Absence Of IL-4 Research Articles

Monnieriside A from Evolvulus linarioides promotes antinociceptive effects in models of inflammatory and postoperative pain in male mice

Monnieriside A (MoA) is a chromone isolated from Evolvulus linarioides. This study investigated the antinociceptive potential of MoA in mice. MoA (0.01–100 mg/kg, i.p.) inhibited nociception in the inflammatory phase of the formalin test without causing motor impairment. MoA (0.1–100 mg/kg, i.p.) also reduced hindpaw mechanical allodynia caused by either intraplantar injection of Complete Freund’s Adjuvant (CFA) or surgical paw incision to simulate postoperative pain. Postoperative antinociception was accompanied by reduced IL-1β levels in the incised paw, assessed by ELISA. The antinociceptive action of MoA (100 mg/kg, i.p.) was preserved in IL-10 knockout mice submitted to paw incision, indicating that IL-10 is not essential to the antinociceptive effect. Interestingly, MoA (100 mg/kg, i.p.) increased the expression of TGF-β in IL-10 knockout mice, which could be a compensation mechanism leading to an antinociceptive effect in the absence of IL-10.

IL-10 Is Critical for Regulation of Cytotoxic CD4+NKG7+ T Cells in Lung Allograft Rejection but Is Not Required for Allograft Acceptance.

Lung transplant remains the primary therapeutic option for patients with end-stage lung disease, but long-term survival rates remain suboptimal compared with other solid organ transplants. Acute cellular rejection (ACR) is a significant challenge in lung transplant recipients, with T cell-mediated mechanisms playing a major role. IL-10 is known for its immunoregulatory function, although its specific role in lung allograft rejection remains unclear. Using the mouse orthotopic lung transplant model, we investigated the role of IL-10 in regulating alloeffector T cell responses. Unexpectedly, we found that IL-10 was not required for early costimulation blockade-induced allograft acceptance. However, IL-10 deficiency or blockade resulted in increased CD4+ T cell numbers, proliferation, graft infiltration, and alloeffector responses. In the absence of IL-10, CD4+ T cell responses predominated over CD8 responses during ACR in contrast to wild-type mice. Type 1 immunity (IFN-γ) responses along with elevated CD4+NKG7+ and CD4+CD107a+ responses predominated during ACR, highlighting a critical regulatory role for IL-10 in modulating CD4+ T cell alloimmune responses. We further demonstrated increased colocalization of NKG7 and CD107a in CD4+ T cells from IL-10-deficient allografts, suggesting coordination in cytotoxic activity. Together, our findings highlight a critical role for IL-10 in regulation of cytotoxic CD4+NKG7+ T cells, an effector population that needs further investigation to elucidate their role in lung allograft rejection.

IL-23 exerts dominant pathogenic functions in Crohn’s disease-ileitis

Crohn’s disease (CD), a main form of Inflammatory Bowel Diseases (IBD) is a chronic inflammatory disorder, mainly affecting the ileum. IL-12 and IL-23 are both targeted by Ustekinumab, a commonly used monoclonal antibody for IBD treatment. However, their specific roles in ileitis have not been extensively explored. Here, we utilized the TnfΔΑRE model of CD-ileitis to probe the functions of IL-12 and IL-23 by employing genetically deficient mice for their respective subunits. Our findings highlight that IL-23, rather than IL-12, plays a pivotal role in the progression of ileitis. IL-23 deficiency resulted in reduced immune cell infiltration in the ileum, and decreased expression of effector cytokines downstream of IL-23 signalling. Interestingly, expanding CD14+ neutrophils were highly expressing Il23ain the inflamed ileum. Furthermore, the deletion of IL-12 conferred modest additional protection only in the absence of IL-23, suggesting potential compensatory mechanisms between these cytokines. Furthermore, our study suggests that IL-23 may function independently of IL-17, as IL-17 deletion exacerbated murine ileitis, consistent with clinical studies in human CD patients using anti-IL-17 inhibitors. This research underscores the significance of targeting IL-23 in CD-ileitis, while the concurrent targeting of both IL-12 and IL-23 should be also considered as an advantageous therapeutic approach.

