Antigen Challenge Research Articles

Amelioration of Cyclophosphamide Induced Immunosupression by the Hydro-Alcoholic Extract of Gymnema Sylvestre Leaves in Albino Rats

Cyclophosphamide (CP), the most commonly used anti-neoplastic agent causes immunosuppression and toxic effects on various organs that are the limiting factors of cancer treatment. It can be hypothesized that addition of new immunopotentiating agents with detoxification properties would have beneficial role in cancer therapy. Many researchers have proved that, if certain plant products are combined with cancer chemotherapeutic agents, reduce toxicities and improve tumour response. In Ayurveda, Gymnema sylvestre is commonly used for diabetes, obesity and asthma. Also it possesses anti-inflammatory, astringent and digestive properties. Reports on the immunostimulatory activity of Gymnema sylvestre leaves are available from some in vitro and in vivo experiments. With this background the present study was undertaken to evaluate the potential beneficial role of hydro-alcoholic extract of Gymnema sylvestre leaves (GSE) on cyclophosphamide induced immunnosupression in rats. In this experiment, five groups (n=6 in each) of wistar albino rats were randomly divided to receive drugs and vehicle orally for 21 days. Gr I and II received vehicle. Gr III, IV and V were administered with Levamisole 50 mg/kg, GSE 25mg/kg and GSE 50 mg/kg respectively. Except Gr I rats, all rats were injected intraperitoneally with Cyclophosphamide (100mg/kg) on day 9th and 16th of drug treatment. The effects on various organ weights, rise in Haemagglutination titre to Sheep RBC Antigen, delayed type of hypersensitivity (DTH) response to Sheep RBC, percentage of neutrophil adhesion to nylon fibre and phagocytic index from carbon clearance test were evaluated. Humoral and cellular immunity were measured from HA titre and DTH response respectively. It has been observed that, GSE 50 mg/kg significantly increased the antibody titre, percentage neutrophil adhesion and phagocytic index in CP induced immunosuppressed rats. It also restored the CP induced changes in organ weights and the DTH response at 24 and 48 hours of antigen challenge. But these effects were not comparable to that of Levamisole. Our study shows that Gymnema sylvestre reduced the CP induced immunotoxicities and therefore, it could be a safe supplement to cyclophosphamide chemotherapy.

A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer.

Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (TRM) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αβ and γδ CD8+ intraepithelial T lymphocytes (IELs) and CD4+ T cells. While the development of intestinal γδ CD8+ IELs has been extensively investigated, the origin and function of intestinal CD4+ T cells have not been clarified. We report that CCR9 signals delivered during naïve T cell priming promote the differentiation of a population of IFN-γ-producing memory CD4+ T cells, which displays a TRM molecular signature, preferentially localizes to the gastrointestinal (GI) tract and associated lymphoid tissue and cannot be mobilized by remote antigenic challenge. We further show that this population shapes the immune microenvironment of GI tissue, thus affecting effector immunity in infection and cancer.

Single Cell Profiling Reveals PTEN Overexpression in Influenza-Specific B cells in Aging HIV-infected individuals on Anti-retroviral Therapy

Memory B cells (MBC) respond to secondary antigen challenge to protect against infection and to boost immunity following vaccinations. Despite effective treatment, chronic HIV infection disturbs MBCs by reducing numbers and altering functionality due to hyper-activation and increased apoptosis leading to suboptimal antibody responses against common infectious agents. We used single cell gene expression analysis to evaluate antigen-specific memory B cells in peripheral blood of virally-suppressed HIV-infected individuals and healthy controls stratified by serum H1N1 antibody response 3 weeks post-administration of the seasonal trivalent inactivated influenza vaccine. We used a fluorescent probe to isolate influenza H1N1-specific B cells and a multiplexed and targeted RT-PCR approach to measure expression levels of 96 genes involved in B cell activation and function. Gene profiling revealed a 4-gene predictive signature containing the phosphoinositide-3 kinase (PI3K) inhibitor, PTEN, for identifying antigen-specific MBC from HIV-infected individuals compared to healthy controls. Gene co-expression analysis showed that in addition to overexpression of PTEN, there was increased co-expression of type I interferon-associated genes with PTEN on single cell level in HIV compared to controls. This study highlights the persistent defects in MBC from HIV-infected individuals and points to the PI3K signaling pathway as a target for potential immune intervention.

Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner.

Chronic viral infections are widespread among humans, with ∼8-12 chronic viral infections per individual, and there is epidemiological proof that these impair heterologous immunity. We studied the impact of chronic LCMV infection on the phenotype and function of memory bystander CD8+ T cells. Active chronic LCMV infection had a profound effect on total numbers, phenotype, and function of memory bystander T cells in mice. The phenotypic changes included up-regulation of markers commonly associated with effector and exhausted cells and were induced by IL-6 in a STAT1-dependent manner in the context of chronic virus infection. Furthermore, bystander CD8 T cell functions were reduced with respect to their ability to produce inflammatory cytokines and to undergo secondary expansion upon cognate antigen challenge with major cell-extrinsic contributions responsible for the diminished memory potential of bystander CD8+ T cells. These findings open new perspectives for immunity and vaccination during chronic viral infections.

Anti-allergic activities of 5,7-dimethoxy-3,4′-dihydroxyflavone via inhalation in rat allergic models

Various studies have shown that flavones have several pharmacological activities including anti-allergy activities. However, the bioavailability of oral flavones is very low, and whether inhaled administration can improve efficacy in respiratory disease models is unclear. In the present study, the anti-allergic activities of inhaling 5,7-dimethoxy-3,4′-dihydroxyflavone (MHF), a synthetic flavonoid, was investigated by comparison with disodium cromoglycate (DSCG) and nedocromil sodium (NS) in rat allergic models. In an anti-DNP-IgE-induced asthmatic model, inhaled MHF dose-dependently inhibited the increase in airway resistance after antigen challenge. In an ovalbumin (OVA)-induced asthmatic model, inhaled MHF showed significant suppression of airway hyperresponsiveness; a decrease in eosinophil and neutrophil counts, IL-4, IL-5 and leukotriene D4 in bronchoalveolar lavage fluid; a reduction in total IgE and OVA-specific IgE levels in serum; and suppression of eosinophil infiltration in lung tissue after antigen challenge. The efficacy of inhaled MHF was comparable to that of NS and DSCG. In conclusion, based on these findings, the report for the first time that that inhaled MHF may be a potential drug for the treatment of allergic asthma.

Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies.

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules.

IL-4 promotes stromal cell expansion and is critical for development of a type-2, but not a type 1 immune response.

IL-4 is critical for differentiation of Th2 cells and antibody isotype switching, but our work demonstrated that it is produced in the peripheral LN under both Type 2, and Type 1 conditions, raising the possibility of other functions. We found that IL-4 is vital for proper positioning of hematopoietic and stromal cells in steady state, and the lack of IL-4 or IL-4Rα correlates with disarrangement of both follicular dendritic cells and CD31+ endothelial cells. We observed a marked disorganization of B cells in these mice, suggesting that the lymphocyte-stromal cell axis is maintained by the IL-4 signaling pathway. This study showed that absence of IL-4 correlates with significant downregulation of Lymphotoxin alpha (LTα) and Lymphotoxin beta (LTβ), critical lymphokines for the development and maintenance of lymphoid organs. Moreover, immunization of IL-4 deficient mice with Type 2 antigens failed to induce lymphotoxin production, LN reorganization, or germinal center formation, while this process is IL-4 independent following Type 1 immunization. Additionally, we found that Type 1 antigen mediated LN reorganization is dependent on IFN-γ in the absence of IL-4. Our findings reveal a role of IL-4 in the maintenance of peripheral lymphoid organ microenvironments during homeostasis and antigenic challenge.

IgG Fc Receptors: Evolutionary Considerations.

