Cells Expressing Activation Markers Research Articles

CD4 T-cell aging exacerbates neuroinflammation in a late-onset mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons in the brain and spinal cord. Accumulating evidence suggests that ALS is not solely a neuronal cell- or brain tissue-autonomous disease and that neuroinflammation plays a key role in disease progression. Furthermore, whereas both CD4 and CD8 T cells were observed in spinal cords of ALS patients and in mouse models of the disease, their role in the neuroinflammatory process, especially considering their functional changes with age, is not fully explored. In this study, we revealed the structure of the CD4 T-cell compartment during disease progression of early-onset SOD1G93A and late-onset SOD1G37R mouse models of ALS. We show age-related changes in the CD4 T-cell subset organization between these mutant SOD1 mouse models towards increased frequency of effector T cells in spleens of SOD1G37R mice and robust infiltration of CD4 T cells expressing activation markers and the checkpoint molecule PD1 into the spinal cord. The frequency of infiltrating CD4 T cells correlated with the frequency of infiltrating CD8 T cells which displayed a more exhausted phenotype. Moreover, RNA-Seq and immunohistochemistry analyses of spinal cords from SOD1G37R mice with early clinical symptoms demonstrated immunological trajectories reminiscent of a neurotoxic inflammatory response which involved proinflammatory T cells and antigen presentation related pathways. Overall, our findings suggest that age-related changes of the CD4 T cell landscape is indicative of a chronic inflammatory response, which aggravates the disease process and can be therapeutically targeted.

Sufficiently activated mature natural killer cells derived from peripheral blood mononuclear cells substantially enhance antitumor activity.

Peripheral blood-derived natural killer (NK) cells spontaneously lyse tumor cells without prior sensitization. However, NK cells in peripheral blood (PBNK cells) are in a resting state and exhibit inhibitory phenotypes and impaired cytotoxicity. Thus, strengthening the cytotoxic effector function of PBNK cells and improving NK cell expansion in vitro for a convenient allogeneic therapy are essential. Pure cytokine activation and expansion of NK cells (super NK [SNK])from peripheral blood mononuclear cells were studied. Markers of activated and inhibited NK cells and cytokine secretion by NK cells were examined using flow cytometry. NK cell antitumor activity in vitro was assessed using lactate dehydrogenase (LDH) cytotoxicity assay and an Incucyte real-time imaging system. Additionally, the function of SNK cells against ascites caused by ovarian cancer in NOD-Prkdc(em26Cd52)il2rg(em26Cd22)/Nju (NCG)mice was determined. In a further investigation of the differences between PBNK and SNK, the mRNA of both cells was sequenced and analyzed. Human peripheral blood mononuclear cells showed selective NK cell expansion upon cytokine activation and culture. Both SNK and PBNK cells expressed activation markers, but at different levels, and SNK cells secreted more cytokines related to cytotoxicity than PBNK cells did. Accordingly, SNK cells exhibited strong antitumor activity ex vivo and improved NCG mice survival after intraperitoneal ovarian cancer transplantation. Mechanistically, SNK cells expressed more genes associated with nucleotide metabolism, fatty acid, and ATP metabolism than PBNK cells. SNK cells derived from peripheral blood mononuclear cells have sufficiently activated mature characteristics and high antitumor activity, rendering them a highly promising and essential therapeutic approach for cancer treatment.

Abstract 13851: CAR-T Cells: A Potential Novel Strategy for Mitigating Fibrosis in Duchenne Muscular Dystrophy

Introduction: Chimeric Antigen T (CAR)-T cells have proven effective preclinically for mitigating cardiac fibrosis in hypertensive cardiomyopathy. Hypothesis: We assessed whether this approach could be applied to Duchenne Muscular Dystrophy (DMD) by targeting Fibroblast Activating Protein (FAP)-expressing activated fibroblasts in cardiac and skeletal muscle fibrosis. Methods: A protocol for the lymphodepletion conditioning of mice was developed to optimize the dosing of cyclophosphamide. Next, immune-magnetically selected T cells were retrovirally transduced to target FAP (FAP-CAR) and quality-controlled by co-culture killing assays using FAP-expressing HEK target cells. Finally, lymphodepleted D2. mdx mice were intravenously injected with 10 7 FAP-CAR T cells (n=9) or 10 7 GFP-T cells (n=9) or Bovine Serum Albumin (BSA, n=8). Cardiac and skeletal muscle end points were assessed 2 and 5 weeks after treatment. Results: In vitro , the engineered CAR-T cells expressed activation markers (CD69, PD-1) and released large amounts of granzyme, perforin and interferon γ upon exposure to FAP-expressing target cells. One IP injection of cyclophosphamide (3mg) 4 days before treatment was safe and induced a lymphodepletion which enabled robust in vivo expansion of FAP-CAR T cell assessed by flow cytometry in lymphoid organs and blood: 10 5 , 2.10 6 and 10 7 detected CAR-T cells at day 3, 5 and 7 post-injections, respectively. This was paralleled by a more than a 2-fold decrease in endogenous FAP expression in skeletal muscles, as assessed by PCR at day 5 post-injections, and measurable FAP-CAR levels in the spleen compared with GFP-T cell controls. In vivo , FAP-CAR T cells significantly improved LV function with an EF change from baseline (Mean±SEM) of 33.1% ± 6.7% in FAP-CAR vs. 3.7% ± 6.4% and -9.6% ± 7.9% in GFP-T and BSA ( p <0.037 and p <0.005, respectively) at 2 weeks. The benefit was maintained at 5 weeks post-treatment. Several markers of fibrosis, particularly collagens 1 and 3, were also down-regulated in heart and muscles. Conclusions: FAP-CAR-T cells might represent a new therapeutic option for mitigating fibrosis in DMD, likely in conjunction with approaches aimed at addressing the root cause of the disease through a re-expression of a functional dystrophin.