544 Interleukin-6 protects renal dysfunction in mouse models of hypertension and salt-sensitive hypertension

OBJECTIVES/GOALS: We are investigating the role of IL-6 in regulating renal function by measuring mean arterial pressure (MAP), renal plasma flow (RPF) and glomerular filtration rate (GFR) in wild type (WT) and IL-6-knockout (KO) mice in established mouse models of angiotensin II (AII)-dependent- hypertension and -salt-sensitive hypertension. METHODS/STUDY POPULATION: Twelve-week-old male WT and KO mice on the C57BL6 background strain were infused with vehicle (V; saline) or angiotensin II (AII; 200 ng/kg/min) for 12-14 days. Half of the AII-treatment groups were maintained on a high salt (HS; 6% NaCl) diet for the duration of the experiment, while the other half of the AII treatment groups and both vehicle groups were fed normal rat chow. MAP was continuously measured by a fluid filled catheter in conscious mice for the duration of the experiment. RPF and GFR were measured on days 12-14 in anesthetized mice by the para-aminohippurate, and fluorescein isothiocynate-Inulin techniques, respectively. All data were analyzed by 2-way ANOVA; *p<0.05 vs. WT, same treatment; #p<0.05 vs.V, same genotype; ^p<0.05, AII vs. AII+HS, same genotype. RESULTS/ANTICIPATED RESULTS: MAP was 31% lower in KO vs WT mice. AII increased MAP (1.2-fold) in WT but not KO mice. HS diet magnified AII-induced increases in MAP in WT and moderately increased MAP in AII-KO mice: [MAP (mmHg): WT+V, 130±7.0; KO+V, 91.0+4.0*; WT+AII, 153±5.0#; KO+AII, 83.0±4.0*; WT+AII+HS, 150±11#; KO+AII+HS, 93.0±4.0#]. AII infusion reduced RPF in the KO but not WT mice. Addition of HS reduced RPF in WT and exacerbated AII-induced reductions in RPF in KO mice [RPF (ml/min/g): WT+V, 1.82±0.23; KO+V, 1.91+0.40; WT+AII, 3.16±0.75#; KO+AII, 1.65±0.42*; WT+AII+HS, 1.10±0.31#^; KO+AII+HS, 1.13±0.XX#^]. The HS diet reduced GFR in AII-infused KO but not WT mice [GFR (µl/min/g): WT+V, 756±XX; KO+V, 788±XX; WT+AII, 1010±63*#; KO+AII, 756±23*; WT+AII+HS, 1100±150#; KO+AII+HS: 540±210*#^]. DISCUSSION/SIGNIFICANCE: The absence of IL-6 in male mice attenuated AII- and/or AII+HS-induced increases in MAP; however, it exacerbated HS-induced reductions in RPF and GFR. These findings suggest inhibiting IL-6 has therapeutic potential as an antihypertensive but not as a renal protective agent in hypertension and salt-sensitive hypertension disease states.

Disruption of IL-17-mediated immunosurveillance in the respiratory mucosa results in invasive Streptococcus pyogenes infection.

Streptococcus pyogenes is a Gram-positive pathogen that causes a significant global burden of skin pyoderma and pharyngitis. In some cases, infection can lead to severe invasive streptococcal diseases. Previous studies have shown that IL-17 deficiency in mice (IL-17-/-) can reduce S. pyogenes clearance from the mucosal surfaces. However, the effect of IL-17 on the development of severe invasive streptococcal disease has not yet been assessed. Here, we modeled single or repeated non-lethal intranasal (IN) S. pyogenes M1 strain infections in immunocompetent and IL-17-/- mice to assess bacterial colonization following a final IN or skin challenge. Immunocompetent mice that received a single S. pyogenes infection showed long-lasting immunity to subsequent IN infection, and no bacteria were detected in the lymph nodes or spleens. However, in the absence of IL-17, a single IN infection resulted in dissemination of S. pyogenes to the lymphoid organs, which was accentuated by repeated IN infections. In contrast to what was observed in the respiratory mucosa, skin immunity did not correlate with the systemic levels of IL-17. Instead, it was found to be associated with the activation of germinal center responses and accumulation of neutrophils in the spleen. Our results demonstrated that IL-17 plays a critical role in preventing invasive disease following S. pyogenes infection of the respiratory tract.

Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms.