Immunoglobulins (Ig), a critical component of the adaptive immune system, are present in all jawed vertebrates and through sophisticated diversification mechanisms are able to recognize antigens of almost infinite diversity. During mammalian evolution, IgG has emerged as the predominant Ig isotype that is elicited upon antigenic challenge, representing the most abundant isotype present in circulation. Along with the IgG molecule, a family of specialized receptors has evolved in mammalian species that specifically recognize the Fc domain of IgG. These receptors, termed Fcγ receptors (FcγRs), are expressed on the surface of effector leukocytes and upon crosslinking by the IgG Fc domain mediate diverse immunomodulatory processes with profound impact on several aspects of innate and adaptive immunity. FcγRs share a high degree of sequence homology among mammalian species and the ancestral locus, where the genes that encode for FcγRs are mapped, can be traced back early in mammalian evolution. FcγRs also share a number of common structural and functional properties among mammalian species and utilize highly conserved motifs for transducing signals upon engagement. Despite the high homology of FcγRs in diverse mammalian species, human FcγRs exhibit unique features relating to the gene organization, expression pattern in the various leukocyte populations, as well as affinity for human IgGs. Such inter-species differences in FcγRs biology between humans and other mammalian species represents a major limitation for the interpretation of in vivo studies on human IgG function using conventional animal models.

Proinflammatory cytokine content in the saliva of children suffering from chronic tonsillitis

We have examined 92 children aged between 6 and 15, suffering from chronic tonsillitis (CT). Tumor necrosis factor (TNF-α) and interleukin 1β and 6 (IL-1β and IL-6) contents have been defined in saliva. The control set comprised 17 healthy children. Cytokine content was defined with the enzyme multiplied immunoassay sets (Vektor Best Ltd., Russia) by enzyme-linked immunosorbent assay. The statistic analysis and data processing were carried out with statistic analysis programs (version 3.2, R Foundation for Statistical Computing, Vienna, Austria). The content of cytokines TNF-α, IL-1, IL-6 in CT children's saliva was high against the healthy children, yet the statistically significant differences were only noted for IL-6. In the CT group the median value of this factor (12.5) was significantly higher than in the control set (6.72) (p=0.01 in Mann-Whitney assessment). IL-6 was chosen as the basic factor for the mathematic model; its combinations in the form of a multi-factor logistic regression were given consideration. From out of the three possible models there was just one that had all the coefficients statistically significantly different from zero (TNF-α - IL-6). It was chosen as the basic diagnostic model for chronic tonsillitis. The created model's sensitivity is 80.4%, while its specificity is 82.4%. The revealed IL-6 dominance in saliva at CT can be is attributable to permanent antigenic challenge characteristic of the toxic allergic CT since, as previously shown, there are living proliferating microorganisms in the palatal tonsil tissues and their blood- and lymph vessels at CT. The conducted ROC-analysis has demonstrated high sensitivity and specificity of the mathematical model, which enabled us to recommend determination of IL-6 in the saliva of the children suffering from CT as an additional diagnostic criterion.

Sustained exposure to prostaglandin D2 augments the contraction induced by acetylcholine via a DP1 receptor-mediated activation of p38 in bronchial smooth muscle of naive mice.

Prostaglandin D2 (PGD2), one of the key lipid mediators of allergic airway inflammation, is increased in the airways of asthmatics. However, the role of PGD2 in the pathogenesis of asthma is not fully understood. In the present study, effects of PGD2 on smooth muscle contractility of the airways were determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In a murine model of allergic asthma, antigen challenge to the sensitized animals caused a sustained increase in PGD2 levels in bronchoalveolar lavage (BAL) fluids, indicating that smooth muscle cells of the airways are continually exposed to PGD2 after the antigen exposure. In bronchial smooth muscles (BSMs) isolated from naive mice, a prolonged incubation with PGD2 (10−5 M, for 24 h) induced an augmentation of contraction induced by acetylcholine (ACh): the ACh concentration-response curve was significantly shifted upward by the 24-h incubation with PGD2. Application of PGD2 caused phosphorylation of ERK1/2 and p38 in cultured BSM cells: both of the PGD2-induced events were abolished by laropiprant (a DP1 receptor antagonist) but not by fevipiprant (a DP2 receptor antagonist). In addition, the BSM hyperresponsiveness to ACh induced by the 24-h incubation with PGD2 was significantly inhibited by co-incubation with SB203580 (a p38 inhibitor), whereas U0126 (a ERK1/2 inhibitor) had no effect on it. These findings suggest that prolonged exposure to PGD2 causes the BSM hyperresponsiveness via the DP1 receptor-mediated activation of p38. A sustained increase in PGD2 in the airways might be a cause of the AHR in allergic asthmatics.

