Presence Of IL-2 Research Articles

IL-21 conditions antigen-presenting human γδ T-cells to promote IL-10 expression in naïve and memory CD4+ T-cells.

Direct interaction between T-cells exerts a major influence on tissue immunity and inflammation across multiple body sites including the human gut, which is highly enriched in 'unconventional' lymphocytes such as γδ T-cells. We previously reported that microbial activation of human Vγ9/Vδ2+ γδ T-cells in the presence of the mucosal damage-associated cytokine IL-15 confers the ability to promote epithelial barrier defence, specifically via induction of IL-22 expression in conventional CD4+ T-cells. In the current report, we assessed whether other cytokines enriched in the gut milieu also functionally influence microbe-responsive Vγ9/Vδ2 T-cells. When cultured in the presence of IL-21, Vγ9/Vδ2 T-cells acquired the ability to induce expression of the immunoregulatory cytokine IL-10 in both naïve and memory CD4+ T-cells, at levels surpassing those induced by monocytes or monocyte-derived DCs. These findings identify an unexpected influence of IL-21 on Vγ9/Vδ2 T-cell modulation of CD4+ T-cell responses. Further analyses suggested a possible role for CD30L and/or CD40L reverse signalling in mediating IL-10 induction by IL-21 conditioned Vγ9/Vδ2 T-cells. Our findings indicate that the local microenvironment exerts a profound influence on Vγ9/Vδ2 T-cell responses to microbial challenge, leading to induction of distinct functional profiles among CD4+ T-cells that may influence inflammatory events at mucosal surfaces. Targeting these novel pathways may offer therapeutic benefit in disorders such as inflammatory bowel disease.

Effects of Injectable Solutions on the Quality of Monocyte-Derived Dendritic Cells for Immunotherapy.

Therapeutic vaccines based on monocyte-derived dendritic cells have been shown to be promising strategies and may act as complementary treatments for viral infections, cancers, and, more recently, autoimmune diseases. Alpha-type-1-polarized dendritic cells (aDC1s) have been shown to induce type-1 immunity with a high capacity to produce interleukin-12p70 (IL-12p70). In the clinical use of cell-based therapeutics, injectable solutions can affect the morphology, immunophenotypic profile, and viability of cells before delivery and their survival after injection. In this sense, preparing a cell suspension that maintains the quality of aDC1s is essential to ensure effective immunotherapy. In the present study, monocytes were differentiated into aDC1s in the presence of IL-4 and GM-CSF. On day 5, the cells were matured by the addition of a cytokine cocktail consisting of IFN-α, IFN-γ, IL-1β, TNF-α, and Poly I:C. After 48 hr, mature aDC1s were harvested and suspended in two different solutions: normal saline and Ringer's lactate. The maintenance of cells in suspension was evaluated after 4, 6, and 8 hr of storage. Cell viability, immunophenotyping, and apoptosis analyses were performed by flow cytometry. Cellular morphology was observed by electron microscopy, and the production of IL-12p70 by aDC1s was evaluated by ELISA. Compared with normal saline, Ringer's lactate solution was more effective at maintaining DC viability for up to 8 hr of incubation at 4 or 22°C.

Cytokines profile in pure neural leprosy.

Pure Neural Leprosy (PNL) is a form of this long time known disease that affects only the peripheral nervous system. Since it is a rare form of the disease, its pathophisiology is still poorly understood. Describe the cytokines profile in patients with PNL. 30 Patients diagnosed with PNL in the Souza Araujo Outpatient Clinic and with cytokines evaluated were selected. They were evaluated by neurologists and diagnosed after a nerve biopsy. Serum levels of IL-1 β, IL-6, IL-10, IL-17, TNF, CCL-2/MCP-1, IFN-ϒ, CXCL-10/IP-10 and TGF-β were evaluates at the moment of the diagnosis. Neural thickening was a common clinical finding in this groups of patients. Small and medium sensitive fibers signs and symptoms were present in 92% of the patients and motor involvement in 53%. 43% of patients presented neuropathic pain and no one had neuritis TGF-beta, IL-17, CCl-2 and IP-10. CCL-2 levels were associated with demyelinating patters and IP-10 and IL-1o were associated with axonal patterns at NCS. PNL patients' cytokine profile appears to be different of other clinical forms of leprosy, with the presence of cytokines described in both tuberculoid and lepromatous leprosy. High levels of CCl-2 may be related to the presence of silent neuritis as well as the presence of IL-10. PNL is unique a form of leprosy, therefore, understanding its immunological profiles essential to better understand the disease itself.