IL-10 Levels Differentially Regulate IFNg Signaling of CART19 and CART22

Introduction: CD19-directed CART (CART19) are highly effective in the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, some pts relapse with CD19 negative disease after CART19, which has led to the development of CD22-directed CART (CART22). While CART22 has produced 70% CR in CD19 negative ALL, this is lower than CART19 and there is less evidence for long-term disease control compared to CART19. Interestingly, toxicity patterns are different with CART22 pts experiencing a later and less severe CRS even at high disease burden. To better understand these differences in efficacy and toxicity we conducted comprehensive serum proteomic profiling on serial samples from pts treated with CART-19 or -22. Methods: Serial serum samples from pts enrolled on two clinical trials of the murine CART19 CTL019 (NCT01626495 & NCT02906371) and CART22 (NCT02650414) were examined. Pts were categorized as having severe (Grade 3 or 4) or minimal CRS (Grade 1 or 2). All statistical analyses were performed using R (v4.2.2) and PRISM (v9). Differential expression analyses were performed between CART19 and CART22 pts. Significance was defined as an absolute fold change > 2, and a false discovery rate (FDR) < 0.05. All in vitro experiments were performed in triplicate. Two-tailed t tests were calculated. Results: 43 pts (CART19 N=26 and CART22 N=17) with 223 unique timepoints were included in this analysis. All CART22 pts had minimal CRS; 13 CART19 pts had minimal and 13 had severe CRS. Both cohorts had high disease burden prior to infusion (CART19 mean = 29.9%, CART22 mean=44.3%, p=0.19). All pts who received CART22 had previously received CD19 directed immunotherapy (CART19 N=16, blinatumomab N=1). At the pre-infusion timepoint, 12 proteins were significantly different between pts in the CART19 and CART22 cohorts. The protein most significantly differentially expressed in patients who subsequently underwent CART22 treatment was the anti-inflammatory cytokine IL-10. IL-10 levels were consistently elevated in patients who received CART22, surpassing levels at all observed timepoints, even when compared with patients treated with CART19 who developed severe CRS (See Fig 1). We therefore hypothesized the IL-10 levels may be impacting the efficacy and CRS associated with CART22. We next tested the impact of IL-10 on IFNγ as a surrogate marker of CRS in a series of in vitro experiments. CART-19 or -22 cells from the same donor were co-cultured with NALM-6 cells in the presence and absence of IL-10. Without IL-10 present, CART22 cells produced higher levels of IFNγ than CART19 cells (8959 vs 6395 pg/mL, p=0.005). Inversely, with IL-10 present, CART19 cells produced more IFNγ than CART22 cells (21006 vs 15157 pg/mL, p=0.05). We evaluated the length of immune synapse (IS) formation, the time between CART cell interaction and blast killing, using live-cell microscopy, and found that CART22 cells had a trend towards a prolonged IS formation relative to CART19 cells (median time to detachment 9.1 min vs. 3.5 min, p=ns). Finally, given the differences in time of onset of CRS following CART19 vs CART22, we looked at levels of IFNγ and its canonical responder CXCL9 between pts with minimal CRS only. As expected, we found a later peak of IFNγ in CART22 pts compared to CART19 pts (Figure 2). Interestingly, despite similar peaks in IFNγ, CXCL9 levels were higher in CART22 pts, implying dysregulation of the IFNγ/CXCL9 axes in CART22 but not CART19 pts. Conclusions: Comparing comprehensive serum proteomic profiling between CART19 and CART22 pts we make several novel observations. First, IL-10 levels are significantly higher in CART22 pts at pre-infusion, suggesting that previous immunotherapy has caused an upregulation of this anti-inflammatory cytokine. Second, we show that IL-10 levels have a differential impact on IFNγ levels in CART19 vs CART22 cells. This may impact both CRS associated with CART cells and efficacy and may contribute to observed differences in IFNγ and CXCL9 levels seen between the two patient populations. These findings are significant for the clinical translation of CART22 and other immunotherapies, as previous immunotherapy may lead to sustained alterations in the cytokine milieu. Furthermore, IL-10 is a targetable cytokine that could be modulated in future studies to improve efficacy of CART22.

Self-Supporting Human Acute Myeloid Leukemia (AML) Bone Marrow Organoids Established from Patient-Derived Xenografts Mimic the Human AML Microenvironment, with Distinct Cytokine Kinetics Correlating with Dynamic Cell Proliferation and Metabolic Shifts