Phase 1/2 Dose-Escalation/Dose-Expansion Study of Anti-CD7 Allogeneic CAR-T Cells (WU-CART-007) in Relapsed or Refractory (R/R) T-Cell Acute Lymphoblastic Leukemia/ Lymphoblastic Lymphoma (T-ALL/LBL)

T-ALL/LBL are challenging hematologic cancers with high rates of relapse and mortality in both children and adults. Despite success in B-cell malignancies, development of CAR-T cell therapy for T cell malignancies has been complicated by induction of fratricide and risk of malignant cell contamination of the drug product in the autologous setting. WU-CART-007 is a CD7-targeted CAR-T cell products with CRISPR/Cas9 deletion of CD7 and T-cell receptor alpha constant (TRAC), to prevent fratricide and enable the use of healthy donor allogeneic T-cells, respectively (Leedom et al. ASH 2021). This off-the-shelf allogeneic CAR-T cell product is being developed for the treatment of CD7 + malignancies. WU-CART-007 1001 (NCT04984356) is a global first-in-human, Phase 1/2 single-agent study of WU-CART-007 in patients (pts) with R/R T-ALL/LBL. The recommended Phase 2 dose (RP2D) of WU-CART-007 is 900 million (M) cells administered on day 1 following lymphodepleting chemotherapy (LDC). Two different LDC regimens have been tested: standard LDC (fludarabine 30 mg/m 2/day x 3 days and cyclophosphamide 500 mg/m 2/day x 3 days), and enhanced LDC (eLDC; fludarabine 30 mg/m2/day x 4 days and cyclophosphamide 1000 mg/m2/day x 3 days).Disease response is assessed by Day 28 bone marrow (BM) aspirate/biopsy, and CT/PET, if applicable, per NCCN Guidelines Version 2.2022; Pharmacokinetics (PK) are measured by ddPCR; samples are collected for immunophenotyping by flow cytometry (FACS). As of July 21, 2023, 18 pts have been dosed with WU-CART-007(n = 11 T-ALL, n = 7 T-LBL); 3 with 100M (DL1), 3 with 300M (DL2), 6 with 600M (DL3), and 6 with 900M (DL4/RP2D) cells in a single infusion. A total of 15 pts received standard LDC while 3 received eLDC at the DL4/RP2D. Median age is 33.5 years (range 20-68). Pts were heavily pretreated with a median of 4 prior lines of therapy (range 2 - 7), 28% (5/18) relapsed following an allogeneic HSCT. Disease burden at baseline consisted of extramedullary disease (EMD) in 28% (5/18) of pts, and a median BM blast count of 60% (range 5-98%) in pts with BM disease (13/18). Overall WU-CART-007 demonstrated manageable safety profile; treatment-related adverse events of ≥ G3 were observed in 8/18 (44%) pts. Cytokine release syndrome (CRS) was observed in 14/18 (78%) pts. Most (72%; 13/18) pts had G1-2 CRS events; a single G3 CRS event was reported which resolved within 72 hours after receiving tocilizumab, dexamethasone, and low-dose vasopressors. Grade 1 ICANS was reported in one patient at DL3, which resolved spontaneously. No GvHD, prolonged T-cell aplasia, or prolonged pancytopenia in the absence of disease were observed. One unrelated DLT lead to cohort expansion at DL3. The majority of deaths were due to disease progression. There were two Grade 5 events not attributed to WU-CART-007, both due to fungal infection. WU-CART-007 showed dose-dependent anti-tumor activity with no responses seen at DL1. In evaluable patients at DL≥ 2 (n=12) Composite Complete Remission Rate (CRc; CR + CRi + CRh): 58% (86% MRD neg), median duration of response 12.3 weeks (1.1 - 29.4); two patients successfully received a consolidating allogeneic-HSCT. At the RP2D, the CRc rate was 60% (3/5; 2 CR, 1CRi). Molecular expansions of CAR-T cells peaked on day 10 in peripheral blood (mean 66,069 copies/ug DNA) and persisted out to day 56. Of all pts tested (n=15), none developed novel anti-HLA antibodies against the donor, and no anti-drug antibodies against the CAR-construct were detected. Phenotypic analysis revealed in vivo WU-CART-007 cells expressed activation markers (KI67, CD38, HLA-DR) and were largely an effector memory CD45RA + (EMRA) CM phenotype (CD45RA +, CD197 +). WU-CART-007 has demonstrated an acceptable safety profile and preliminary evidence of anti-leukemic activity. This program advances CAR-T cell therapy in heavily pre-treated patients with R/R T-ALL/LBL. Enrollment is ongoing.