Human γδ T-cell receptor repertoire is shaped by influenza viruses, age and tissue compartmentalisation.

BackgroundAlthough γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T‐cell receptor (TCR) clonal expansions. We dissected anti‐viral functions of human γδ T cells towards influenza viruses and defined influenza‐reactive γδ TCRs in the context of γδ‐TCRs across the human lifespan.MethodsWe performed 51Cr‐killing assay and single‐cell time‐lapse live video microscopy to define mechanisms underlying γδ T‐cell‐mediated killing of influenza‐infected targets. We assessed cytotoxic profiles of γδ T cells in influenza‐infected patients and IFN‐γ production towards influenza‐infected lung epithelial cells. Using single‐cell RT‐PCR, we characterised paired TCRγδ clonotypes for influenza‐reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues.ResultsWe provide the first visual evidence of γδ T‐cell‐mediated killing of influenza‐infected targets and show distinct features to those reported for CD8+ T cells. γδ T cells displayed poly‐cytotoxic profiles in influenza‐infected patients and produced IFN‐γ towards influenza‐infected cells. These IFN‐γ‐producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9‐TCRγ, capable of pairing with numerous TCR‐δ chains, suggesting their significant role in γδ T‐cell immunity. Neonatal γδ T cells displayed extensive non‐overlapping TCRγδ repertoires, while adults had enriched γ9δ2‐pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ‐pairings characterised by large clonal expansions, a profile also prominent in adult tissues.ConclusionHuman TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge.

Prevention of Hearing Loss by Alteration of the Systemic Immune System

Although congenital sensorineural hearing loss (SHL) in the bilateral cochleae mainly results from genetic abnormalities, chronic SHL progressing in later life is often influenced by systemic immune disturbances, including autoimmunity, chronic inflammation, and immunosenescence. We have investigated the relationship between the inner ear and systemic immunity and reviewed the possibilities to prevent SHL, including autoimmune SHL and age-related SHL. We also demonstrated two lymphocyte populations, interleukin 1 receptor type II (IL-1R2)-positive T cells (T1R2) and naturally occurring regulatory T cells (nTregs) in CD4+ T cells, which increase with aging, suppress host immune function and promote organ degeneration. Alterations in systemic immunity by fewer microbial antigen challenges in the living environment, elimination of immune suppressive lymphocytes, or immune rejuvenation with a reconstituted thymus may contribute not only to renew the cochlear function in SHL, but also to extend the healthy life of functional organs in a vigorous and youthful body, one of humanity’s greatest dreams.

Regulatory role of capsaicin-sensitive peptidergic sensory nerves in the proteoglycan-induced autoimmune arthritis model of the mouse

ObjectiveThe regulatory role of capsaicin-sensitive peptidergic sensory nerves has been shown in acute inflammation, but little is known about their involvement in T/B-cell driven autoimmune arthritis. This study integratively characterized the function of these nerve endings in the proteoglycan-induced chronic arthritis (PGIA), a translational model of rheumatoid arthritis.MethodsPeptidergic afferents were defunctionalized by resiniferatoxin (RTX) pretreatment in BALB/c mice, PGIA was induced by repeated antigen challenges. Hind paw volume, arthritis severity, grasping ability and the mechanonociceptive threshold were monitored during the 17-week experiment. Myeloperoxidase activity, vascular leakage and bone turnover were evaluated by in vivo optical imaging. Bone morphology was assessed using micro-CT, the intertarsal small joints were processed for histopathological analysis.ResultsFollowing desensitization of the capsaicin-sensitive afferents, ankle edema, arthritis severity and mechanical hyperalgesia were markedly diminished. Myeloperoxidase activity was lower in the early, but increased in the late phase, whilst plasma leakage and bone turnover were not altered. Desensitized mice displayed similar bone spurs and erosions, but increased trabecular thickness of the tibia and bony ankylosis of the spine. Intertarsal cartilage thickness was not altered in the model, but desensitization increased this parameter in both the non-arthritic and arthritic groups.ConclusionThis is the first integrative in vivo functional and morphological characterization of the PGIA mouse model, wherein peptidergic afferents have an important regulatory function. Their overall effect is proinflammatory by increasing acute inflammation, immune cell activity and pain. Meanwhile, their activation decreases spinal ankylosis, arthritis-induced altered trabecularity, and cartilage thickness in small joints.