Hallmarks of acute myeloid leukemia (AML) include (1) formation of a self-supporting and self-regulating tumor microenvironment (TME) in the bone marrow (BM) rendering pathological niches that orchestrate retention, proliferation and survival of AML blasts, and (2) a dynamic metabolism that promotes therapy resistance and disease progression. Efforts to understand AML biology and enhance current therapies are hampered by the paucity of state-of-the-art human models that can recreate the three-dimensional (3D) BM TME and recapitulate the heterogeneity, complexity, dynamics, and metabolic shifts observed in human disease. Previously, we described the development of the first long-term self-propagating serum- and cytokine-free human BM organoid generated from an AML patient-derived xenograft (PDX) that captured metabolic shifts during AML progression. Here, we characterize the dynamic changes observed in the self-constituted TME with respect to AML proliferation, metabolism, and upon reconstitution of secondary organoids. BM was collected from NSG mice engrafted with human primary AML cells (PDX-AML model) or naïve control mice and seeded into polyurethane scaffolds in serum- and cytokine-free medium at 4e6 cells per scaffold, optimized previously. Scaffolds were recharged with autologous PDX-AML BM on day (D) 13 and cultured until D70. Organoids were assessed for (1) inter- and intra-scaffold AML kinetics, (2) the TME, (3) metabolic composition and (4) propagation into secondary organoids. AML cell kinetics from organoid culture supernatants reflected those of scaffold-extracted populations (n=3). CD33 +CD44 + AML cells expanded and/or were maintained from 7% (D0) to >10% (D70), being >15% at most culture timepoints. Confocal microscopy revealed reconstitution of typical AML-TME niche interactions within organoids, including expression of fibronectin, VCAM-1 and N-Cadherin, and formation of proliferative niches composed of AML cells with heterogeneous Osteopontin- and Osterix-expressing support cells. Human AML-supportive cytokines were consistently observed in organoid supernatants (N=3), with SDF-1, IL-1α, IL-8, GM-CSF, FGF-2 and Osteopontin present throughout the culture; some cytokines had biphasic kinetics with factors critical for TME formation present early (e.g. Osteopontin, GM-CSF), and those required for AML proliferation dominant later (e.g. FLT3L, IL-8), coinciding also with metabolic shifts during culture. Despite the absence of IL-6, single molecule RNA fluorescence in situ hybridization confirmed IL-6 expression by niche-resident AML blasts suggesting autocrine signaling networks beyond those identified in supernatants. Cells from D70 organoids were able to re-establish secondary organoids (n=2) which also exhibited long-term (D70) AML maintenance (>11% blasts), and regenerated the AML TME. Previously, we identified 3 culture stages defined by metabolic shifts: 1) pre/early recharge (D4-D19), (2) intermediate (D25-D40) and, (3) late (from D55) where organoids exhibited highest proliferation. To further characterize these shifts, intracellular metabolites were extracted from organoids (n=5) at D10, D25, D40, D55 and D70, analyzed with gas-chromatography mass spectrometry (GC-MS) and evaluated through unsupervised hierarchical clustering and multivariate analysis. Significance Analysis of Microarrays (SAM) analysis demonstrated dysregulation of alanine, serine, isoleucine, glycolate, hexanoic acid and glycolysis when comparing stage 1 with stage 2, and in isoleucine, leucine, serine, succinate and tryptophan metabolism when comparing stage 2 with stage 3. When proliferative and non-proliferative timepoints were compared, amino acid and glycolysis-driven metabolic switches were identified, typical of AML metabolism. We have characterized a long-term 3D human AML BM biomimicry in serum- and cytokine-free conditions with a self-organizing TME that recapitulates many features of AML BM, including robust human AML cell maintenance within niches, cytokine and metabolic kinetics to support different phases of progression, and the ability to propagate into secondary organoids. This dynamic, physiologically-relevant ex vivo platform can enable studies in human AML pathogenesis/progression and is poised to test novel therapeutics directed towards AML cells, the TME and metabolic targets.

Co-Expression of an IL-15 Superagonist Facilitates Self-Enrichment of GD2-Targeted CAR-NK Cells and Mediates Potent Cell Killing in the Absence of IL-2.

In contrast to T lymphocytes, natural killer (NK) cells do not require prior sensitization but are rapidly activated upon encountering virally infected or neoplastic cells. In addition, NK cells can be safely applied in an allogeneic setting, making them important effector cells for the development of off-the-shelf therapeutics for adoptive cancer immunotherapy. To further enhance their therapeutic potential, here, we engineered continuously expanding NK-92 cells as a clinically relevant model to express a humanized second-generation chimeric antigen receptor (CAR) with a composite CD28-CD3ζ signaling domain (hu14.18.28.z) that targets the disialoganglioside GD2, which is expressed at high levels by neuroblastoma cells and other tumors of neuroectodermal origin. In a separate approach, we fused an IL-15 superagonist (RD-IL15) to the GD2-CAR via a P2A processing site. Lentivirally transduced NK-92/hu14.18.28.z and NK-92/hu14.18.28.z_RD-IL15 cells both displayed high and stable CAR surface expression and specific cytotoxicity toward GD2-positive tumor cells. GD2-CAR NK cells carrying the RD-IL15 construct in addition expressed the IL-15 superagonist, resulting in self-enrichment and targeted cell killing in the absence of exogenous IL-2. Furthermore, co-culture with RD-IL15-secreting GD2-CAR NK cells markedly enhanced proliferation and cytotoxicity of bystander immune cells in a paracrine manner. Our results demonstrate that GD2-CAR NK cells co-expressing the IL-15 superagonist mediate potent direct and indirect antitumor effects, suggesting this strategy as a promising approach for the further development of functionally enhanced cellular therapeutics.

