CD25 Treg Research Articles

A 5-Lipoxygenase Inhibitor, Zileuton, Modulates Host Immune Responses and Improves Lung Function in a Model of Severe Acute Respiratory Syndrome (SARS) Induced by Betacoronavirus.

Exacerbated inflammatory responses are a hallmark of severe coronavirus disease 2019 (COVID-19). Zileuton (Zi) is a selective inhibitor of 5-lipoxygenase, an enzyme involved in the production of several inflammatory/pro-resolving lipid mediators. Herein, we investigated the effect of Zi treatment in a severe acute respiratory syndrome (SARS) model. Mouse hepatitis virus (MHV)3-infected mice treated with Zi significantly improved the clinical score, weight loss, cardiopulmonary function, and survival rates compared with infected untreated animals. The protection observed in Zi-treated mice was associated with a lower inflammatory score, reduced dendritic cell-producing tumor necrosis factor (TNF), and increased neutrophil-producing interleukin (IL)-10 in the lungs three days after infection (dpi). At 5 dpi, the lungs of treated mice showed an increase in Th2-, Treg CD4+-, and Treg CD8+-producing IL-10 and reduced Th1 infiltrating cells. Furthermore, similar results were found upon Zi treatment after SARS-CoV-2 infection in transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2), significantly improving the clinical score, weight loss, and lung inflammatory score compared with untreated animals. Our data suggest that Zi protects against developing severe lung disease during SARS induced by betacoronavirus without affecting the host's capacity to deal with infection.

Evaluation of IL-6, FoxP3 Treg lymphocytes, intestinal barrier biomarkers and the use of synbiotics in obese adolescents: a pilot study.

This prospective pilot study evaluated inflammatory and intestinal barrier biomarkers and the effects of a synbiotic in obese adolescents. Eighteen obese and 20 eutrophic adolescents were evaluated for body composition using bioimpedance analysis (BIA), body mass index (BMI), IL-6 and lipopolysaccharide (LPS) serum levels, CD4 and FoxP3 Treg lymphocytes and monocytes. Synbiotic supplementation for 60 days was also evaluated for these parameters only in obese adolescents. We observed an increase in CD4 lymphocyte (18.0 ± 12.4 vs. 8.9 ± 7.5; p < 0.01), IL-6 (0.30 ± 0.06 vs. 0.20 ± 0.06; p = 0.02) and LPS (0.18 ± 0.15 vs. 0.08 ± 0.05; p < 0.01) levels in obese compared to eutrophic adolescents. After synbiotic supplementation, FoxP3 Treg lymphocytes increased (14.0 ± 6.7 vs. 9.9 ± 5.4; p = 0.02) in obese adolescents. Obese adolescents presented a state of microinflammation and intestinal barrier breakdown, and synbiotic supplementation increased the expression of FoxP3 Treg lymphocytes, an anti-inflammatory regulator. Whether the increase in FoxP3 Treg lymphocytes may have an impact on inflammation and outcomes in obese adolescents deserves further evaluation.

Effect of Concurrent Chemoradiotherapy on Regulatory T Cells,CD8/Treg Ratio,PD1 and CTLA-4 in Patients with Cervical Cancer