Transfusion with Stored Antigen-Negative Blood Impairs T Cell Cross Priming to Red Cell Alloantigen in a Subsequent Transfusion

BACKGROUND: Donor red blood cell (RBC) alloantigens are efficiently cross-presented by recipient antigen presenting cells (APCs), thereby priming recipient naive CD8 T cells; however, this process and its downstream immunological consequences are poorly understood. RBC antigen cross presentation has been linked to cytotoxic T cell responses driving bone marrow graft rejection, but in other systems it causes tolerance. The conventional dendritic cell (cDC) subset, cDC1, is required for cross-presentation of RBC antigen. Macrophages are insufficient for cross-presentation in the absence of cDC1s, but do promote RBC antigen cross-presentation by cDCs in vitro. After prolonged storage, a portion of transfused RBCs are rapidly cleared by the spleen. In murine models, transfused stored RBCs have multiple effects on splenic APCs, including architectural distortion, dendritic cell activation, and macrophage depletion via ferroptosis due to increased erythrophagocytosis. Thus, the antigen presentation machinery controlling alloimmunization to RBC antigens may be influenced by the storage duration of previously transfused RBCs.AIMS: To better understand how recent transfusions may influence the immunogenicity of future transfusions, we used a mouse model to determine whether prior transfusion of stored, antigen-matched RBCs influences CD8 T cell cross priming to antigen mismatched RBCs in a subsequent transfusion.METHODS: In an in vivo cross-priming assay, OVA-expressing RBCs from transgenic HOD mice (with RBC surface expression of hen egg lysozyme, ovalbumin (OVA), and Duffy antigens) were the transfused antigen source and naïve OT-1 CD8 T cells, which express an MHC class I restricted OVA-specific TCR transgene, were the responders. Magnetically-selected CD8+ OT-1 cells, labeled with CellTrace Far Red (CTFR), were adoptively transferred into MHC identical (H-2Kb) UBC-GFP C57BL6/J recipients. After 16 hours, these mice were transfused with fresh or stored (12 days in CPDA-1), leukoreduced UBC-GFP RBCs or PBS. Five hours after the initial transfusion, recipients were transfused again with fresh leukoreduced HOD RBCs. To determine the effect of stored RBC transfusion on T cell priming to a non-RBC associated antigen, groups of mice carrying adoptively transferred OT-1 T cells were transfused with fresh or stored RBCs and then challenged 5 hours later with soluble OVA protein (10 µg, IV). After 4 days, splenic OT-1 T cells were analyzed for proliferation (by CTFR dye dilution) and activation (by CD44 and CD122 expression).RESULTS: Proliferation and activation of OT-1 T cells in response to challenge with soluble OVA protein were significantly increased in mice that had been previously transfused with stored RBCs compared to those transfused with fresh RBCs (Fig. 1A). In contrast, OT-1 T cell activation in response to HOD RBC transfusion was decreased in mice that had previously received a stored RBC transfusion, as compared to those receiving fresh RBCs or PBS (Fig. 1B).CONCLUSION: The increased T cell priming after soluble OVA challenge in the mice previously transfused with stored vs. fresh RBCs may be related to the increased expression of co-stimulatory molecules on DCs after stored RBC transfusion. However, T cell priming to the RBC-associated antigen requires additional factors, which are disrupted by previous transfusion of stored, but not fresh, RBCs. Splenic red pulp macrophages potentiate OT-1 T cell cross priming by dendritic cells in vitro and become severely depleted after stored RBC transfusion in vivo. Thus, macrophage help may be needed for T cell cross priming to an RBC antigen in vivo, and stored RBC transfusions may disrupt macrophage function, thereby affecting the immunogenicity of subsequent RBC alloantigen exposure.Figure 1. Effect of fresh and stored RBC transfusion on priming after antigenic challenge with soluble OVA or HOD RBCs.Representative contour plots show proliferation-induced dye dilution and CD44 expression on OT-1 cells in the spleen 4 days after transfusion of fresh (left) or stored (right) RBCs followed 5 hours later with IV injection of soluble OVA protein (A) or HOD RBCs (B). Dot plots at the right of representative contour plots show compiled data from two independent experiments quantifying the percentage of activated and proliferated cells (CTFR diluted, CD44hi) in the OT-1 subgate (mean ± SEM, n = 4-9). *P < 0.05, **P < 0.01, unpaired t test. DisclosuresNo relevant conflicts of interest to declare.