A chemically inducible IL-2 receptor signaling complex allows for effective in vitro and in vivo selection of engineered CD4+ T cells

Engineered Tcells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of invivo cytokine support can lead to poor engraftment of transferred Tcells, including regulatory Tcells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary Tcells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ Tcells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ Tcells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, invivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both invitro enrichment and invivo engraftment and activation, features likely beneficial across multiple gene-edited Tcell applications.

Elucidating Inflammation-induced Cell Fate Decisions in Primary Human Tregs

Abstract Adoptive regulatory T-cell (Treg) therapy is an emerging therapeutic paradigm for promoting immune tolerance in transplant and autoimmune disease settings. Prior investigations demonstrate that murine Tregs can undergo epigenetic reprogramming within chronically inflamed tissue environments, resulting in acquisition of proinflammatory functions and the capacity to exacerbate tissue damage. Despite the ramifications of Treg lineage decommitment for cell therapy applications, inflammation-induced human Treg cell fate decisions remain poorly understood. Here, we present a robust in vitromodel of IL6, IL1β, and IL23-driven Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3conserved non-coding sequence (CNS)2 enhancer re-methylation, diminished in vitrosuppressive function, and elevated proinflammatory cytokine expression. To gain insight into the gene regulatory networks enabling the loss of Treg identity, we generated single-cell transcriptomic and chromatin accessibility profiles of primary human Tregs maintained in the presence or absence of IL6, IL1β, and IL23. Unsupervised clustering revealed a dysfunctional Treg population with an epigenetic signature consistent with murine Treg to “exTreg” conversion, including altered chromatin accessibility at the IFNγ, IL17A, and FOXP3CNS2 loci. Inference of transcription factor (TF)-associated changes in chromatin accessibility indicated a key role for E26 transformation-specific (ETS) family members. Ongoing experiments aim to identify specific TF modules that can be targeted to better safeguard the function and stability of Treg therapeutics.

Role of Src-PI3K signaling activation in regulation of STAT5b-deficient NK cell function in response to IL-2

BackgroundMutations in the Signal Transducer and Activator of Transcription (STAT)5b, are associated with low numbers of NK cells and poor cytolytic function. IL-2 enhances cytotoxicity and requires STAT5b as a downstream effector. It can also induce Src protein activation to promote lytic granule convergence, an essential step in mobilizing lytic granules to the immunologic synapse for cytotoxicity. Subsequently, after lytic granule convergence and conjugation of the NK cell to the target cell activation of PI3K signaling pathway activation leads to target lysis. We have previously shown that IL-2 can partially rescue NK cell cytotoxicity in STAT5b-deficient cells. However, how IL-2 stimulation induces PI3K activity outside of IL-2/STAT5b signaling is not entirely clear. Using STAT5b-deficient NK cells (p. Q41X) and STAT5b-KD NK cell lines as a human model,we elucidate the role of the non-canonical IL-2 signaling pathway through Src, MEK, ERK, and pS6 activation. MethodsWe used multiparametric flow cytometry, functional NK cell assays (standard Cr51 release assay), and microscopy to elucidate the effect of non-canonical IL-2 signaling in human STAT5-deficient NK cells (p.Q41X) and conditional doxycycline STAT5-KD YTS and NK92 NK cell lines. STAT5b expression was evaluated by Western blot. Src, MEK, ERK1/2, and pS6 activationwere evaluated in primary NK cells and STAT5-KD NK cell lines by WB and FC in presence or absence of IL-2. ResultsThe STAT5b-KD NK cell lines mimics the abnormalities observed in primary STAT5b-deficient NK cells (p.Q41X). Mutant primary and cell lines showed abnormal NK cell maturation, decreased convergence of lytic granules to the microtubule-organizing center (MTOC) leading to impaired NK cell cytotoxic capacity partially restored after IL-2 stimulation. We measure downstream mediators of PI3 K activation (MEK, ERK, and pS6) after IL-2 and observed increase levels of pAKT and pERK compared to HD andWT with lower levels of pS6. The activation of these protein correlated with lytic granule convergence and restored cytotoxicity suggesting that IL-2 through Src activates PI3 K by way of pAKT and pERK. ConclusionsOur results suggest restored granule convergence and partial improvement in NK cell killing happens through the non-canonical IL-2 pathway by Src-PI3K-MEK-ERK activation.