To investigate the significance of chemoradiotherapy on regulatory T (Treg) cells, CD8 / Treg ratio, squamous cell carcinoma antigen (SCC), PD1, and CTLA-4 in the peripheral blood of cervical cancer (CC)patients. A retrospective study was performed 56 cervical cancer patients treated with concurrent chemoradiotherapy from September 06, 2019 to April 19, 2021 were selected, in patients who underwent surgery. Flow cytometry was used to determine the levels of regulatory T cells, CD8 / Treg ratio, squamous cell carcinoma antigen, PD1, and CTLA-4 in the peripheral blood of patients before and after concurrent therapy, Differences in relative level values before and after treatment were calculated using statistical protocols such as the paired samples t-test. The proportion of CD4+CD25+CD127low Treg in CD4+T cells was (15.96±4.29) % in cervical cancer patients and (9.76±4.21) % in healthy controls, and the difference between the two groups was statistically significant (P < 0.05). In different age groups, Treg, CD8 levels, CD4/CD8 ratio and CD8/Treg ratio before and after radiotherapy and chemotherapy had no significant relationship with age and pathological types (P > 0.05), but CD8/Treg ratio was higher in patients with adenocarcinoma than in patients with squamous cell carcinoma after radiotherapy and chemotherapy, and the difference was statistically significant (Z = -2.076 P = 0.038). For postoperative patients, CD8 levels were lower before and after chemoradiotherapy than after chemoradiotherapy (T = -2.320 P = 0.020). In terms of PD1, regardless of age, pathological type, the level of PD1 after radiotherapy and chemotherapy was higher than that before chemotherapy, and the difference was statistically significant. The level of adenocarcinoma (53.50±10.16) % was significantly higher than that of squamous carcinoma (43.72±11.89) % (T = -2.609 P = 0.011). The PD1 level of patients with cervical cancer radical resection (41.64±13.29) % was lower than that of patients without cervical cancer radical resection (46.84±10.61) %, the difference was statistically significant (T = 2.187 P = 0.031). The PD1 level of patients without pelvic lymph node metastasis (48.84±10.04) was significantly higher than that of patients with pelvic lymph node metastasis (42.96±10.85), and the difference was statistically significant (T = -2.019 P = 0.049), There were no significant differences in vascular positivity, invasion depth, stump positivity, pelvic lymph node positivity and Treg level, CD8 level, CTLA4 level, SCC, CD4/CD8 ratio, CD8/Treg ratio (ALL P > 0.05). The level of Treg cells in patients with cervical cancer is significantly higher than that in healthy people, and it does not decrease immediately after radiotherapy and chemotherapy. Peripheral blood Treg, PD1, CD8 and CD8/Treg can reflect the immune function of the body, which may provide a certain reference for immunotherapy.

Causal role of immune cells in schizophrenia: Mendelian randomization (MR) study

BackgroundComplex immune-brain interactions that affect neural development, survival and function might have causal and therapeutic implications for psychiatric illnesses. However, previous studies examining the association between immune inflammation and schizophrenia (SCZ) have yielded inconsistent findings.MethodsComprehensive two-sample Mendelian randomization (MR) analysis was performed to determine the causal association between immune cell signatures and SCZ in this study. Based on publicly available genetic data, we explored causal associations between 731 immune cell signatures and SCZ risk. A total of four types of immune signatures (median fluorescence intensities (MFI), relative cell (RC), absolute cell (AC), and morphological parameters (MP)) were included. Comprehensive sensitivity analyses were used to verify the robustness, heterogeneity, and horizontal pleiotropy of the results.ResultsAfter FDR correction, SCZ had no statistically significant effect on immunophenotypes. It was worth mentioning some phenotypes with unadjusted low P-values, including FSC-A on NKT (β = 0.119, 95% CI = 0.044 ~ 0.194, P = 0.002), DN (CD4-CD8-) NKT %T cell (β = 0.131, 95% CI = 0.054 ~ 0.208, P = 9.03 × 10− 4), and SSC-A on lymphocytes (β = 0.136, 95% CI = 0.059 ~ 0.213, P = 5.43 × 10− 4). The causal effect of SCZ IgD on transitional was estimated to 0.127 (95% CI = 0.051 ~ 0.203, P = 1.09 × 10− 3). SCZ also had a causal effect on IgD+ %B cell (β = 0.130, 95% CI = 0.054 ~ 0.207, P = 8.69 × 10− 4), and DP (CD4+CD8+) %T cell (β = 0.131, 95% CI = 0.054 ~ 0.207, P = 8.05 × 10− 4). Furthermore, four immunophenotypes were identified to be significantly associated with SCZ risk: naive CD4+ %T cell (OR = 0.986, 95% CI = 0.979 ~ 0.992, P = 1.37 × 10− 5), HLA DR on CD14− CD16− (OR = 0.738 (95% CI = 0.642 ~ 0.849, P = 2.00 × 10− 5), CD33dim HLA DR+ CD11b− AC (OR = 0.631, 95% CI = 0.529 ~ 0.753, P = 3.40 × 10− 7) and activated & resting Treg % CD4 Treg (OR = 0.937, 95% CI = 0.906 ~ 0.970, P = 1.96 × 10− 4).ConclusionsOur study has demonstrated the close connection between immune cells and SCZ by genetic means, thus providing guidance for future clinical research.