Immune Response-Dependent Assembly of IMP Dehydrogenase Filaments.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to xanthosine monophosphate, the rate-limiting step in de novo guanosine monophosphate (GMP) synthesis. In cultured cells, IMPDH polymerizes into micron-scale filamentous structures when GMP synthesis is inhibited by depletion of purine precursors or by various drugs, including mycophenolic acid, ribavirin, and methotrexate. IMPDH filaments also spontaneously form in undifferentiated mouse embryonic stem cells and induced pluripotent stem cells, hinting they might function in various highly proliferative cell types. Therefore, we investigated IMPDH filament formation in human and murine T cells, which rely heavily on de novo guanine nucleotide synthesis to rapidly proliferate in response to antigenic challenge. We discovered extensive in vivo IMPDH filament formation in mature T cells, B cells, and other proliferating splenocytes of normal, adult B6 mice. Both cortical and medullary thymocytes in young and old mice also showed considerable assembly of IMPDH filaments. We then stimulated primary human peripheral blood mononuclear cells ex vivo with T cell mitogens phytohemagglutinin (PHA), concanavalin A (ConA), or antibodies to CD3 and CD28 for 72 h. We detected IMPDH filaments in 40–60% of T cells after activation compared to 0–10% of unstimulated T cells. Staining of activated T cells for the proliferation marker Ki-67 also showed an association between IMPDH filament formation and proliferation. Additionally, we transferred ovalbumin-specific CD4+ T cells from B6.OT-II mice into B6.Ly5a recipient mice, challenged these mice with ovalbumin, and harvested spleens 6 days later. In these spleens, we identified abundant IMPDH filaments in transferred T cells by immunofluorescence, indicating that IMPDH also polymerizes during in vivo antigen-specific T cell activation. Overall, our data indicate that IMPDH filament formation is a novel aspect of T cell activation and proliferation, and that filaments might be useful morphological markers for T cell activation. The data also suggest that in vivo IMPDH filament formation could be occurring in a variety of proliferating cell types throughout the body. We propose that T cell activation will be a valuable model for future experiments probing the molecular mechanisms that drive IMPDH polymerization, as well as how IMPDH filament formation affects cell function.

Pluripotent Cell-Derived Off-the-Shelf TCR-Less CAR-Targeted Cytotoxic T Cell Therapeutic for the Allogeneic Treatment of B Cell Malignancies