IL-4 acts through aryl hydrocarbon receptor to antagonize TLR7 induced double negative 2 B cells in lupus

Abstract We recently showed that in systemic lupus erythematosus (SLE), IL-4R signaling is a powerful antagonist that can effectively suppress the development of activated naïve (aNAV) and CD11c +T-bet +IgD −CD27 −double negative 2 (DN2) B cells promoted by both type I and type II IFNs. In the present study, we used the BXD2 mouse model of lupus to determine the mechanism of IL-4 in suppressing the development of DN2 B cells in vivo. Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentages of CD11c +T-bet +IgD −B cells. Feature-barcoding single cell RNA-sequencing analysis showed that IL-4 modulated B cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23 +CD21 −follicular B cells. IL-4 induced the gene encoding interleukin-4-induced1 (IL4i1), an enzyme that metabolizes aromatic amino acids and this was associated with the upregulation of aryl hydrocarbon receptor (AhR) and downstream genes. Metabolomics analysis revealed IL-4 induced AhR agonistic metabolites in B cells including kynurenine (Kyn), indole-3-acetic acid, and indole-3-lactic acid. In the absence of IL-4, Kyn and a potent AhR agonist, formylindolo[3,2-b]carbazole (FICZ), significantly suppressed TLR7 plus IFNβ-induced DN2 B-cell development in vitro. Our results suggest that IL-4 acts through the IL4i1-AhR pathway to inhibit B-cell regulatory response to TLR7 and type I IFN. Identification of small molecular metabolites that act directly in B cells to induce homeostasis may lead to development of orally dosed metabolome modulating therapeutics efficacious in the treatment of SLE. This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311.

Abstract LB096: IL15-engineered tumor infiltrating lymphocytes (cytoTIL15TM) exhibit activity against autologous tumor cells from multiple solid tumor indications without IL2

Abstract Tumor infiltrating lymphocyte (TIL) therapy has shown promising results in the treatment of metastatic melanoma. However, TIL therapy has conventionally required co-administration of IL2, which is associated with toxicity in patients. We previously showed that melanoma TILs engineered to express membrane-bound IL15 (mbIL15) under the control of the ligand acetazolamide (ACZ) can achieve IL2-independent expansion during manufacturing, antigen-independent persistence in vitro and anti-tumor efficacy in vivo. In the current study, we extend the cytoTIL15 cell therapy product concept to indications beyond melanoma including non-small cell lung cancers (NSCLC), triple-negative breast cancers (TNBC) and head and neck squamous cell carcinomas (HNSCC), tumor types which represent significant unmet medical needs, particularly in the post-checkpoint inhibitor refractory setting. TILs from primary NSCLC, HNSCC and TNBC were engineered to express mbIL15 in the presence of ACZ and expanded in the absence of IL2 using our proprietary rapid expansion protocol (REP). CytoTIL15 cells were predominantly CD8 positive, enriched for mbIL15 expression and maintained T cell receptor variable beta chain (TCRVβ) diversity throughout expansion. In vitro antigen- and cytokine-independent survival and polyfunctionality of cytoTIL15 cells was measured from cultures that included ACZ. To assess anti-tumor activity, cytoTIL15 cells were co-cultured with autologous patient-derived cell lines (PDc) or tumor digests from patient-derived xenografts (PDx), and cytotoxicity and IFNγ release into supernatant was measured. In vitro, cytoTIL15 cells + ACZ exhibited similar or increased polyfunctionality compared to unengineered TIL + IL2. Unlike unengineered TILs, cytoTIL15 cells + ACZ persisted in an antigen-free setting without IL2, were cytotoxic to autologous PDc and released IFNγ in response to autologous PDx tumor digest. Taken together, these data show that IL2-independent, fully functional cytoTIL15 cells can successfully be generated from tumors such as NSCLC, HNSCC & TNBC, which afflict large numbers of patients. Citation Format: Kyle D. Pedro, Rachel Burga, Alonso Villasmil Ocando, Meghan Langley, Gauri Kulkarni, Zheng Ao, Benjamin Primack, Theresa Ross, Violet Young, Jeremy Tchaicha, Michelle Ols, Jan Ter Meulen. IL15-engineered tumor infiltrating lymphocytes (cytoTIL15TM) exhibit activity against autologous tumor cells from multiple solid tumor indications without IL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB096.

Concomitant inhibition of PPARγ and mTORC1 induces the differentiation of human monocytes into highly immunogenic dendritic cells

Monocytes can differentiate into macrophages (Mo-Macs) or dendritic cells (Mo-DCs). The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the differentiation of monocytes into Mo-Macs, while the combination of GM-CSF/interleukin (IL)-4 is widely used to generate Mo-DCs for clinical applications and to study human DC biology. Here, we report that pharmacological inhibition of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the presence of GM-CSF and the absence of IL-4 induces monocyte differentiation into Mo-DCs. Remarkably, we find that simultaneous inhibition of PPARγ and the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) induces the differentiation of Mo-DCs with stronger phenotypic stability, superior immunogenicity, and a transcriptional profile characterized by a strong type I interferon (IFN) signature, a lower expression of a large set of tolerogenic genes, and the differential expression of several transcription factors compared with GM-CSF/IL-4 Mo-DCs. Our findings uncover a pathway that tailors Mo-DC differentiation with potential implications in the fields of DC vaccination and cancer immunotherapy.