Periprosthetic effusions surrounding breast expander: a flow cytometric, immunohistochemical and molecular characterization.

The synthesis of the periprosthetic capsule during implant-based breast reconstruction is the result of a coordinate cascade of inflammatory events ending in a fibrous tissue deposition around the expander or implant. Although the development of small volumes of fluid is one of the complications of prosthetic-based breast reconstruction, the characterization of the periprosthetic effusions coupled with the micro-textured devices, that have been recently introduced after the recall of macro-textured ones, is still lacking. The investigation of these periprosthetic effusions and paired capsules in terms of immunological content were the primary and secondary aims of the present study, respectively. For this, 68 women, 41 of whom had periprosthetic effusions at the time of expander replacement with implant, were recruited. For each case, capsule and healthy dermal tissues were taken and for women with periprosthetic effusion, peripheral blood was also collected. Periprosthetic effusions and peripheral blood were characterized by cytometry while capsules and dermal tissues by immunohistochemistry and Nanostring analysis. The results showed an increase of Th1, Th2 lymphocytes and a HLA-DR+bright CD16+ cells (likely representing monocytes-derived macrophages) in periprosthetic effusions in respect to peripheral blood. These pro-inflammatory cells were counterbalanced by the gain of suppressive CD4 Treg cells. In the corresponding capsules, immunohistochemistry revealed the absence of Th1 cells and the presence of tissutal FOXP3 Treg. No significant difference in expression of inflammatory-related genes between capsules and dermal tissues was present. These results suggest the presence of a Treg-controlled inflammation in both periprosthetic effusions and capsules.

Diverse intratumoral heterogeneity and immune microenvironment of two HPV-related cervical cancer types revealed by single-cell RNA sequencing.

Cervical squamous cell carcinoma (SCC) and adenocarcinoma (AD) are the main histological types of human papillomavirus-related cervical cancer. However, there are few reports on cell type-specific molecular differences between SCC and AD. Here, we used unbiased droplet-based single-cell RNA sequencing to elucidate the cellular differences between SCC and AD in tumor heterogeneity, and tumor microenvironment (TME). A total of 61 723 cells from three SCC and three AD patients, were collected and divided into nine cell types. Epithelial cells exhibited high intra- and interpatient heterogeneity and functional diversity. Signaling pathways, such as epithelial-to-mesenchymal-transition (EMT), hypoxia and inflammatory response were upregulated in SCC, while cell cycle-related signaling pathways were highly enriched in AD. SCC was associated with high infiltration of cytotoxicity CD8 T, effector memory CD8 T, proliferativenatural killer (NK), and CD160+ NK cells as well as tumor-associated macrophages (TAMs) with high major histocompatibility complex-II genes. AD exhibited a high proportion of naive CD8 T, naive CD4 T, Treg CD4, central memory CD8, and TAMs with immunomodulatory functions. Additionally, we also observed that the majority of cancer-associated fibroblasts (CAFs) were from AD, and participated in inflammation regulation, while SCC-derived CAFs exhibited similar functions to tumor cells, such as EMT and hypoxia. This study revealed the widespread reprogramming of multiple cell populations in SCC and AD, dissected the cellular heterogeneity and characteristics in TME, and proposed potential therapeutic strategies for CC, such as targeted therapy and immunotherapy.