Time Limited Exposure to a ROR1 Targeting Bispecific T Cell Engager (NVG-111) Leads to Durable Responses in Subjects with Relapsed Refractory Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL)

Background: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is absent or expressed at low levels in normal adult tissues but overexpressed in a range of malignancies including CLL and MCL. NVG-111 is a humanized first in class, tandem scFv, ROR1xCD3 bispecific T cell engager. NVG-111 mediates potent killing of ROR1 + tumors by engaging a unique epitope on the Frizzled domain of ROR1 and redirecting T cell activity via the CD3 binder engineered for attenuated cytokine release. Methods: In this phase 1, first-in-human, dose-escalation study (ClinicalTrials.gov identifier: NCT04763083), NVG-111 was evaluated in relapsed/refractory CLL and MCL subjects with an ECOG PS of 0-2 who received ≥2 prior lines of treatment including a Bruton tyrosine kinase inhibitor (BTKi). Bayesian continual reassessment method with overdose control was implemented to guide escalation over a dose range of 0.3-45ug/day. Each subject received at least one dose of NVG-111, administered as a continuous intravenous infusion (cIV) over 21 days followed by 7 days off drug (=1 cycle). NVG-111 was administered in combination (N=8) with ibrutinib to subjects who had achieved a partial response to >1 year of ibrutinib therapy, or as monotherapy in subjects that progressed after covalent BTKi/B-cell lymphoma 2 inhibitor (BCL2i) (N=4). Primary objective was safety, with the secondary objectives being efficacy (overall response rates [ORR], minimal residual disease (MRD) measured by ERIC-compliant flow cytometry and duration of response [DoR]). Serum cytokine levels were determined using a human high sensitivity cytokine premixed magnetic Luminex assay and T cell function was longitudinally evaluated using cytometry by time of flight (CyTOF) analysis. Results: Between May 2021 and July 2023 12 subjects (10 males and 2 females, median age 60 years) completed a maximum of 6 cycles of treatment with NVG-111 (median=3 cycles [range 1-6 cycles]). Pharmacokinetic and pharmacodynamic data demonstrated systemic NVG-111 exposure and exposure-dependent NVG-111 targeting of ROR1 on circulating tumor cells. Adverse events (AEs) related to NVG-111 occurred in 10 subjects (83%), with the most common being nausea (58%), headaches (67%), and fatigue (33%), majority of which were grade 1 or 2. Grade 1(71%) or 2(29%) cytokine release syndrome (CRS) occurred in 58% of subjects during week 1 of cycle 1 except in one subject who had a second grade 1 CRS in cycle 2. Grade 3 dose limiting toxicities (DLTs) occurred in three subjects (25%) consisting of transient elevation of liver enzymes (ALT and AST) in one subject dosed at 3ug/day, immune effector cell-associated neurotoxicity syndrome-like symptoms (ICANS) in one subject dosed at 30ug/day and headache in one subject dosed at 45ug/day. All AEs were fully reversible with no late emergent grade 3 or greater toxicities. Evidence of T cell activation was observed in all 12 subjects with peak cytokine levels at the 30µg/day dose levels (mean±SEM): TNFα, 43±17pg/ml; IL6, 566±541pg/ml; IFNɣ, 18±12pg/ml; and IL10, 103±56pg/ml. Furthermore, T cell profiling showed an increase in cytotoxic CD8 + T cell activation marker expression during each NVG-111 treatment cycle without evidence of T cell exhaustion. Efficacy was evaluable in 11 subjects with objective clinical responses observed in 55% (6/11), including two subjects with clear evidence of single agent activity. Amongst these, three CLL subjects were rendered MRD negative in peripheral blood and one MCL subject achieved complete metabolic response by the Lugano criteria. Two subjects had stable disease but in one of these subjects there was a >50% reduction in peripheral blood lymphocytosis and a 40% reduction in lymphadenopathy. Despite time limited exposure to NVG-111, the restricted mean survival time for DoR is currently 13.6 months (SEM 3.07), the median is not yet calculable with response ongoing in four subjects. The median progression-free survival currently is 18.7 months (95% CI 2.6 - not calculable). Conclusion: These data provide clinical proof of concept for selective targeting of ROR1 with a TCE leading to objective evidence of antitumor activity with encouraging response durability even in CLL patients known to have defective T cell function. The safety profile was consistent with the mechanism of action. Further evaluation of this promising molecule is ongoing.

TCR-Engineered Lymphocytes Targeting NY-ESO-1: In Vitro Assessment of Cytotoxicity against Tumors.

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.

Exploring TCR-like CAR-Engineered Lymphocyte Cytotoxicity against MAGE-A4.

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.

Legend or Truth: Mature CD4+CD8+ Double-Positive T Cells in the Periphery in Health and Disease.

The expression of CD4 and CD8 co-receptors defines two distinct T cell populations with specialized functions. While CD4+ T cells support and modulate immune responses through different T-helper (Th) and regulatory subtypes, CD8+ T cells eliminate cells that might threaten the organism, for example, virus-infected or tumor cells. However, a paradoxical population of CD4+CD8+ double-positive (DP) T cells challenging this paradigm has been found in the peripheral blood. This subset has been observed in healthy as well as pathological conditions, suggesting unique and well-defined functions. Furthermore, DP T cells express activation markers and exhibit memory-like features, displaying an effector memory (EM) and central memory (CM) phenotype. A subset expressing high CD4 (CD4bright+) and intermediate CD8 (CD8dim+) levels and a population of CD8bright+CD4dim+ T cells have been identified within DP T cells, suggesting that this small subpopulation may be heterogeneous. This review summarizes the current literature on DP T cells in humans in health and diseases. In addition, we point out that strategies to better characterize this minor T cell subset's role in regulating immune responses are necessary.