DIAGNOSTIC CRITERIA OF ABDOMINAL SEPSIS IN PATIENTS WITH POSTOPERATIVE PERITONITIS CAUSED BY FAILURE OF THE GASTROINTESTINAL TRACT SUTURES

The aim is to study changes in nonspecific resistance and cytokine regulation in patients with postoperative peritonitis with abdominal sepsis.
 Materials and methods. In the Ivano-Frankivsk Regional Clinical Hospital of the Ivano-Frankivsk Regional Council hospital in the surgery department 456 patients were treated with postoperative peritonitis during 2000 -2021. Immunological studies were conducted in 66 patients with various degrees of severity of abdominal sepsis and its complications.
 Research results. In patients with abdominal sepsis with postoperative peritonitis caused by the failure of sutures of the gastrointestinal tract, the initial level of CD11a+ is 1.6 times lower than that of control group patients. On the first day after relaparotomy, the level of CD11a+ cells in the blood increased by 1.8 times. On the 7th day of the postoperative period, this indicator corresponded to the control one.
 The initial level of CD16+ cells in the blood, as well as their content on the first day of the postoperative period, did not differ significantly from the control indicators. However, on the seventh day after the operation, this indicator was twice as high as in the control group. In patients with abdominal sepsis with postoperative peritonitis during the entire observation period, the expression of HLA-DR+ molecules on immunocompetent cells did not differ from that in the control group.
 Violation of immune recognition mechanisms was confirmed by the low expression level of HLA-DR+ molecules on immunocompetent cells, which practically did not change in the postoperative period and was twice as low as in the control group during all observation periods.
 The initial level of IL-2 was equal to the control group. On the first day after the operation, the concentration of IL-2 in the blood decreased by half, compared to the control level. On the seventh day after the operation, a further decrease in the concentration of IL-2 in the blood was observed by 2.2 times compared to the control level.
 The concentration of IL-4 in the plasma of patients with abdominal sepsis with postoperative peritonitis caused by the failure of the sutures of the gastrointestinal tract, who died in the postoperative period, was not determined during the entire observation period.
 The plasma content of IL-6 at the initial level was 1.7 times higher than that of the control group. This indicator was twice the control level after relaparotomy. By the seventh day of the postoperative period, its increase in 4.5 times compared to practically healthy individuals.
 Thus, the failure of sutures is accompanied by an imbalance of cytokine regulation of the immune response: a permanent decrease in the plasma concentration of IL-2, against the background of the absence of IL-4 in the blood, is combined with a progressive increase in the content of IL-6 in the blood, which creates an additional cytokine signal of suppression of the immune response. Such changes are regarded as an inadequate reaction of the immune system to a potential infection of the abdominal cavity.
 Conclusions. These changes in non-specific resistance and cytokine regulation, in particular such indicators as CD11a, CD162, CD95, CD16, HLA-DR+, IL-2, IL-4, IL-6 showed their high diagnostic informativ and sensitivity in predicting the development of abdominal sepsis in patients with postoperative peritonitis caused by failure of sutures of the gastrointestinal tract.

The role of IL-10 in regulating inflammation and gut microbiome in mice consuming milk kefir and orally challenged with S. Typhimurium.

Kefir has been suggested as a possible bacterial prophylaxis against Salmonella and IL-10 production seems to be crucial in the pathogenesis of salmonellosis in mice. This study evaluated the role of IL-10 in the inflammation and gut microbiome in mice consuming milk kefir and orally challenged with Salmonella enterica serovar Typhimurium. C57BL wild type (WT) (n = 40) and C57BL IL-10-/- (KO) (n = 40) mice were subdivided into eight experimental groups either treated or not with kefir. In the first 15 days, the water groups received filtered water (0.1 mL) while the kefir groups received milk kefir (10% w/v) orally by gavage. Then, two groups of each strain received a single dose (0.1 mL) of the inoculum of S. Typhimurium (ATCC 14028, dose: 106 CFU mL-1). After four weeks, the animals were euthanized to remove the colon for further analysis. Kefir prevented systemic infections only in IL-10-/- mice, which were able to survive, regulate cytokines, and control colon inflammation. The abundance in Lachnospiraceae and Roseburia, and also the higher SCFA production in the pre-infection, showed that kefir has a role in intestinal health and protection, colonizing and offering competition for nutrients with the pathogen as well as acting in the regulation of salmonella infectivity only in the absence of IL-10. These results demonstrate the role of IL-10 in the prognosis of salmonellosis and how milk kefir can be used in acute infections.