A KIR x CD8 targeting bispecific modulator enhances regulatory CD8 T cell functions, and reduces inflammation in models of autoimmune disease

Abstract INTRODUCTION: We have characterized a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation results in their oligoclonal expansion and elimination of pathogenic CD4 T cells. Here we describe a bispecific CD8 Treg modulator that activates autoimmune patient derived CD8 Treg resulting in the cytolytic elimination of pathogenic CD4 T cell in vitro, ex vivo, and in vivo. METHODS: We tested target and cell binding of a novel bispecific CD8 Treg modulator by Octet and flow cytometry. The functional impact was evaluated using cytotoxicity assays and supernatant analysis in vitro, using Celiac patient derived biopsies ex vivo, and in acute GVHD studies using human PBMCs. RESULTS: We evaluated the efficacy and mechanism of action of a novel bispecific CD8 Treg modulator directed toward CD8 and inhibitory KIR2DL1/2/3. The modulator enhanced cytolytic CD8 Treg functions in vitro using autoimmune patient PBMCs, increased activated CD4 T cell death, and decreased pro-inflammatory cytokines. In Celiac patient derived duodenal tissues, the modulator increased CD8 Treg prevalence and reduced epithelial cell death after gliadin peptide stimulation. In acute GVHD studies, CD8 Treg modulator treated mice had CD8 Treg expansion in a dose dependent fashion and reduced inflammation. CONCLUSIONS: A novel KIR x CD8 targeting bispecific modulator restores cytolytic function of CD8 Treg, resulting in a decrease of pathogenic CD4 T cells. We postulate that the bispecific CD8 T cell modulator can reduce autoreactive CD4 T cell prevalence to reestablish immune balance and may represent a selective and broadly applicable therapeutic approach for the treatment of autoimmune disease.

Pre-clinical pharmacologic and tolerability characterization of a novel KIRxCD8 targeting bispecific CD8 Treg modulator

Abstract We have characterized a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. Here we describe the binding and specificity profiles of a novel bispecific CD8 Treg modulator in vitro, ex vivo, and in vivo, and initial assessment of tolerability and pharmacology in a humanized mouse model. We evaluated the binding, pharmacokinetics (PK), and early tolerability of the bispecific CD8 Treg modulator targeting CD8 and KIR2DL1/2/3. Despite single arm binding to NK cells via the KIR targeting arm and CD8 T cells by the CD8 targeting arm of the bispecific, we did not detect activation of immune cells or increased pro-inflammatory cytokines in vitro, nor did we observe any contribution of NK cells to the elimination of pathogenic CD4 T cells. In healthy mice, the CD8 Treg modulator had a PK profile consistent with antibody molecules; treatment delayed disease onset in an acute GVHD setting. In humanized CD34-engrafted NSG-IL-15 transgenic mice, supporting engraftment of NK cells, no activation of immune cells in peripheral blood or terminal tissues or induction of proinflammatory cytokines in the serum was observed following a single dose of the CD8 Treg modulator. The recently described CD8 Treg network appears dysfunctional in autoimmune diseases. The KIRxCD8 targeting bispecific modulator targets this network, engages and activates CD8 Treg and reduces inflammation without increasing unwanted immune cell activation or pro-inflammatory cytokines, both in vitro and in vivo. Our early data suggest that KIR-mediated enhancement of CD8 Treg function using a bispecific modulator is a broadly applicable and a promising CD8 Treg specific therapeutic modality for the treatment of autoimmune disease.