▪The advent of off-the-shelf chimeric antigen receptor (CAR) T cell therapeutics is widely recognized to be a major potential advancement for the treatment of cancer. Several obstacles currently hamper the broad use of CAR T cells, including the inherent variability and cost of manufacturing of autologous cellular populations, the absolute requirement for precise genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity. Here we present pre-clinical data for FT819, a first-of-kind off-the-shelf human induced pluripotent stem cell (hiPSC)-derived CAR T cell product. FT819 is defined by the precise genetic engineering of multiple targeting events at the single cell level to create a clonal master iPSC line. The engineered features include the targeted integration of a novel, modified CD19 CAR into the T cell receptor α (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease (GvHD), and the expression of a high-affinity, non-cleavable form of CD16 (hnCD16) to deliver an adjustable system to address tumor antigen escape.Through a proprietary cellular reprogramming platform, peripheral blood derived T cells are converted to hiPSCs, engineered to contain the modified CD19 CAR targeted into the TRAC locus and hnCD16, and clonally selected to create a master hiPSC line (TRAC-TiPSC, FT819). Molecular characterization of the TRAC-TiPSC master cell line by 5‘ junction, 3‘ junction and internal sequence PCR confirmed homology directed repair and bi-allelic targeting of the CD19 CAR into the TRAC locus. The origin of the clonal master cell bank was confirmed to be a TCRαβ T cell by PCR-mediated detection of TCRδ locus deletion and methyl-seq analysis of the TCRα locus. Flow cytometric analysis demonstrated the maintenance of a uniform population of hiPSCs (>95% SSEA4/TRA-1-81/OCT4/NANOG) and expression of hnCD16 transgene (>95% CD16).Utilizing our stage-specific T cell differentiation protocol, we demonstrate that the TRAC-TiPSCs yield TRAC-iT cells with uniform expression of the CAR (>95%), complete elimination of TCR surface expression and clinically enabling expansion through the manufacturing process (>50,000 fold). To confirm the lack of alloreactivity conferred by the deletion of endogenous TCR expression, mixed lymphocyte reactions were performed using TRAC-iT, primary TCR+ T cells and primary TCR+CAR+ T cells as responders and HLA-mismatched peripheral blood mononuclear cells (PBMCs) as targets. In comparison to primary T cells and primary CAR-T cells, TRAC-iT did not respond and proliferate in response to TCR stimulation or HLA-mismatched PBMCs indicating that the risk of GvHD was alleviated.In vitro functional studies established that TRAC-iT possess a potent cytotoxic T lymphocyte response to CD19 antigen challenge in a similar manner to peripheral blood CAR T cells as demonstrated by expression of markers indicative of degranulation (CD107a/b, Granzyme B), T cell activation (CD69, CD25), and production of INFγ, TNFα and IL2. Importantly, TRAC-iT targeted tumor in an antigen specific manner as verified by lysis of CD19+, but not CD19-, tumor cell lines as seen by in vitro cytolytic assays (50% killing E:T; TRAC-iT = 1:8, primary CAR-T = 1:4).In vivo studies demonstrated that TRAC-iT cells effectively control tumor progression in a mouse model of acute lymphoblastic leukemia Nalm6 (TRAC-iT versus no treatment, p<0.0001). To validate the capability of TRAC-iT to simultaneously target multiple antigens, TRAC-iT was co-cultured with mixtures of CD19+CD20+ and CD19-CD20+ tumor cells in the presence of anti-CD20 monoclonal antibody, Rituxan. In vitro cytolytic assays demonstrate that only TRAC-iT cells can effectively identify and eliminate CD19-CD20+ tumor cells when combined with Rituxan. Importantly, the antibody-dependent cellular-cytotoxicity did not appear to interfere with CAR function as TRAC-iT maintained its directed cytotoxic capacity. Collectively, these preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf product than can be effectively and safely used in the treatment of B cell malignancies in the allogeneic setting. DisclosuresClarke:Fate Therapeutics Inc.: Employment. Chang:Fate Therapeutics Inc.: Employment. Husain:Fate Therapeutics Inc.: Employment. Lai:Fate Therapeutics Inc.: Employment. Peralta:Fate Therapeutics Inc.: Employment. Stokely:Fate Therapeutics Inc.: Employment. Abujarour:Fate Therapeutics Inc.: Employment. Dinella:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Pribadi:Fate Therapeutics Inc.: Employment. Chu:Fate Therapeutics Inc.: Employment. Truong:Fate Therapeutics Inc.: Employment. Sabouri-Ghomi:Fate Therapeutics Inc.: Employment. Meza:Fate Therapeutics Inc.: Employment. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Sadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Perinatal antibiotic exposure alters composition of murine gut microbiota and may influence later responses to peanut antigen

BackgroundAccumulating evidence suggests that the gut microbiota shapes developmental processes within the immune system. Early life antibiotic use is one factor which may contribute to immune dysfunction and the recent surge in allergies by virtue of its effects on gut microbiota.Objective and methodsAs a first step towards determining whether a relationship exists between perinatal antibiotic induced changes in the gut microbiota and the later development of a peanut allergy, we exposed newborn mice to either the broad-spectrum antibiotic vancomycin or to a vehicle for 6 weeks and then used a novel murine model of peanut allergy.ResultsEarly-life treatment with vancomycin resulted in a significant shift in the gut microbiota community characterized by a reduction in the abundance of firmicutes and preponderance of inflammatory proteobacteria. Mice with an antibiotic-altered microbiota, showed a localized allergic-like response characterized by ear swelling and scratching following intra-dermal peanut antigen challenge. Likewise, circulating IgE levels were increased in antibiotic-treated mice, but no evidence of a systemic allergic or anaphylactic-like response was observed. Importantly, we utilized the naturally occurring pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), rather than the more commonly used cholera toxin, as an adjuvant together with the peanut antigen.ConclusionOur data suggest that early antibiotic exposure promotes a shift in the gut microbiota community that may in turn, influence how mice later respond to a TNF-α + antigen challenge. However, further studies verifying the capacity of microbiota restoration to protect against allergic responses will be needed to confirm a causal role of antibiotic-induced microbiota variations in promoting allergic disease phenotypes.