Abatacept Enhances Natural Killer Cell Cytotoxicity Following Haploidentical HCT: A Novel Platform to Dissociate GVL from Gvhd

Background: Abatacept (Abata, CTLA4 Ig), has been explored for the past 3 decades for blockade of CD28-B7 costimulation pathway in organ transplantation. One of the disadvantages of Abata has been its sparing effect on natural killer (NK) cells, resulting in NK-mediated rejection in preclinical models. However, we explored this caveat of abatacept to augment NK cell mediated anti-leukemia effect without invoking T cell mediated GVHD, where unmodified donor lymphocyte infusions (DLI) were administered 6 hours following administration of abatacept in-vivo (Abata-DLI) to patients with advanced malignancies undergoing haploidentical (Haplo)-HCT on days +7, +21 and +35. An early and rapid proliferation of mature NK cells was observed, which correlated with an improved relapse-free survival, without an increase in GVHD. To explore the mechanistic pathway behind this phenomenon, we studied the effect of Abata on cytotoxicity as well as cytokine profile of NK cells both in-vitro as well as in-vivo (CTRI:REF/2021/08/046552). Methods: NK cells and monocytes were isolated from PBMC of healthy donors. NK cells ± monocytes, were incubated with IL2, IL15 and IL12/15/18 with or without Abata for 72 hours and 7 days. They were assessed for cytotoxicity and IFN-g and perforin expression against K562 cell lines at effector: target (E:T) ratio of 5:1 and 1:1 . In HCT recipients, samples were obtained at day +20 (baseline), +30, +45, +60, and 6, 12 and 18 months. The samples were cryopreserved and both cytotoxicity and cytokine assays were carried out in batches after thawing. Cytokine profile was analysed in total NK cells and in NKG2A+ and NKG2C+ subsets. Results: The study was carried out on 11 healthy donors and 20 haplo-HCT recipients (15 with malignant diseases receiving Abata-DLI and 5 with non-malignant diseases receiving Abata without DLI). NK cells from healthy donors showed significant increase in cytotoxicity at 72 hours in presence of Abata with IL2 and IL15 (Figure1, p<0.01) at E:T ratio of 5:1 as well as 1:1 at both 72 hours as well as 7 days, but not with IL12/15/18. The effect was independent of monocytes. Perforin tended to be upregulated at both 72 hours and 7 days with Abata and IL15. In patients receiving Abata-DLI, cytotoxicity of NK cells was significantly increased at all time points compared to baseline at E:T ratio of 5:1 and 1:1 (Fig 2, P < 0.01). NK-cytotoxicity was significantly higher than the corresponding donor samples at days +45 and +60 (p<0.05). This was particularly so in 11 patients without any event compared to 4 patients with relapse/GVHD. To evaluate if Abata-DLI had greater impact on NK-cytotoxicity, the data was compared with 5 patients who received Abata alone. This showed significantly greater cytotoxicity at days +45 and +60 in the Abata-DLI group, which was more pronounced, when only those without events were analysed from the Abata-DLI group (p<0.01). There was no significant change in cytokine profile in those receiving Abata alone, but in Abata-DLI group, IFN-g was significantly upregulated in the NKG2C+ subset at day +60 at 1:1 ratio. Perforin expression was significantly higher at days+45 and +60 at both 5:1 and 1:1 in the Abata-DLI cohort, for both NKG2C+ and NKG2A+ subsets. In the Abata-DLI group NKG2C+ subset showed significantly higher IFN-g at days+45 and +60 and upregulation of perforin at all time points. 3 patients transplanted with active leukemia, assessed beyond day +60, showed greater cytotoxicity compared to the corresponding donors at 6-18 months. Conclusion: In this study, Abata was shown to increase cytotoxicity of NK cells, independent of monocytes, in the presence of IL2 and IL15, but not with IL12/15/18. This was observed at both high as well as low E:T ratios, with significant upregulation of perforin. In Haplo-HCT with Abata-DLI, there was marked increase in NK-cytotoxicity with upregulation of both IFN-g and Perforin, when compared to baseline as well as in comparison to those receiving Abata without DLI. Our findings suggest that Abatacept directly augments NK-cytotoxicity and provides rationale for exploring Abata-augmented NK cell therapy as a novel approach to augment NK-mediated GVL effect without invoking T cell mediated GVHD

CD19-Specific CAR NK Cells Coexpressing IL-21 Exhibit Superior Expansion and Antitumor Activity Against CD19-Postive Lymphoma