TGF-β promotes the resolution of acute hepatitis B virus infection

Abstract An estimated 296 million people live with a chronic hepatitis B virus (HBV) infection, resulting in approximately 800,000 annual deaths. Thus, despite the availability of an effective vaccine, HBV remains a global health concern. Most children and a fraction of adults infected with HBV develop a persistent liver infection that yields lifelong complications. It is believed that the immune tolerogenic environment of the liver and the dysfunction of T and B cells contribute to HBV persistence in these individuals. Here, an adeno-associated virus (AAV)-based mouse model of HBV infection was utilized to investigate the underlying mechanisms of immune dysfunction that impede viral clearance. HBV infection was initiated in mice by AAV-HBV transduction, and TGF-β was depleted to determine the role of this cytokine in HBV persistence. As TGF-β is often associated with negative regulation of the immune response, we hypothesized that its inhibition might improve HBV-specific T and B cell responses. However, the lack of TGF-β resulted in impaired HBV surface antigen (HBsAg) clearance and diminished anti-HBsAg antibody levels, revealing a positive role for TGF-β in the antibody response to HBV. This effect of TGF-β on the antibody response to HBsAg was unique to AAV-HBV transduction, as a similar increase in anti-HBsAg was not observed by TGF-β depletion following infection with recombinant vesicular stomatitis virus expressing HBsAg. We also observed differences in B cell activation marker expression during HBsAg clearance in mice transduced with AAV-HBV and depleted of TGF-β. Together, these findings highlight the impact of TGF-β on the B cell response to HBV and have begun to define a unique role for this cytokine in promoting HBV clearance. Supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI148354. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abstract 1869: Preclinical evaluation of mosunetuzumab for the treatment of relapsed/refractory chronic lymphocytic leukemia

Abstract Background: CD20 x CD3 T-cell engaging bispecific antibodies, such as Mosunetuzumab (Mosun), have shown promising efficacy in Non-Hodgkin’s lymphoma. However, their role in treating chronic lymphocytic leukemia (CLL) is still uncertain. CLL, a clonal disease of mature B cells, is associated with high B/T cell ratios and diminished T cell activation and function, including the inability to form strong immunosynapses (Ramsay et al, J Clinical Investigation, 2008). Herein, we explore whether Mosun could overcome these barriers and elicit strong T-cell mediated B-cell killing in CLL samples. Methods: PBMCs from CLL patients and healthy donors were exposed to a titration of Mosun for 48 and 72 hours and analyzed for B cell death with flow cytometry. Markers of T cell activation and exhaustion were quantified in CD4 and CD8T cells and within their CD45RA/CCR7 subsets. The concentrations of secreted cytokines were measured by ELISA. To study the impact of high B/T ratio on B cell killing, B cells were isolated from healthy donor PBMC and added back to autologous PBMC to attain B/T ratios seen in CLL patient samples. Results: After 48 hours with 0.04ug/ml Mosun, the percentage of dead B cells in healthy-donor samples (n=9) exceeded that in CLL samples (n=7) by four-fold (54.2 + 17.9 vs: 12.9 + 17.5%, p=0.0004). Under the same conditions, significantly more T cells expressed activation markers in healthy donors than in CLL patients, with 58.8 +17.1% of healthy donor CD8T cells expressing both CD69 and CD25 compared to and 31.4 +21.4% of CLL CD8T cells (p=0.013). The mean concentration of Granzyme B secreted by healthy donor samples upon Mosun treatment was over ten times higher than by CLL patient samples. Reconstitution of a healthy donor sample to a B/T ratio of 10 resulted in decreased B cell killing (29.7% dead B cells vs: 70.5% in un-reconstituted) without affecting T cell activation (62.9% CD69+CD25+ CD8T cells versus 56.2% in un-reconstituted). Conclusions: Although single-agent Mosun treatment can activate T-cells in CLL patient samples and result in B-cell death, the response is suboptimal due to a number of factors, including deficient T cell activation and high B/T ratio. Ongoing work is focusing on evaluating drug partners to debulk CLL B cells or prime CLL T cells for better B-cell killing by Mosun. Citation Format: Kathy Barrett, Kai Lu, Hyun Yong Jin, Rosie Millen, Marcus Lefebure, Yanwen Jiang. Preclinical evaluation of mosunetuzumab for the treatment of relapsed/refractory chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1869.