IL-7 receptor blockade blunts antigen-specific memory T cell responses and chronic inflammation in primates

Targeting the expansion of pathogenic memory immune cells is a promising therapeutic strategy to prevent chronic autoimmune attacks. Here we investigate the therapeutic efficacy and mechanism of new anti-human IL-7Rα monoclonal antibodies (mAb) in non-human primates and show that, depending on the target epitope, a single injection of antagonistic anti-IL-7Rα mAbs induces a long-term control of skin inflammation despite repeated antigen challenges in presensitized monkeys. No modification in T cell numbers, phenotype, function or metabolism is observed in the peripheral blood or in response to polyclonal stimulation ex vivo. However, long-term in vivo hyporesponsiveness is associated with a significant decrease in the frequency of antigen-specific T cells producing IFN-γ upon antigen restimulation ex vivo. These findings indicate that chronic antigen-specific memory T cell responses can be controlled by anti-IL-7Rα mAbs, promoting and maintaining remission in T-cell mediated chronic inflammatory diseases.

SOX4 controls invariant NKT cell differentiation by tuning TCR signaling.

Natural killer T (NKT) cells expressing the invariant T cell receptor (iTCR) serve an essential function in clearance of certain pathogens and have been implicated in autoimmune and allergic diseases. Complex effector programs of these iNKT cells are wired in the thymus, and upon thymic egress, they can respond within hours of antigenic challenges, classifying iNKT cells as innate-like. It has been assumed that the successful rearrangement of the invariant iTCRα chain is the central event in the divergence of immature thymocytes to the NKT cell lineage, but molecular properties that render the iTCR signaling distinct to permit the T cell lineage diversification remain obscure. Here we show that the High Mobility Group (HMG) transcription factor (TF) SOX4 controls the production of iNKT cells by inducing MicroRNA-181 (Mir181) to enhance TCR signaling and Ca2+ fluxes in precursors. These results suggest the existence of tailored, permissive gene circuits in iNKT precursors for innate-like T cell development.

Neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced FGF-2 in a mouse model of asthma with mixed inflammation.

Bronchial asthma is traditionally characterized by chronic allergic inflammation, including eosinophilia and elevated Th2 cytokines. Recently, IL-17-derived neutrophil infiltration was shown to correlate with asthma severity and airway remodelling. To investigate the role of IL-17-derived neutrophils in airway remodelling in chronic bronchial asthma. We utilized house dust mite antigen-induced mouse models of asthma. Intranasal sensitization and chronic antigen challenge caused a mixed allergic inflammation that included eosinophils and neutrophils (Mix-in group). We neutralized IL-17 and fibroblast growth factor (FGF-2) and investigated the mechanism of airway remodelling in the Mix-in group. The Mix-in group displayed neutrophilic infiltration and high levels of IL-17 in lung tissue. The Mix-in group also exhibited more bronchial smooth muscle hyperplasia. IL-17 neutralization decreased the magnitude of all of these effects in the Mix-in group. Antibody arrays revealed an increase in FGF-2 in the Mix-in Group relative to the Eo-ip group, and FGF-2 elevation was associated with smooth muscle hypertrophy/hyperplasia. High concentrations of neutrophil elastase enhanced E-cadherin/β-catenin signalling in bronchial epithelial cells. Neutrophil elastase inhibitor treatment decreased FGF-2 production and E-cadherin/β-catenin signalling, which inhibited smooth muscle hyperplasia. The IL-17/neutrophil axis may play an important role in airway remodelling by contributing to smooth muscle hypertrophy/hyperplasia in mixed allergic inflammation and accordingly represents an attractive therapeutic target for severe asthma.