Background Chimeric antigen receptor (CAR) expressing NK cells induce robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. IL-15 and IL-21 promote NK cell expansion, survival, and possess both overlapping and distinctive functions. CAR NK cells engineered to express IL-15 have shown long-term persistence and potent antitumor activity, however, the pleiotropic effects of IL-21 on CAR NK cell-mediated tumor rejection are lacking. Therefore, we compared the therapeutic potency of CD19-specific human NK cells that constitutively express IL-15 or IL-21. Methods The codon-optimized minigene encoding cytokine IL-21 or IL-15, linked with CD19-CAR containing 4-1BB costimulatory domain by a P2A sequence were synthesized and sub-cloned into the pSFG retroviral vector yielding the BBz.IL21 or BBz.IL15 retroviral construct. To produce CD19-CAR retrovirus, 293T/17 cells were transfected with a combination of plasmids containing CAR constructs, RDF plasmid and PegPam3 plasmid. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. The expression of CAR, phenotype, degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 3 post transduction. To measure IL-15 and IL-21 production, supernatants were collected from CAR NK cells cultured in the absence of IL-2 for 24h and evaluated by ELISA. Results After transduction, all constructs were stably expressed by NK cells, with construct containing IL-21 (71.23 ± 4.05 %, n = 3) demonstrating higher transduction efficiency compared with the BBz.IL15 (61.13 ± 3.43 %), but slightly lower than the BBz construct (79.63 ± 3.83 %). High levels of IL-21 were found in the supernatant of BBz.IL21 NK cells after resting 24h (3464 ± 199 pg/ml), while no detectable IL-21 was observed on BBz and BBz.IL15 NK cells. NK cells expressing BBz.IL15 produced significantly more IL-15 than BBz.IL21 and BBz NK cells as baseline (BBz.IL15: 213 ± 14 pg/ml, BBz.IL21: 61.86± 4 pg/ml, BBz: 60.56 ± 11 pg/ml). Given that IL-15 or IL-21 could potentially induce antigen-independent NK cell proliferation, we compared the ability of each NK-cell group to maintain growth in the presence of IL-2. The expansion of CAR NK cells expressing IL-21 was significantly higher than that of CAR NK cells expressing IL-15, while the number of BBz.IL15 NK cells and BBz NK cells were comparable (fold expansion in 5 days: BBz.IL21: 26.6 fold, BBz.IL15: 15.9 fold, BBz: 17.2 fold), suggesting a synergistic effect of IL-21 and IL-2 on CAR NK cell expansion. BBz.IL21 NK cells shared a similar immunophenotypic profile to BBz.IL15 NK cells, as the expression of inhibitory receptors PD1, LAG3, TIM3 and NKG2A, as well as activation receptors NKG2D and CD69 were comparable. CAR engagement by CD19-postive Raji cells specifically induced cytokines production by CD19-CAR NK cells but not by control NK cells. IL-21 coexpression significantly enhanced IFN-γ and TNF-α production compared with CAR NK cells as well as CAR NK cells expressing IL-15 (BBz.IL21 vs. BBz.IL15, P < 0.05; BBz.IL21 vs. BBz, P < 0.05; BBz.IL15 vs. BBz, P > 0.05). Moreover, a higher percentage of degranulated NK cells was observed on BBz.IL21 NK cells compared to BBz.IL15 NK cells and BBz NK cells. To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. CAR NK cells expressing IL-21 showed superior cytotoxicity, compared to CAR NK cells expressing IL-15. Conclusion CD19-CAR NK cells engineered to express IL-21 gene produced high levels of IL-21 during cultivation and enabled greater ex vivo expansion of CAR NK cells compared with CAR NK cells expressing IL-15. IL-21 coexpression induced a significant increase in IFN-γ, TNF-α production and degranulation compared with IL-15 coexpression, resulting in enhanced antitumor activity of CD19-CAR NK cells against CD19-postive lymphoma.

4-1bb Enrichment Enhances Adoptive T Cell Therapy for Hematological Malignancies Using a Novel Approach for Ex Vivo Immune Cell Priming with DC/Tumor Fusion Vaccine

Introduction: While CAR T cells have transformed outcomes for patients with hematologic malignancies, application to patients with acute myeloid leukemia (AML) is limited by lack of surface antigens that differentiate between malignant and normal hematopoietic precursors. We have developed a personalized vaccine created by fusion of patient-derived tumor with autologous dendritic cells (DCs). The vaccine presents an array of tumor antigens stimulating polyclonal immunity. In a phase II clinical trial of patients with AML in first remission, vaccination with DC/AML fusions resulted in expansion of leukemia specific T cells associated with durable disease remission. The ex vivo generation of vaccine educated T cells provides a powerful substrate of effector cells for adoptive immunotherapy that are selective and capture tumor heterogeneity. This novel adoptive cell therapy enhances T-cell potency with potential to mediate disease regression. Enrichment of vaccine-educated T cells for activated antigen-specific effector cells via agonistic 4-1bb antibody-based selection further enhances for activation and memory cell phenotype; superior T cell efficacy was observed in AML and MM models. Methods: DC/tumor fusion vaccines were generated from C57BL/6J mice DCs and syngeneic C1498 mCh/luc + AML cells or C57BL/KaLwRij and syngeneic 5TGM1 mCh/luc + MM cells, respectively. T cells isolated from spleen were co-cultured with autologous irradiated DC/tumor fusions in presence of IL-2/7/15. Vaccine-educated T cells underwent selection with agonistic 4-1bb (3H3) followed by expansion with anti-CD3/CD28 activation beads. T cells were phenotyped for markers of activation (CD25/CD69), immune checkpoint (PD1/LAG3/TIM3), and memory (CD44+CD62L-) and for enrichment (anti-rat H&L). Cytotoxicity was evaluated on luminescent assay. Mice engrafted with C1498 mCh/luc + received the T cell therapy 7 days later. Disease progression and survival was monitored for 100 days by BLI. Results: In the C1498 model, vaccine-educated T cells demonstrated evidence of immune activation (CD25+CD69+) and memory (CD44+CD62L-) phenotype compared to unstimulated naïve T-cell controls (TN) (4-fold, p=0.1143; 1.3-fold, p=0.0195, respectively). Vaccine-educated T cells selected based on 4-1bb expression showed 9-fold higher expression of activation markers (p=0.0034) while memory markers were 4.4-fold higher compared to TN (p=0.0274). Selection enriched for 4-1bb + vaccine-educated T cells resulting in enhanced antigen-specific recognition as measured by induction of IFNg expression. Tumor specificity and activation was maintained following CD3/CD28-mediated expansion. The 4-1bb positive vaccine-educated T cells showed 11-fold enhanced cytotoxicity compared to TN at 10:1 E:T (P=0.0005), while vaccine-educated T cells showed 4-fold increase (P=0.0226). Phenotypic and functional analysis support 4 days as the optimal duration of time for T-cell vaccine education. Cell fold-expansion supports 3-5 days as the optimal duration for anti-CD3/CD28 expansion. In vivo, 60% of mice treated with 4-1bb + vaccine-educated cells were alive at 60 days vs 20% treated with unselected vaccine-educated cells. In the 5TGM1 model, activation, memory, immune checkpoint phenotype and cytotoxicity were increased in vaccine-educated T cells selected for 4-1bb expression. Conclusion: Vaccine-educated T cells represent a unique platform for adoptive immunotherapy for AML further enhanced by selection for 4-1bb expression by agonistic antibody. T-cell stimulation by fusion vaccine and subsequent enrichment by agonistic 4-1bb selection enhances cytotoxicity, activation and memory cell phenotype. Thus, ex vivo vaccine stimulation and 4-1bb selection presents a novel approach for cell-based immunotherapy for AML.