The role of NFAT in CD8+Foxp3+ Tregs

Abstract CD8 +Foxp3 +regulatory T cells (CD8 Treg) correspond to a very low frequency population which can be observed during GVHD at early stage or in some cancer types. However, in vitro, around 30–60% of CD8 can express Foxp3 after induction culture with TCR stimulation and TGF-β (iCD8Treg). Why in healthy mice and humans this cell population has very low or non-frequency been still an unresolved question. In vitro, iCD8Tregs can suppress effector cells in a similar way as iCD4Treg. However, in vivo, in the IBD model, we observed that in contrast to iCD4Treg, iCD8Treg lose the Foxp3 expression and consequently their suppressive function. Also, iCD8Treg increase the IFN-γ production. These results suggest that iCD8Treg are not able to maintain the Foxp3 expression in comparison with iCD4Treg cells. What signaling pathway is responsible for the Foxp3 losing in iCD8Treg? We found a positive correlation between Foxp3 maintenance and cyclosporin A (CSA) concentration, and a reduction of IFN-γ in culture in iCD8Treg, but not for iCD4Treg. We evaluated the effect of CSA, an NFAT inhibitor, on the Tregs induction culture (with TGF-β) in the CD8 cells and found that there was no increase of Foxp3 expression. These results suggest that NFAT signaling plays a role in the Foxp3 maintenance but not in the induction of the iCD8Treg and this role is different from iCD4 Tregs.

Human CAR19 CD8 +iTreg exceed CAR19 CD8 CTLs both in suppressing GVHD lethality and eliminating CD19 +Nalm-6 lymphoma cells in vivo

Abstract Simultaneously achieving a graft versus leukemia (GVL) effect while suppressing graft versus host disease (GVHD) would facilitate the development of immunotherapies for hematopoietic stem cell transplantation. Here, we show that human peripheral blood CD8+CD25− T-cells cultured with IL-2, TGF-β and rapamycin generates CD8 induced Tregs (iTregs). CD8 iTregs expressed FoxP3, CD25 and higher CD103 and CD39, but lower Helios, CTLA-4, and TIGIT than CD4 Tregs. While in vitro suppressor function was modestly reduced with CD8 iTreg, in vivo suppression of GVHD lethality was comparable to that of CD4 Treg. In vitro, cytolytic activity with an αCD3×αCD19 bispecific engager against CD19+ Nalm-6 lymphoma cells was comparable to CD8 CTLs. We subsequently generated CD8 iTreg and CTLs expressing αCD19scFv (CAR19). Following CAR19-EGFR sort purification, CAR19 CD8 iTreg and CAR19 CTL had similar expression of perforin, IFNγ and TNFα. In a 48h Incucyte killing assay against CD19+Nalm-6 targets, using CAR19 CD8 iTreg and CAR19 CTLs generated from the same donors, no significant differences were noted; CD8 iTreg killing was perforin-dependent and granzyme-independent. No killing was observed against CD19 koNalm-6 targets. Unexpectedly, CAR19 CD8 iTreg were found to be significantly more effective at limiting tumor burden in vivo and significantly delayed tumor-related mortality in a xenogeneic Nalm-6 tumor model compared to CAR19 CTLs (p = 0.0018). Mechanistic studies are ongoing. Together, these findings highlight distinct advantages of CD8 iTreg as a concurrent immunotherapy and provide a rationale for CD8 iTreg based CAR therapies to prevent GVHD and promote GVL. Supported by grants from NIH (R01 HL11879-07 and 2P01 CA065493-25) as well as funding from the Childrens' Cancer Research Fund

Distinct Patterns in Treg and T-helper Subset Reconstitution After Autologous Hematopoietic Stem Cell Transplantation in Juvenile-onset Systemic Sclerosis Patients