Disseminated purulent peritonitis outcome affects NKT cell phenotype

The aim of our study was to investigate the main characteristics of peripheral blood NKT cell phenotype in patients with disseminated purulent peritonitis (DPP) in dynamics of postoperative period, depending on the disease outcome. Fifty-two patients with acute surgical diseases and injuries of the abdominal organs complicated by DPP, and 68 healthy individuals in control group, were examined. Blood sampling was performed before surgery (preoperative period), as well as on the day 7, 14 and 21 of postoperative period. All patients with DPP were divided into two groups depending on disease outcome in postoperative period: patients with favorable disease outcome (n = 34); and patients with unfavorable outcome (n = 18). Study of the phenotype of blood NKT lymphocytes was performed by flow cytometry using direct immunofluorescence of whole peripheral blood samples with monoclonal antibodies. The low relative and absolute level of NKT cells was observed in DPP patients regardless of outcome disease in preoperative period. At the same time, the absolute level of NKT cells returned to normal only in patients with favorable DPP outcome and only by day 21 after surgery. Patients with favorable DPP outcome by the end of examination period had normalized quantity of mature NKT-lymphocytes and significantly decreased level of cytotoxic cells which was apparently associated with migration of such cell subsets to site of inflammation. A reduced level of non-classical (expressing CD8 marker) mature and cytokine-producing NKT cells was detected only in patients with favorable DPP outcome in preoperative period which returned to normal by the end of postoperative period. At the same time, patients with unfavorable disease outcome had reduced quantity of NKT cells of these subsets by day 21 of postoperative treatment. Patients with favorable outcome had high level of mature and cytotoxic CD11b+ NKT cells already in the preoperative period, while patients with unfavorable DPP outcome had increased level of cytotoxic CD11b+ NKT cells only by day 21 after surgery. The proportion of NKT cells expressing activation markers (CD28 and CD57) was reduced in patients in preoperative period that returned to normal immediately after surgery with favorable outcome, while it recovered with unfavorable outcome closer to the end of postoperative examination. The defined features of NKT cell phenotype in patients with unfavorable DPP outcome characterize disturbances in subset ratio and mechanisms of functioning of this cell fraction. This determines a need to develop immunotherapeutic methods aimed at stimulating immunoregulatory activity of NKT cells.

Ex Vivo Generation and Infusion of Anti-Minor Histocompatibility Antigen Expanded T Cells for Patients Who Relapse after Allogeneic HLA-Matched Stem Cell Transplantation

Rationale Long-term clinical remissions of hematologic malignancies after allogeneic hematopoietic stem cell transplantation (HSCT) largely rely on the graft-versus-leukemia effect (GVL). In a fully HLA-matched setting, the GVL effect is mediated primarily by T cell immune responses against host minor histocompatibility antigens (MiHAs) found on the surface of malignant cells. Upon relapse of leukemia following HSCT, donor lymphocyte infusions (DLI) have unfortunately failed to exert efficient GVL activity. This is likely due to the insufficient number of anti-MiHA T cells within DLIs and to the risk of graft-vs-host disease (GVHD) that prevents the administration of high numbers of T cells. To generate potent and selective anti-leukemia activity, we have developed a strategy for the ex vivo selection and expansion of T cells targeting MiHAs preferentially expressed on hematopoietic cells (Perreault, Leukemia 2016). Thus, these cells should display augmented GVL immune reactivity while harboring a low risk of causing GVHD. To our knowledge, this is the first clinical study targeting several different molecularly defined MiHAs and on different HLA molecules. Patients and Methods In an open-label, multi-center phase 1 clinical trial (CR-MIHA-001; NCT 03091933), patients with hematologic malignancies who relapsed after an allogeneic HLA-matched HSCT were treated with sibling donor-derived, ex vivo- expanded T cells selected and expanded for reactivity toward tissue-restricted recipient MiHAs (GLIDE: Guided Lymphocyte Immunopeptide Derived Expansion). After donor cell collection, patients received fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 for 3 days followed by infusion of GLIDE, consisting of donor T-lymphocytes expanded ex vivo following exposure to dendritic cells coated with immunogenic peptides (MiHAs) present on the surface of host hematopoietic cells, at a single target dose of 4x107 viable T-cells/m2. A second dose of GLIDE, aiming at 3-5 times the original dose, could be administered to patients at a minimum of 6 weeks after the first infusion. GLIDE cell characteristics were evaluated using multi-parameter flow cytometry and TCR sequencing. Patients were followed up for one year post-GLIDE infusion for toxicity and clinical outcome measures. Results Ten patients, median age of 58 (range 33-69), 4 females/6 males with AML (n=7), NHL (1), CLL (1), and myelofibrosis (1) were treated with GLIDE. These patients had relapsed at a median of 12 months (range 5-126 mo) after allogeneic HSCT. GLIDE cells were personalized to target single MiHAs in 9 patients and two MiHAs in 1 patient. Overall, GLIDE cell products were directed at 9 different MiHAs on 6 different HLA-class 1 molecules (HLA-A02:01, A24:02, B07:02, B18:01, B44:02, and B44:03). A first dose of GLIDE was administered at 4.0x107 CD3+ cells/m2 to 8 patients, and 2 patients received slightly lower doses (2.6 and 3.7 x 107 CD3+ cells/m2). Final GLIDE products consisted mainly of T-cells (median: 98,7%), with CD8>CD4>NKT>NK cells. Most CD8+ T cells were effector memory and central memory T cells (Figure 1). At the end of the expansion period, CD8+ T cells expressed activation markers (CD25+ = 67%) with low levels of PD1+, PDL1+, CD57+ and KLRG1+ as markers of exhaustion. Not surprisingly, GLIDE cells had a more restricted TCR β chain profile than initial lymphopheresis cells. Six patients received a second dose of GLIDE (mean: 12 x 107 CD3+ cells/m2) at 53 days after the first infusion (median, range: 48-118 days). There were no infusional toxicities, and cells were well-tolerated by patients. Only one patient developed thrombocytopenia and subsequent chronic GVHD. There were no other adverse events potentially related to GLIDE. According to TCR β sequencing data, T cells present in the adoptively transferred anti-MiHA GLIDE product persisted in peripheral blood up to one year after infusion. Survival at one year was observed in 4 patients with 3 patients in sustained remission (Figure 2). Conclusion This pilot study demonstrates the feasibility of anti-MiHA T cell ex vivo generation (GLIDE) and safety of its administration to patients relapsing after allogeneic HSCT, as well as the clinical potential of these cells. The persistent remissions and low occurrence of GVHD support further development of GLIDE to target acute myeloid leukemia and other hematologic malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pregnancy imparts distinct systemic adaptive immune function.