Interleukin-6 upregulates extracellular matrix gene expression and transforming growth factor β1 activity of tendon progenitor cells

BackgroundProlonged inflammation during tendon healing and poor intrinsic healing capacity of tendon are causal factors associated with tendon structural and functional degeneration. Tendon cells, consisting of mature tenocytes and tendon progenitor cells (TPC) function to maintain tendon structure via extracellular matrix (ECM) synthesis. Tendon cells can succumb to tissue cytokine/chemokine alterations during healing and consequently contribute to tendon degeneration. Interleukin-(IL-)1β, IL-6 and TNFα are key cytokines upregulated in injured tendons; the specific effects of IL-6 on flexor tendon-derived TPC have not been discerned.MethodsPassage 3 equine superficial digital flexor tendon (SDFT)-derived TPC were isolated from 6 horses. IL-6 impact on the viability (MMT assay with 0, 1, 5 and 10 ng/mL concentrations), migration (scratch motility assay at 0, 10ng/mL concentration) of TPC in monolayer culture were assessed. IL-6 effect on tendon ECM and chondrogenic gene expression (qRT-PCR), TGFβ1 gene expression and activity (ELISA), and MMP-1, -3 and − 13 gene expression of TPC was evaluated.ResultsIL-6 decreased TPC viability and migration. IL-6 treatment at 10 ng/mL significantly up-regulated TGFβ1 gene expression (6.3-fold; p = 0.01) in TPC, and significantly increased the TGFβ1 concentration in cell culture supernates. IL-6 (at 10 ng/mL) significantly up-regulated both tendon ECM (COL1A1:5.3-fold, COL3A1:5.4-fold, COMP 5.5-fold) and chondrogenic (COL2A1:3.9-fold, ACAN:6.2-fold, SOX9:4.8-fold) mRNA expression in TPC. Addition of SB431542, a TGFβ1 receptor inhibitor, to TPC in the presence of IL-6, attenuated the up-regulated tendon ECM and chondrogenic genes.ConclusionIL-6 alters TPC phenotype during in vitro monolayer culture. Pro- and anti-inflammatory roles of IL-6 have been implicated on tendon healing. Our findings demonstrate that IL-6 induces TGFβ1 activity in TPC and affects the basal TPC phenotype (as evidenced via increased tendon ECM and chondrogenic gene expressions). Further investigation of this biological link may serve as a foundation for therapeutic strategies that modulate IL-6 to enhance tendon healing.

Allogeneic Veto-CAR CD8 T Cells Enabling Prolonged Graft Survival, Anti-Tumor Specificity, and Low Risk of Graft Versus Host Disease

¥ Equal contribution Studies in mice have demonstrated that CD8 T cells exhibit marked veto activity against host T cells with anti-graft specificity, enhancing engraftment after non-myeloablative T cell depleted mismatched bone marrow transplantation. To reduce the risk of GVHD associated with allogeneic CD8 veto T cells, these studies used naïve CD8 T cells stimulated against 3rd party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. Recently, we showed that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin-immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen (Or-Geva et al., Leukemia, 2019). Based on these insights, human anti-viral CD8 central memory veto cells were successfully generated from CMV and EBV positive donors. Donor PBMC collected by leukapheresis were initially depleted of CD45RA+, CD4+ and CD56+ cells, and co-cultured with dendritic cells from the same donor, pulsed with a mixture of CMV, EBV, Adeno and BKV viral peptides. Within 12 days of culture, starting with 3 days of cytokine deprivation, more than 3x10 9 highly homogenous CD8 T cells, predominantly expressing a central memory phenotype could be routinely harvested. These cells exhibited marked veto activity in-vitro with more than a 3 log-depletion of alloreactivity as measured by limiting dilution analysis. Based on these results, a first in human phase 1-2 clinical trial is currently in progress, testing the safety of such veto cells in the context of non-myeloablative haploidentical T cell-depleted hematopoietic stem cell transplantation (HSCT). Preliminary results indicate that infusion of 5-10x10 6 veto cells / kg supports engraftment with a low risk of GVHD (Champlin et al. Blood, 2022). Most attempts to use allogenic chimeric antigen receptor (CAR) T cells involve gene editing, knocking down the TCR to avoid GVHD, and eliminating MHC expression to avoid graft rejection. Our veto cell preparation could potentially offer an attractive platform for attaining allogeneic CAR T cells capable of deleting host anti-donor T cell clones by virtue of their veto activity, with a very low risk for GVHD and without the need of gene editing. Using our current protocol for production of anti-viral central memory veto CD8 T cells, we first evaluated transduction efficacy at different time points during the veto production using a retroviral CAR directed against the Her-2 antigen (N29). The scFv N29 was attached to a costimulatory determinant of CD28 and a T cell activating domain followed by a GFP reporter gene. Our results suggested that optimal CAR transduction was attained on day 5 of culture. The veto cell product harvested on day 12 exhibited 92% CD8 T cells, of which 72% expressed the transduced N29 CAR. Notably, the transduced cells continued to exhibit veto activity and functional assays showed killing of target cells in a dose dependent manner. Next, we performed similar experiments transducing veto Tcm with a clinical grade gammaretroviral vector encoding a second-generation 4-1BB co-stimulated CD19-specific CAR. A typical experiment, shown in Fig.1, demonstrates a transduction level of 66.7 % (Fig. 1B,) and confirms the central memory phenotype of the transduced cells (Fig. 1C). Notably, upon infusion of 3x10 6 veto-CD19CAR into NSG mice implanted 3 days earlier with 0.25x10 6 Nalm-6-Luc+ tumor cells (CD19+), a marked eradication of tumor cells (Fig.1D) and survival benefit (Fig.1E) were found, while infusion of 3x10 6 non-transduced veto cells were ineffective. Based on these results, we plan to initiate clinical trials using haploidentical VETO-CD19CAR for the treatment of B-cell malignancies, in combination with hematopoietic transplantation and as off the shelf cellular therapy. .