Systemic sclerosis (SSc) is a refractory autoimmune disease. In recent years, autologous hematopoietic stem cell transplantation (ASCT) has been performed to treat SSc. However, the reconstitution of immune components post-ASCT in SSc have not been fully described.We tested a total of 13 samples from 4 consenting SSc patients (median age: 18.5-years-old, 3F/1M) receiving IRB-approved immunoablation (TBI 600cGy) with lung/kidney shielding and Cytoxan (n = 3) or Thiotepa (n = 1) +/– Alemtuzumab followed by ASCT (CD34+ selected). Peripheral blood samples were collected at baseline, and 3, 6, 12, 24-months post-ASCT. By flow cytometry, we focused on the FOXP3+Treg and T-helper subset reconstitution.All 4 subjects experienced significant improvement in several clinical and quality of life parameters, especially in their skin disease evident within weeks after ASCT. T-cell recovery was evident by 6-months reaching pre-ASCT levels by 2-years post-ASCT (Fig. 1A). There was an increased percentage of FOXP3+Tregs in total CD4+T cells from 3- to 6-months post-ASCT (Fig. 1B), leading to an increase in the ratio of Treg/Tcon (Fig. 1B). The higher proportions of proliferating Tregs (Fig. 1C) likely led to their increased numbers. This trend was sustained until the last evaluation at 2-years post-ASCT (Fig. 1C). In contrast, CD4+Tcons showed a relatively lower proliferation rate, leading to a higher ratio of Ki67+Treg/Ki67+Tcon compared to pre-ASCT values (Fig. 1C). Notably, there was an increased proportion of Tregs expressing the skin-homing marker cutaneous lymphocyte-associated antigen (CLA) at 3-months lasting until the last test at 2-years post-ASCT (Fig. 1D), whereas CLA+/CD4+Tcons were less plentiful.The reconstitutions of T-helper (Th) subsets were also examined according to their chemokine receptor expression. Gradually decreased numbers of Th2, Th9, and Th17 were observed in the peripheral pool (Fig. 1E,1F). Nevertheless, the absolute number of Th1 and Th22 cells returned to levels seen pre-ASCT without any recurrence or flare of SSc symptoms.In summary, an increased number of circulating FOXP3+Tregs was observed leading to an elevated Treg/Tcon ratio in the peripheral pool. Skin-homing CLA+Treg subset was further enriched correlating with improved skin scores in all patients. The decrease of the Th2/Th9/Th17 subsets in the peripheral pool post-ASCT possibly contributes to the clinically less severe autoimmune phenotype. [Display omitted]

The functional role of Helios in CD4+ T cells

Abstract The transcription factor Helios is expressed in the majority of Foxp3+ Regulatory T cells (Treg), where it plays an essential role in Treg function. Loss of Helios in the Treg population induces systemic autoimmunity, marked by CD4 and CD8 hyperproliferation, enhanced germinal center formation, and systemic immune activation. Helios is also expressed in a small subset of conventional CD4 +T cells. However, when Helios is deleted in all CD4 cells, conventional and Treg, by crossing the mice to CD4Cre, this phenotype is abrogated, indicating that Helios must also play a significant role in the function of conventional CD4 +T cells. In order to examine specific subsets of CD4 +memory cells, we performed single cell transcriptome analysis of CD44 himemory cells from WT mice and mice with the CD4 specific deletion of Helios. Characterization of the CD44 hiHelios +population showed that Helios expression changed dramatically with age, so cells from young (5–6 weeks) and old (5–6 months) mice were included in the analysis. Both longitudinal (young vs old) and cross-sectional (WT vs KO) comparisons were made, with emphasis placed on cross-sectional comparisons at old age. The sequencing results indicated significant transcriptomic and numeric shifts in multiple subpopulations of the analyzed splenocytes, including the almost complete loss of one cluster in both the young and old Helios deficient mice. Taken together, the observed transcriptomic and clustering changes may explain the role Helios plays in the abrogation of autoimmunity in our model and may provide a starting point for us to analyze its function in other biological pathways. This work was supported by the Intramural Research Program of NIAID, NIH This work was supported by the Intramural Research Program of NIAID, NIH

Protective effects of oridonin against osteoporosis by regulating immunity and activating the Wnt3a/β-catenin/VEGF pathway in ovariectomized mice