Pregnancy represents a state of systemic immune activation that is primarily driven by alterations in circulating innate immune cells. Recent studies have suggested that cellular adaptive immune components, T cells and B cells, also undergo changes throughout gestation. However, the phenotypes and functions of such adaptive immune cells are poorly understood. Herein, we utilized high-dimensional flow cytometry and functional assays to characterize T-cell and B-cell responses in pregnant and non-pregnant women. Peripheral blood mononuclear cells from pregnant (n=20) and non-pregnant (n=25) women were used for phenotyping of T-cell and B-cell subsets. T-cell proliferation and B-cell activation were assessed by flow cytometry after in vitro stimulation, and lymphocyte cytotoxicity was evaluated by using a cell-based assay. Statistical comparisons were performed with linear mixed-effects models. Pregnancy was associated with modestly enhanced basal activation of peripheral CD4+ T cells. Both CD4+ and CD8+ T cells from pregnant women showed increased activation-induced proliferation; yet, a reduced proportion of these cells expressed activation markers compared to non-pregnant women. There were no differences in peripheral lymphocyte cytotoxicity between study groups. A greater proportion of B cells from pregnant women displayed memory-like and activated phenotypes, and such cells exhibited higher activation following stimulation. Maternal circulating T cells and B cells display distinct responses during pregnancy. The former may reflect the unique capacity of T cells to respond to potential threats without undergoing aberrant activation, thereby preventing systemic inflammatory responses that can lead to adverse perinatal consequences.

Effective Combination Immunotherapy with Oncolytic Adenovirus and Anti-PD-1 for Treatment of Human and Murine Ovarian Cancers.

Simple SummaryThis study was conducted to find a new, more efficient, treatment for ovarian cancer. A combination of an oncolytic adenovirus (TILT-123) with immune checkpoint inhibitors was employed to treat ex vivo patient samples and was found statistically significantly more effective than control treatments ex vivo and showed potent efficacy towards in vivo tumor growth.Ovarian cancer (OvCa) is one of the most common gynecological cancers and has the highest mortality in this category. Tumors are often detected late, and unfortunately over 70% of OvCa patients experience relapse after first-line treatments. OvCa has shown low response rates to immune checkpoint inhibitor (ICI) treatments, thus leaving room for improvement. We have shown that oncolytic adenoviral therapy with Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (aka. TILT-123) is promising for single-agent treatment of cancer, but also for sensitizing tumors for T-cell dependent immunotherapy approaches, such as ICI treatments. Therefore, this study set out to determine the effect of inhibition of the immune checkpoint inhibitors (ICI), in the context of TILT-123 therapy of OvCa. We show that simultaneous treatment of patient derived samples with TILT-123 and ICIs anti-PD-1 or anti-PD-L1 efficiently reduced overall viability. The combinations induced T cell activation, T cells expressed activation markers more often, and the treatment caused positive microenvironment changes, measured by flow cytometric assays. Furthermore, in an immunocompetent in vivo C57BL/6NHsda mouse model, tumor growth was hindered, when treated with TILT-123, ICI or both. Taken together, this study provides a rationale for using TILT-123 virotherapy in combination with TILT-123 and immune checkpoint inhibitors together in an ovarian cancer OvCa clinical trial.

Inflammatory-mediated taste dysfunction – Is NO the key?

Diabetes mellitus (DM), also called diabetes, is a chronic metabolic disorder, with complications that include failure of immune responses, such as immune cell proliferation and activation. The current study investigated changes in natural killer (NK) cell activity under diabetic conditions. To demonstrate the influence of hyperglycemia on NK cell activity, type 1 and 2 diabetes models were constructed using C57/BL6 mice. NK cells isolated from mice with either type 1 or 2 diabetes showed lower cytotoxic activity against cancer cells than NK cells isolated from healthy control mice, and the longer the duration since induction of diabetes, the lower was the cytotoxic activity. Moreover, in the diabetic mouse models, fewer NK cells expressed activation markers, such as NKG2D and NKp46, compared to NK cells from control mice. Production of the apoptosis inducer, granzyme B, was also lower in NK cells from diabetic mice than control mice. These results demonstrate that high glucose conditions influence NK cell activity.