Specific PD-1 Deletion on Regulatory T Cells Leads to Enhanced Anti-Tumor Responses

Programmed cell death 1 (PD-1) is a T cell inhibitory receptor and the most extensively exploited therapeutic target of checkpoint immunotherapy in cancer. Ligation of PD-1 by its ligands PD-L1 or PD-L2 leads to inhibition of CD4 + and CD8 + T cell responses. However, very little is known about the specific role of PD-1 in regulatory T (Treg) cells. Previous studies have shown that PD-1 engagement during induction of Treg polarization by TCR/CD28 signaling in the presence of IL-2 and TGF-β promoted the generation and expansion of Treg cells with potent suppressive function. It has also been shown that patients with high numbers of PD-1 + Treg cells have unfavorable responses to PD-1 checkpoint immunotherapy. Moreover, patients who developed hyperprogressive disease after PD-1 blocking immunotherapy exhibited enhanced Treg cell proliferative ability compared to patients with non-hyperprogressive disease. In the present study, we sought to understand the role of PD-1 on Treg cells in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with FoxP3Cre-YFP ( Pdcd1 f/fFoxP3Cre) resulting in specific deletion of PD-1 in Treg cells. Under steady-state conditions, the percentage of Treg cells in the spleen and lymph nodes remained comparable between Pdcd1 f/fFoxP3Cre and Pdcd1wt/wtFoxP3Cre control mice. However, PD-1-deficient Treg cells had a more activated phenotype compared to their counterparts in control mice, with higher expression of CD69, ICOS, and PD-L1. Furthermore, Treg cells in Pdcd1 f/fFoxp3Cre mice expressed increased levels of GITR, CTLA-4, ICOS, and PD-L1 compared to endogenous CD4 + T conventional cells (CD4 +Foxp3 -, Tconv) and CD8 + T cells. To investigate the implications of these immunological changes in anti-tumor responses, we used the MC38 colon adenocarcinoma syngeneic mouse model. We found a smaller tumor size in Pdcd1 f/fFoxP3Cre mice compared to their control counterparts. Treg cells from spleens and tumor-draining lymph nodes (tdLNs) showed minimal differences in their activation state, expression of checkpoint markers, and proliferation levels, as determined by direct ex vivo assessment. Similarly, CD4 + Tconv and CD8 + T cells from spleens and tdLNs showed a very low level of activation that was comparable between the two experimental groups. In the tumor site, however, Pdcd1 f/fFoxp3Cre tumor-bearing mice had a significant reduction in the fractions of CD4 + Tconv and Treg cells and a significant increase in the fraction of CD8 + T cells compared to control tumor-bearing mice. Detailed analysis revealed that in tumor-bearing Pdcd1 f/fFoxp3Cre mice intratumoral naive and central memory CD4 + T cells were reduced whereas CD4 + T effector cells were increased. Similarly, in Pdcd1 f/fFoxp3Cre tumor-bearing mice we noticed a reduction in naïve and central memory CD8 + T cells but a significant expansion in the effector CD8 + T cells . No significant differences were detected in the expression of activation markers in tumor infiltrating Treg cells between the two groups. In contrast, in Pdcd1 f/fFoxp3Cre tumor-bearing mice, tumor infiltrating CD4 + Tconv cells displayed an activated phenotype with upregulation of PD-L1, ICOS, TIM3, CD69, and CTLA-4, downregulated PD-1, and increased IFN-γ production. Tumor infiltrating CD8 + T cells also displayed an activated phenotype with upregulation of activation markers such as CD69, ICOS, PD-L1, TIM3, and LAG3 but reduced PD-1 expression. Notably, both CD4 + Tconv and CD8 + T cells proliferated more robustly in the Pdcd1 f/fFoxp3Cre tumor-bearing mice as determined by the high expression of the proliferation marker Ki67. These results indicate that selective ablation of PD-1 in Treg cells confers a less suppressive immunological environment and allows for expansion and activation of CD4 + and CD8 + T cells, leading to anti-tumor responses from CD4 + and CD8 + T effector cells.

