition of ATG-F-treated cells to autologous PBMCs stimulated with antiCD3/antiCD28 antibodies resulted in significant (50-75%) inhibition of cell proliferation (p<0.001), measured by CFSE and Ki67 staining. Moreover, CD69 cell expression and interferon-γ (IFNγ) proinflammatory cytokine secretion were reduced by 50-60% and 65-90%, respectively, in the presence of ATG-F-activated Treg cells (p<0.01).Importantly, addition of cyclosporine A to the induction culture with ATG-F interfered with the ATG-induced regulatory phenotype acquisition, suggesting the involvement of interleukin-2 in ATG-mediated activity.In order to purify the tolerizing population, sorting of CD4+CD25+CD127-low cells (considered as viable regulatory T cells) from ATG-F-treated culture was performed. Sorted cells demonstrated greater suppressive potency than bulked pre-sorted cell population when added to autologous stimulated PBMCs. Of note, Treg-depleted fraction was still able to suppress the proliferation, albeit less efficiently then sorted Treg cells, suggesting that ATG-F is capable to induce multiple immune suppressive cell populations that should be further defined.To explore the possible involvement of soluble factor(s)-mediated mechanisms, in addition to the involvement of cell to cell contact mechanism, conditioned medium (CM) produced by ATG-F-primed cells was applied on stimulated autologous PBMCs. Addition of CM produced by ATG-F-treated cells, but not IgG-treated cells, resulted in significant suppression (30-65%, p<0.01) of T cell proliferation and activation, indicating the presence of soluble factors secreted by ATG-F-primed suppressive cells. Indeed, significant dose- and time-dependent induction of TGFβ secretion was observed in ATG-treated cells. To this end, addition of TGFβ receptor kinase inhibitor SB-431542 interfered with suppressive activity of ATG-F-primed cells, enabling partial rescue of proliferation and IFNγ secretion in response to antiCD3/antiCD28 activation. Similar results were obtained with anti-TGFβ neutralizing antibodies.Finally, characterization of phenotype and frequencies of regulatory immune populations in peripheral blood of 26 patients undergoing allogeneic transplantation with conditioning regimen including ATG-F (15mg/kg) was performed. Consistent with our ex vivo results, transient increase in percent of circulating CD4+CD25+CD127-low cells was detected in the ATG-F treated patients on days 14, 21 and 28 after HSCT. Furthermore, elevated levels of TGFβ were detected in the patients' plasma at day 28 and remaining high at day 60 post HSCT.Our results demonstratethat in vitro treatment with ATG-F is capableto induce functional Treg cells. Suppressive ability of ATG-F-induced cells was both contact and soluble-factors dependent and was partially promoted by TGFβ. Patients undergoing allogeneic HSCT with ATG-F-including conditioning demonstrated increased frequencies of circulating Treg cells and elevated plasma levels of TGFβ. Altogether, our data further support the use of ATG-F, a potent inducer of Treg cells, for prevention of GVHD post HSCT and potentially other therapeutic applications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Feasibility and safety of cCD20 RNA CAR-bearing T cell therapy for the treatment of canine B cell malignancies

CD19 chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating human hematological malignancies. However, the full potential of CAR technology has not been reached in solid malignancies and limitations exist in utilizing genetically identical murine models to interrogate the safety and efficacy of anti-tumor immune therapies. Canines are an outbred population that naturally develop spontaneous disease. Here we have established a treatment model for CAR T cell therapy in outbred dogs with spontaneous B cell lymphoma. We developed a protocol to isolate, activate, and express CAR in canine T cells. We characterized a methodology utilizing anti-cCD3 antibody with a K562 antigen-presenting cell (APC) line that expresses human CD32 and canine CD86 to activate canine lymphocytes. This method yields robust (50-120x) expansion of cCD4+ or cCD8+ T cells from normal or lymphoma-diseased dogs. Canine T cells were then engineered utilizing mRNA electroporation to express a CAR targeting the B cell antigen CD20. An anti-canine CD20 (cCD20) CAR was selected for proof of principle studies to establish feasibility of efficient CAR expression and redirected effector function in canine T cells from a healthy and lymphoma-diseased dogs. The cCD20-Z CAR construct was efficiently, but transiently, expressed in canine T cells after mRNA electroporation. cCD20 CAR T cells exhibited specific lysis and IFN-γ secretion in response to cCD20+ target cells. These data indicate successful establishment of a method to isolate, activate, and express a fully functional CAR in canine T cells for use in adoptive immunotherapy. These results rationalized testing of this CAR approach in canines with advanced CD20+ malignancies. In an IACUC approved protocol, we administered three doses of autologous cCD20-Z RNA CAR+ T cells to a companion canine with advanced B cell lymphoma. Treatment was well-tolerated and no evidence of serious adverse events was observed. The anti-tumor effect, however, was transient, suggesting a need for product optimization. Future study will establish a lenti- or retroviral-based method to modify canine T cells to promote T cell persistence and durable anti-tumor effects in vivo. In conclusion, our study establishes the methodologies and feasibility of anti-cCD20 CAR therapy in canines with spontaneous B cell lymphoma that may represent a future clinical option for such companion pets. This work also lays the foundation for study of a large, outbred animal model that better resembles spontaneous, human malignancies to allow for improved assessment for potential toxicity risks prior to translation into human studies.