Type 1 and 2 diabetes are associated with reduced natural killer cell cytotoxicity

Diabetes mellitus (DM), also called diabetes, is a chronic metabolic disorder, with complications that include failure of immune responses, such as immune cell proliferation and activation. The current study investigated changes in natural killer (NK) cell activity under diabetic conditions. To demonstrate the influence of hyperglycemia on NK cell activity, type 1 and 2 diabetes models were constructed using C57/BL6 mice. NK cells isolated from mice with either type 1 or 2 diabetes showed lower cytotoxic activity against cancer cells than NK cells isolated from healthy control mice, and the longer the duration since induction of diabetes, the lower was the cytotoxic activity. Moreover, in the diabetic mouse models, fewer NK cells expressed activation markers, such as NKG2D and NKp46, compared to NK cells from control mice. Production of the apoptosis inducer, granzyme B, was also lower in NK cells from diabetic mice than control mice. These results demonstrate that high glucose conditions influence NK cell activity.

CXCL8 Associated Dendritic Cell Activation Marker Expression and Recruitment as Indicators of Favorable Outcomes in Colorectal Cancer.

Accumulating evidence suggests that tumor-infiltrating immune cells (TICs) in the tumor microenvironment (TME) serve as promising therapeutic targets. CXCL8 (IL-8) may also be a potential therapeutic target in cancer. CXCL8 is a potent chemotactic factor for neutrophils, myeloid-derived suppressor cells (MDSCs) and monocytes, which are considered immunosuppressive components in cancer-bearing hosts. Here, we identified the TME-related gene CXCL8 in a high-ImmuneScore population that contributed to better survival in colorectal cancer (CRC) patients from The Cancer Genome Atlas (TCGA) database. An integrated gene profile and functional analysis of TIC proportions revealed that the dendritic cell (DC) activation markers CD80, CD83, and CD86 were positively correlated with CXCL8 expression, suggesting that CXCL8 may be functional as antitumor immune response status in the TME. The gene signature was further validated in independent GSE14333 and GSE38832 cohorts from the Gene Expression Omnibus (GEO). To test the differential contributions of immune and tumor components to progression, three CRC cell lines, CT26, MC38 and HCT116, were used. In vitro results suggested no significant growth or survival changes following treatment with an inhibitor of the CXCL8 receptor (CXCR1/2) such as reparixin or danirixin. In vivo treatment with danirixin (antagonists of CXCR2) promoted tumor progression in animal models established with CT26 cells. CXCR2 antagonism may function via an immune component, with CXCR2 antagonist treatment in mice resulting in reduced activated DCs and correlating with decreased Interferon gamma (IFN-γ) or Granzyme B expressed CD8+ T cells. Furthermore, CXCL8 induced DC migration in transwell migration assays. Taken together, our data suggested that targeting the CXCL8-CXCR2 axis might impede DC activation or recruitment, and this axis could be considered a favorable factor rather than a target for critical antitumor effects on CRC.

AhR Ligands Modulate the Differentiation of Innate Lymphoid Cells and T Helper Cell Subsets That Control the Severity of a Pulmonary Fungal Infection.

In agreement with other fungal infections, immunoprotection in pulmonary paracoccidioidomycosis (PCM) is mediated by Th1/Th17 cells whereas disease progression by prevalent Th2/Th9 immunity. Treg cells play a dual role, suppressing immunity but also controlling excessive tissue inflammation. Our recent studies have demonstrated that the enzyme indoleamine 2,3 dioxygenase (IDO) and the transcription factor aryl hydrocarbon receptor (AhR) play an important role in the immunoregulation of PCM. To further evaluate the immunomodulatory activity of AhR in this fungal infection, Paracoccidioides brasiliensis infected mice were treated with two different AhR agonists, L-Kynurenin (L-Kyn) or 6-formylindole [3,2-b] carbazole (FICZ), and one AhR specific antagonist (CH223191). The disease severity and immune response of treated and untreated mice were assessed 96 hours and 2 weeks after infection. Some similar effects on host response were shared by FICZ and L-Kyn, such as the reduced fungal loads, decreased numbers of CD11c+ lung myeloid cells expressing activation markers (IA, CD40, CD80, CD86), and early increased expression of IDO and AhR. In contrast, the AhR antagonist CH223191 induced increased fungal loads, increased number of pulmonary CD11c+ leukocytes expressing activation markers, and a reduction in AhR and IDO production. While FICZ treatment promoted large increases in ILC3, L-Kyn and CH223191 significantly reduced this cell population. Each of these AhR ligands induced a characteristic adaptive immunity. The large expansion of FICZ-induced myeloid, lymphoid, and plasmacytoid dendritic cells (DCs) led to the increased expansion of all CD4+ T cell subpopulations (Th1, Th2, Th17, Th22, and Treg), but with a clear predominance of Th17 and Th22 subsets. On the other hand, L-Kyn, that preferentially activated plasmacytoid DCs, reduced Th1/Th22 development but caused a robust expansion of Treg cells. The AhR antagonist CH223191 induced a preferential expansion of myeloid DCs, reduced the number of Th1, Th22, and Treg cells, but increased Th17 differentiation. In conclusion, the present study showed that the pathogen loads and the immune response in pulmonary PCM can be modulated by AhR ligands. However, further studies are needed to define the possible use of these compounds as adjuvant therapy for this fungal infection.