HBHA-IGRA and cytotoxic mediators release assays for the diagnosis of cervical tuberculous lymphadenitis.

Cervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is a serious health problem in Tunisia. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis (CNTL). Here, we evaluated the performance of heparin-binding hemagglutinin (HBHA) interferon-gamma (IFN-γ) release assay (IGRA) for the diagnosis of CTL. In addition, we evaluated granzyme B, granulysin, and perforin release assays as CTL biomarkers and assessed their potential contribution to improve HBHA-IGRA performance. Peripheral blood mononuclear cells from CTL-suspected patients were stimulated with HBHA, early secreted antigenic target 6 (ESAT-6), or purified protein derivative (PPD) for 24 h in the presence of IL-7. Cytotoxic mediators and IFN-γ release were assessed by enzyme-linked immunosorbent assay. Receiver operating characteristic curves were used to evaluate the capacity of HBHA, ESAT-6, and PPD to discriminate between CTL (n = 27) and CNTL (n = 21). After applying bivariate and multivariate analyses, IFN-γ responses to HBHA appeared to offer the best distinction between CTL and CNTL, with an area under the curve of 0.9947, associated with 95.24% and 100% sensitivity and specificity, respectively. A principal component analysis showed clear clustering of the CTL versus the CNTL groups. This clustering was mainly attributed to HBHA-induced IFN-γ, PPD-induced granzyme B, and PPD-induced IFN-γ. These results thus suggest that the HBHA-IGRA provides high diagnostic accuracy for CTL versus CNTL, with high sensitivity and specificity. Combining HBHA-induced IFN-γ and PPD-induced granzyme B improves the accuracy to identify CTL.IMPORTANCECervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is currently a major health problem in Tunisia and in several regions around the world. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis. This study demonstrates the added value of the heparin-binding hemagglutinin-interferon-gamma release assay as an immunoassay in the context of CTL.

Anti-CD19/CD8 bispecific T cell engager for the potential treatment of B cell malignancies

The administration of blinatumomab was accompanied by several adverse effects, including activation of regulatory T-cells and cytokine storm. The objective of this study was to produce and evaluate a novel αCD8/CD19 BiTE (αCD8/CD19) with the potency to directly target CD8+T-cells. In-silico studies were utilized for determining proper folding, receptor binding, and structural stability of αCD8/CD19 protein. Western blotting and indirect surface staining were used to evaluate the size accuracy and binding potency of the purified protein. Functionality was assessed for granzyme B production, cytotoxicity, and proliferation. TheαCD8/CD19recombinant protein was produced in the CHO-K1 cell line with a final concentration of 1.94 mg/l. The αCD8/CD19 bound to CD8+and CD19+cell lines and induced significant granzyme B production, cytotoxic activity and proliferation potential in the presence of IL-2 and tumor target cells. The maximum CD8+T-cell biological activity was observed on the 10th day with 10:1 effector-to-target ratio.

Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1×1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

Urinary IL-6 and IL-8 as predictive markers in bladder urothelial carcinoma: A pilot study.

Cytokines are known to be a key a factor in numerous malignancies and to exert an important regulatory role in the tumor microenvironment. Interest has grown in understanding how cytokines modulate the tumor microenvironment and which cytokines may serve as markers of the tumor process; however, a complete picture of the cytokine landscape in bladder cancer remains unclear. Fresh urine specimens with sufficient volume were collected at random intervals. The urine concentrations of IL-8 (CXCL8), CCL18, and CXCL9 were determined using the standard commercially available enzyme immunoassay. The urine concentrations of IL-6 were determined using the high sensitivity enzyme immunoassay kit. Urinary cytokine concentrations were normalized with urinary creatinine concentrations. Significantly elevated concentrations of IL-6 and IL-8 were detected in the urine from patients with urothelial carcinoma on follow-up compared to patients with benign follow-up. The presence of both IL-6 and IL-8 in the urine samples from the high grade urothelial carcinoma (HGUC) cohort revealed a clear discrimination when compared to samples from patients with benign follow-up. The presence of the combination of both IL-6 and IL-8 had a sensitivity of 90.0% and a specificity of 81.25%. Similar data were obtained when receiver operating characteristic analysis was performed on both IL-6 and IL-8 concentrations in the urine from patients with HGUC vs. the hematuria cohort. The presence of IL-6 and IL-8 in urine specimens may have predictive value for urothelial carcinoma. However, a large longitudinal study is required to statistically eliminate confounding factors and support this theory.