Knockout of IL-6 mitigates cold water-immersion restraint stress-induced intestinal epithelial injury and apoptosis.

Interleukin-6 (IL-6) is essential for maintaining intestinal epithelial homeostasis. Although cold water-immersion restraint (CWIR) stress is commonly used to induce in vivo gastric injury, it also affects intestinal epithelial permeability. Although IL-6 is increased in response to acute physiological and psychological stress, its exact effects on the pathophysiology of the intestinal epithelium in response to acute CWIR stress remain unknown. We used IL-6 knockout (KO) mice with acute CWIR modeling to investigate the effect of IL-6 deficiency on intestinal epithelial morphology and pathological damage using histological staining assays under the acute stress. We detected jejunal epithelial apoptosis using TUNEL and standard molecular experiments. CWIR caused intestinal epithelial damage, which was alleviated by the absence of IL-6, as evidenced by morphological changes and goblet cell and intestinal permeability alteration. IL-6 KO also reduced CWIR-mediated inflammatory levels and improved stress defense. Meanwhile, IL-6 deficiency decreased the intestinal epithelial apoptosis induced by CWIR administration. This IL-6 KO-led effect depended more on mitochondrial AIF signaling rather than the traditional caspase pathway. As a result, we concluded that acute CWIR-induced severe intestinal damage and jejunal epithelium apoptosis could be alleviated by IL-6 deficiency, implying a protective effect of IL-6 deficiency on the intestines under acute stress. The findings shed new light on treating CWIR-induced intestinal disorders by inhibiting IL-6 signaling.

Abstract C072: Cytokine modulation can suppress an EMT-like phenotype in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of only 11% and approximately half of patients present with distant metastasis at the time of diagnosis. PDAC is accompanied by pronounced alterations in stromal responses and immune surveillance programs, which are now recognized as some of the major drivers in PDAC tumor evolution. The role of immune modulators in driving metastatic potential in PDAC is poorly understood. We find that low expression levels of cytokine IL-23 in patients with PDAC correlate with poor differentiation status of these tumors, and thus predict poor outcomes. Consistent with this observation, we have found that a loss of host-derived IL-23 in a mouse model of pancreatic cancer promotes an EMT-like phenotype. The tumor morphology is altered and appears to be less differentiated. Additionally, we see a significant increase in the number of tumor cells expressing Zeb1 and a decrease in E-cadherin expression. We also find an increase in metastatic capacity in the absence of IL-23 in the tumor stroma. Single cell RNA sequencing of tumors injected orthotopically into IL-23 knockout mice reveals clusters of tumor cells with increases in EMT-associated genes such as Zeb1 and Vimentin and concurrent decreases in gastric identity genes. Overall, our findings indicate that IL-23 plays a role in suppressing an EMT-like phenotype in pancreatic cancer with one mechanism being through regulation of gastric lineage genes. Citation Format: Whitney Bell, Nancy Kren, Yan Wang, Yuliya Pylayeva-Gupta. Cytokine modulation can suppress an EMT-like phenotype in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C072.

DISORDERS OF IMMUNE RESPONSE IN PATIENTS WITH ACUTE NECROTIC PANCREATITIS

Summary. Aim. To study the cytokine regulation of the immune response in patients with severe acute necrotic pancreatitis (ANP).
 Material and methods. A prospective cohort study of 22 patients with severe ANP who were treated in the clinic during 2014-2020 has been performed. We studied changes in blood cells expressing clusters CDIIa+, CD162+, CD95+, CD16+, HLA-DR+ molecules, levels of interleukins IL-2, IL-4, IL-6. The efficacy of treatment was determined by the duration of the organ failure, the level of postoperative complications and mortality.
 Results. Low expression of CD11a+ and CD162+ on immunocompetent cells together with two time reduction of the content of CD95+ cells was observed in patients with severe ANP, which caused a decrease in the concentration of IL-6 at the same time with a slight increase in IL-4. A permanent reduction of the content of CD11a+-, CD162+- and CD95+-leukocytes simultaneously with a drop of the concentration of IL-4 and an excessive increase in the levels of IL-2 and IL-6 was associated with a negative course of the disease.
 Conclusions. The imbalance of cytokine regulation of the immune response develops in patients with severe ANP. Progressive and excessive increase of plasma concentrations of IL-2 and IL-6 occurs as well as complete absence of IL-4 occurs in patients with an adverse course of the disease.