Activated Lymphocytes and Increased Risk of Dermatologic Adverse Events during Sorafenib Therapy for Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma is the second cause of cancer-related death worldwide. Of those advanced-stage patients who are treated with sorafenib, those who develop early dermatologic adverse events have a better prognosis. These events are possibly immune-related. Therefore, we analyzed the phenotype of 52 sorafenib-treated patients’ circulating lymphocytes throughout treatment. We found that different co-stimulatory and immune exhaustion markers, such as Programmed cell death protein 1 (PD-1) and DNAX accessory molecule 1 (DNAM-1) amongst others, correlate with the probability of developing these adverse events, both before and during the treatment. We also compared the phenotype of those lymphocytes expressing DNAM-1 with those that do not, and while NK DNAM-1-expressing cells have a co-stimulatory phenotype, T DNAM-1-expressing cells are immune-suppressors. Overall, we set a rationale for the combination of sorafenib and immune-targeted therapies; and for the use of immune markers (such as DNAM-1) for patients’ prognosis evaluation.Advanced hepatocellular carcinoma patients treated with sorafenib who develop early dermatologic adverse events (eDAEs) have a better prognosis. This may be linked to immune mechanisms, and thus, it is relevant to assess the association between peripheral immunity and the probability of developing eDAEs. Peripheral blood mononuclear cells of 52 HCC patients treated with sorafenib were analyzed at baseline and throughout the first eight weeks of therapy. T, B, Natural Killer cells, and their immune checkpoints expression data were characterized by flow cytometry. Cytokine release and immune-suppression assays were carried out ex vivo. Cox baseline and time-dependent regression models were applied to evaluate the probability of increased risk of eDAEs. DNAM-1, PD-1, CD69, and LAG-3 in T cells, plus CD16 and LAG-3 in NK cells, are significantly associated with the probability of developing eDAEs. While NK DNAM-1+ cells express activation markers, T DNAM-1+ cells induce immune suppression and show immune exhaustion. This is the first study to report an association between immune checkpoints expression in circulating immune cells and the increased incidence of eDAEs. Our results support the hypothesis for an off-target role of sorafenib in immune modulation. We also describe a novel association between DNAM-1 and immune exhaustion in T cells.

Дифференцировка моноцитов крови и особенности цитокинового статуса у больных туберкулезом легких

In clinical manifestation of pulmonary tuberculosis, alveolar macrophages perform as a reservoir where they accumulate mycobacteria and lose their effector functions due to the pathological conversion of macrophage pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, which provides transition to chronic infection. The study hypothesis suggested that the cytokine status, as evaluated by leukocyte secretion of cytokines in vitro, influences the monocyte polarization in the blood during their migration to the inflammatory focus, thereby determining differentiation and pathways of macrophage activation in tissues.The aim of this work was to assess the immunophenotype of blood monocytes and the in vitro secretion of immunoregulatory cytokines by mononuclear peripheral blood leukocytes from patients with different clinical forms of pulmonary tuberculosis taking into account the pathogen sensitivity to major anti-tuberculosis drugs.Methods. 65 patients with newly diagnosed pulmonary tuberculosis were evaluated. The study material was venous blood and peripheral blood mononuclear leukocytes. Monocyte immunophenotype was determined in whole blood by flow cytometry on a Cytoflex flow cytometer (Becman Coulter, USA) with monoclonal antibodies (eBioscience, USA). Results were processed with a CytExpert 2.0 software. The number of cells expressing surface markers, CD14, CD163, CD204 and HLA-DR, was determined. Content of cytokines (IL-2, IL-10, TGFβ) in supernatants of cell cultures was measured by enzyme-linked immunosorbent assay (ELISA). Results of the study were processed with a SPSS v.11.0 standard software package.Results. The study results suggested that with an overall decrease in the number of circulating CD14-positive blood monocytes in patients with pulmonary tuberculosis regardless of its clinical form, high expression of cell activation markers remained both for the pro-inflammatory M1 phenotype (HLA-DR-positive monocytes) and the anti-inflammatory M2 phenotype (CD163-positive monocytes). In disseminated tuberculosis, the number of anti-inflammatory CD204-positive monocytes, M2 macrophage precursors, increases indicating predomination of the immunosuppressive response. In vitro analysis of the cytokine status showed that tuberculosis progression is accompanied by inhibition of effector immune responses and increases in anti-inflammatory cytokine concentrations in vitro. These changes may be equally either a cause or a consequence of deficient IL-2 secretion. We also found that the secretion of mediators with suppressor effects (IL-10, TGFβ) varied depending on both the clinical form of tuberculosis and the pathogen sensitivity to anti-TB drugs; IL-10 hypersecretion was observed in patients with drug-sensitive, infiltrative tuberculosis whereas TGFβ hypersecretion was observed in disseminated, drug-resistant tuberculosis.Conclusion. Features of blood monocyte differentiation in patients with pulmonary tuberculosis allowed us to conclude that monocytes, the macrophage precursors, start expressing markers for different functions of M1 and M2 macrophages with polarization toward the M2 immunophenotype already in the bloodstream. Therefore, in the development of pulmonary tuberculosis, cytokine regulation mechanisms become involved in suppressing the activation of innate immunity, which possibly causes chronic inflammation in the lungs and formation of Mtb-induced immunodeficiency.