DAP12 Deficiency in Liver Allografts Results in Enhanced Donor DC Migration, Augmented Effector T Cell Responses and Abrogation of Transplant Tolerance

Liver interstitial dendritic cells (DC) have been implicated in immune regulation and tolerance induction. We found that the transmembrane immuno-adaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulated conventional liver myeloid (m) DC maturation and their in vivo migratory and T cell allostimulatory ability. Livers were transplanted from C57BL/6(H2(b) ) (B6) WT or DAP12(-/-) mice into WT C3H (H2(k) ) recipients. Donor mDC (H2-K(b+) CD11c(+) ) were quantified in spleens by flow cytometry. Anti-donor T cell reactivity was evaluated by ex vivo carboxyfluorescein diacetate succinimidyl ester-mixed leukocyte reaction and delayed-type hypersensitivity responses, while T effector and regulatory T cells were determined by flow analysis. A threefold to fourfold increase in donor-derived DC was detected in spleens of DAP12(-/-) liver recipients compared with those given WT grafts. Moreover, pro-inflammatory cytokine gene expression in the graft, interferon gamma (IFNγ) production by graft-infiltrating CD8(+) T cells and systemic levels of IFNγ were all elevated significantly in DAP12(-/-) liver recipients. DAP12(-/-) grafts also exhibited reduced incidences of CD4(+) Foxp3(+) cells and enhanced CD8(+) T cell IFNγ secretion in response to donor antigen challenge. Unlike WT grafts, DAP12(-/-) livers failed to induce tolerance and were rejected acutely. Thus, DAP12 expression in liver grafts regulates donor mDC migration to host lymphoid tissue, alloreactive T cell responses and transplant tolerance.

Class A dioscorins of various yam species suppress ovalbumin-induced allergic reactions

Context: Dioscorins, the primary storage proteins in yam tubers, of different species exhibited varying immunomodulatory activities in mice. We inferred that this might be attributed to the various isoforms in the yam tubers.Objective: We aimed to investigate the antiallergic potential of the Class A dioscorins of various yam species using the ovalbumin (OVA)-induced murine allergy model.Materials and methods: We purified the recombinant Class A dioscorins (rDioscorins) of various yam species from Escherichia coli and evaluated their antiallergic potential by enzyme-linked immunosorbent assay.Results: The Class A rDioscorins of various yam species suppressed allergic reactions by significantly decreasing the serum IgE and histamine levels. The serum IFN-γ and IgG2a levels significantly increased in all rDioscorin-treated mice. The splenocytes of the rDioscorin-treated mice also exhibited upregulated IFN-γ secretion in response to ConA stimulation. By contrast, the serum IL-5 levels decreased to basal levels in mice treated with Class A rDioscorins and the amount of IL-5 produced by splenocytes decreased in response to ConA stimulation.Discussion: The Class A rDioscorins suppress allergic reactions, possibly through modulating an imbalanced Th1/Th2 immune response to OVA by promoting Th1 cell responses. Furthermore, the Class A rDioscorins of various yam species exhibited similar immunomodulatory activities in OVA-sensitized mice, which were different from the activities demonstrated by native dioscorins, suggesting that distinct immunomodulatory effects of native dioscorins on mice were attributed to the various isoforms in the yam tubers.Conclusion: The Class A dioscorins of various yam species exhibit antiallergic activity and are potential immunotherapeutic agents for treating IgE-mediated hypersensitivity.