SAT166 Elevated Insulin Autoantibody During COVID19 Illness Associated With Significant Hypoglycemia In A Type 1 Diabetic

Abstract Disclosure: D.A. Shewmon: None. D.C. Preston: None. Background: Insulin antibodies are a rare cause of hypoglycemia in diabetes mellitus type 1 (DM1). Exacerbation of this phenomenon by COVID-19 has not been previously reported. Clinical Case: A 46 year old, 60 kg female with DM1 of 19 year duration with hypoglycemic unawareness and retinopathy, who was admitted for flu-like symptoms, a blood glucose of 600 mg/dL in the absence of DKA, and COVID-19 by RT-PCR test. Hospitalization was complicated by 48 hours of hypoglycemia in the absence of insulin treatment, requiring continuous 10% dextrose infusion. Four months earlier the patient had been admitted for an undiagnosed non-COVID flu-like illness, with hypoglycemia for 60 hours in the absence of insulin treatment. Her glucose control was stable on 5 units of insulin per day at five and nine days after admission, and 25 units per day at 35 days after admission. At both admissions, the workup included a normal cortrosyn stimulation test, an undetectable C-peptide, a normal IGF-2 level, and a careful investigation to rule out evidence for self-administration of insulin. However insulin antibodies were elevated at 1.17 +/- 0.56 U/mL (n=4, normal <0.4) during the non-COVID illness, and 5.0 U/mL and 5.4 U/mL, during and 35 days after the COVID-19 illness, respectively. Thus insulin antibodies were 13 times the upper limit of normal during COVID-19 illness versus 2.9 times the upper limit of normal during the non-COVID-19 illness. Viral infection is one of the most significant environmental factors that may cause overstimulation of the immune system and has been shown to be associated with autoantibody generation and increase, in addition to autoimmune diseases. Known cytokine profiles on COVID-19 infections include IL-1, IL-6, IL-12, IFN-gamma, and TNF-alpha. The presence of IL-1 and TNF-alpha in particular, are known to be key pro-inflammatory cytokines in myriad autoimmune disorders. In vitro data suggest that IL-1 is also an important effector cytokine in DM1, through a number of direct (i.e., beta cell toxicity) and indirect means (i.e., by marking beta cells for Fas-dependent destruction by autoreactive cytotoxic T-lymphocytes). Conclusion: Our data could represent another example of the exacerbation of autoimmunity associated with COVID-19. The increases in the incidence of both autoantibodies and autoimmune diseases seen with COVID-19 suggest one of the conceptual frameworks for creating hypotheses regarding the nature of the Long COVID syndrome. Presentation: Saturday, June 17, 2023

Hypertonic Salt Solution Enhances Inflammatory Responses in Cultured Splenic T-Cells from Dahl Salt-Sensitive Rats but Not Dahl Salt-Resistant Rats.

This study aimed to delineate the effect of sodium chloride on the induction of inflammatory responses and the development of hypertension in Dahl salt-sensitive (SS) and salt-resistant (SR) rats. Splenocytes were isolated from the spleens of SS and SR rats, and cultured on anti-CD3-coated plates for 5 days. The cultured splenic T-cells were challenged with a hypertonic salt solution (0, 20, or 40 mM) in the absence or presence of IL-6 (0, 20, or 60 ng/mL), TGF-β (0, 5, or 15 ng/mL), or IL-23 (0, 10, or 30 ng/mL), and analyzed via ELISA, flow cytometry, and immunofluorescence. The hypertonic salt solution potentiated IL-17A production, as well as the differentiation of Th17 cells via IL-6/TGF-β/IL-23, exclusively in SS rats. However, it did not affect IL-10 production or the differentiation of Treg cells in any of the groups. Furthermore, it potentiated the signal of RORγt in IL-6-treated splenic T-cells from SS rats. To summarize, cultured splenic T-cells exhibited enhanced inflammatory responses on exposure to a hypertonic salt solution in SS rats only, which indicated that sodium chloride and inflammatory cytokines synergistically drove the induction of pathogenic Th17 cells and the development of hypertension in this group only.

Therapeutic efficacy of the resorcylic acid lactone LL-Z1640-2 for adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) remains incurable. The NF-κB and interferon regulatory factor 4 (IRF4) signalling pathways are among the critical survival pathways for the progression of ATL. TGF-β-activated kinase 1 (TAK1), an IκB kinase-activating kinase, triggers the activation of NF-κB. The resorcylic acid lactone LL-Z1640-2 is a potent irreversible inhibitor of TAK1/extracellular signal-regulated kinase 2 (ERK2). We herein examined the therapeutic efficacy of LL-Z1640-2 against ATL. LL-Z1640-2 effectively suppressed the in vivo growth of ATL cells. It induced in vitro apoptosis and inhibited the nuclear translocation of p65/RelA in ATL cells. The knockdown of IRF4 strongly induced ATL cell death while downregulating MYC. LL-Z1640-2 as well as the NF-κB inhibitor BAY11-7082 decreased the expression of IRF4 and MYC at the protein and mRNA levels, indicating the suppression of the NF-κB-IRF4-MYC axis. The treatment with LL-Z1640-2 also mitigated the phosphorylation of p38 MAPK along with the expression of CC chemokine receptor 4. Furthermore, the inhibition of STAT3/5 potentiated the cytotoxic activity of LL-Z1640-2 against IL-2-responsive ATL cells in the presence of IL-2. Therefore, LL-Z1640-2 appears to be an effective treatment for ATL. Further studies are needed to develop more potent compounds that retain the active motifs of LL-Z1640-2.

Antigen-specific cytokine profiles for pulmonary Mycobacterium avium complex disease stage diagnosis.

Controlling pulmonary Mycobacterium avium complex (MAC) disease is difficult because there is no way to know the clinical stage accurately. There have been few attempts to use cell-mediated immunity for diagnosing the stage. The objective of this study was to characterize cytokine profiles of CD4+T and CD19+B cells that recognize various Mycobacterium avium-associated antigens in different clinical stages of MAC. A total of 47 MAC patients at different stages based on clinical information (14 before-treatment, 16 on-treatment, and 17 after-treatment) and 17 healthy controls were recruited. Peripheral blood mononuclear cells were cultured with specific antigens (MAV0968, 1160, 1276, and 4925), and the cytokine profiles (IFN-γ, TNF-α, IL-2, IL-10, IL-13, and IL-17) of CD4+/CD3+ and CD19+ cells were analyzed by flow cytometry. The response of Th1 cytokines such as IFN-γ and TNF-α against various antigens was significantly higher in both the on-treatment and after-treatment groups than in the before-treatment group and control (P < 0.01-0.0001 and P < 0.05-0.0001). An analysis of polyfunctional T cells suggested that the presence of IL-2 is closely related to the stage after the start of treatment (P = 0.0309-P < 0.0001) and is involved in memory function. Non-Th1 cytokines, such as IL-10 and IL-17, showed significantly higher responses in the before-treatment group (P < 0.0001 and P < 0.01-0.0001). These responses were not observed with purified protein derivative (PPD). CD19+B cells showed a response similar to that of CD4+T cells. There is a characteristic cytokine profile at each clinical stage of MAC.

Cell and cytokine analyses from bronchoalveolar lavage in non-critical COVID-19 pneumonia.

Cell and cytokine analyses from bronchoalveolar lavage (BAL) in non-critically ill patients with COVID-19 pneumonia are poorly described. This study focused on patients hospitalized in the non-intensive care unit for either suspected COVID-19 pneumonia or persistent respiratory symptoms following proven COVID-19 pneumonia. Overall, 54 patients who underwent BAL between April 2020 and February 2021 for suspected or follow-up of proven COVID-19 pneumonia were included. Based on SARS-CoV-2 polymerase chain reaction test results and clinical follow-up, three pulmonary disease groups were defined: non-COVID-19 (n = 20), acute COVID-19 (n = 13), and post-COVID-19 (n = 24) pneumonia patients. Cytological and cytokine analyses were performed on BAL fluid (IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ, HGF, and TGF-β), with investigators blinded to the patient groups. Lymphocytic alveolitis with plasmocytes was observed in acute COVID-19 pneumonia, returning to normal post-COVID-19. The highest cytokine levels were observed in COVID-19 patients, with significantly increased IFN-γ, IL-10, and HGF levels compared to non-COVID-19 patients, while significantly decreased IL-6, IL-8, IL-10, IFN-γ, TNF-α, and HGF levels were noted in post-COVID-19 patients. In COVID-19 patients, correlations between IL-10, TNF-α and IFN-γ concentrations were found. Lymphocytic alveolitis with plasmacytosis was found in non-critical COVID-19 pneumonia This alveolitis is associated with the presence of IL-6, IL-8, IL-10, TNF-α, IFN-γ and HGF. Alveolitis and cytokines levels decreased in post-COVID-19 pneumonia.

Role of Interleukin(IL)-6 in NK Activity to Hypoxic-Induced Highly Invasive Hepatocellular Carcinoma(HCC) Cells.

Natural killer (NK) cell dysfunctions against hepatocellular carcinoma (HCC) in a hypoxic environment. Many solid tumors are present in a hypoxic condition, which changes the effector function of various immune cells. The transcription of hypoxic-inducible factors (HIFs) in cancer cells make it possible to adapt to their hypoxic environment and to escape the immune surveillance of NK cells. Recently, the correlation between the transcription of HIF-1α and pro-inflammatory cytokines has been reported. Interleukin (IL)-6 is higher in cancers with a highly invasive ability, and is closely related to the metastasis of cancers. This study showed that the expression of HIF-1α in HCC cells was associated with the presence of IL-6 in the environment of HCC-NK cells. Blocking of IL-6 by antibody in the HCC-NK interaction changed the production of several cytokines including TGF-β, IL-1, IL-18 and IL-21. Interestingly, in a co-culture of HIF-1α-expressed HCC cells and NK cells, blocking of IL-6 increased the production of IL-21 in their supernatants. In addition, the absence of IL-6 significantly enhanced the cytotoxic ability and the expression of the activating receptors (NKG2D, NKp44, and NKG2C) in NK cells to HIF-1α-expressed HCC cells. These effects might be made by the decreased expression of HIF-1α in HCC cells through the inhibited phosphorylation of STAT3. In conclusion, the absence of IL-6 in the interaction of HIF-1α-expressed HCC cells and NK cells could enhance the antitumor activity of NK cells to HCC cells.

The Role of IL-17 in the Pathogenesis of Oral Squamous Cell Carcinoma.

Elucidating the inflammatory mechanisms underlying formation and progression of oral squamous cell carcinoma (OSCC) is crucial for discovering new targeted therapeutics. The proinflammatory cytokine IL-17 has proven roles in tumor formation, growth, and metastasis. The presence of IL-17 is demonstrated in both in vitro and in vivo models, and in OSCC patients, is mostly accompanied by enhanced proliferation and invasiveness of cancer cells. Here we review the known facts regarding the role of IL-17 in OSCC pathogenesis, namely the IL-17 mediated production of proinflammatory mediators that mobilize and activate myeloid cells with suppressive and proangiogenic activities and proliferative signals that directly induce proliferation of cancer cells and stem cells. The possibility of a potential IL-17 blockade in OSCC therapy is also discussed.