This study was performed with the aim of investigating the effect of oridonin (ORI) on estrogen deprivation-induced osteoporosis in mice and its mechanism. Animal experiments were used in this work to validate the anti-osteoporotic efficacy of ORI. Morphometric analysis was performed by micro-CT. A special protein meter was used to detect the content of immunoglobulin lgM, immunoglobulin lgG, complement C3 and C4 in the serum of mice. The expression of CD4+CD25+Foxp3+ Treg cell and CD4+/CD8+ lymphocyte subsets in mice was detected by flow cytometry. ELISA was used to detect the content of insulin-like growth factor (IGF-1), tumor necrosis factor (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6). In addition, key signaling molecules in the Wnt3a/β-catenin signaling pathway were detected by Western blotting. The results showed that compared with the model group, the contents of calcium and phosphorus in the femurs of mice in the ORI groups were increased, and the spleen coefficient was decreased. The ALP activity in the serum of mice in the high and medium dose ORI groups was decreased, and the uterine coefficient was increased. ORI significantly increased the maximum bending load and the maximum bending stress of the femurs of mice, increased the number of trabeculae, and repaired the bone microstructure. At the same time, ORI could significantly increase the levels of immunoglobulin (lgG and lgM) and complement (C3 and C4), increase the activity of peritoneal macrophages in mice, increase the expression of CD4+CD25+Foxp3+ Tregs and CD4+/CD8+ in the spleen, increase the content of IGF-1, reduce the content of TNF-α, IL-1 and IL-6 and increase the expression levels of VEGF, Wnt3a, p-GSK3β/GSK3β and β-catenin/Lamin in the femoral tissue. These results indicated that ORI might regulate the expression of VEGF through the Wnt3a/β-catenin signaling pathway, improve the immunity of mice, maintain the balance of the immune system, and promote angiogenesis, thereby improving the bone mineral density and bone tissue morphology of mice and playing an anti-osteoporotic role.

Abstract 4336: Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system

Abstract The PD-1/PD-L1 signaling pathway is essential for immune control and maintaining immune system balance. PD-1 and PD-L1 proteins exert most functions in cells and tissues by undergoing modifications and forming dynamic complexes – effects that cannot be explored by genomics, transcriptomics, or conventional immunostaining methods. Numerous cancer therapies are being developed that affect PD-1/PD-L1 signaling, and tools to study the PD-1/PD-L1 axis are, therefore, essential. One prerequisite for successfully establishing diagnostic assays is reproducibility, standardization, and, ideally, high sample throughput via automation. Here, we aimed to introduce a new technological solution to allow for the simultaneous detection of PD-L1 and PD-1 interaction and the adjacent immune cell context in tissue samples on the Lunaphore’s automated microfluidic staining platform. The assay for detecting PD-1-PD-L1 interaction was developed and technically validated by Navinci Diagnostics. Although the detection of interaction alone provided important information on the immune status in the tumor microenvironment, we believed that PD-1/PD-L1 readout would be most powerful in the context of its cellular ecosystem. Therefore, the PD-1/PD-L1 assay was combined with the analysis of immune and cancer cells to determine their involvement in this signaling. The feasibility of combining multiplex immunofluorescence with proximity ligation assay was conducted and confirmed on human tonsils. The spatial profiling panel included immune markers for all T-cells (CD3), helper T-cell (CD4), cytotoxic T-cells (CD8), B-cells (CD19/20) and activation/exhaustion markers granzyme B (activated cytotoxic T-cell), Ki67 (proliferating cells), and LAG3 (malignant B cells, CD8 T cells, CD4 Tregs). We evaluated, implemented, and optimized the assays on the Lunaphore staining instrument to reduce labor time and cost for the end users. In the next phase, the potential of our assay to be benchmarked as a diagnostic tool in clinical praxis will be thoroughly tested and validated on diagnostic tissue samples from NSCLC patient cohorts. We believe this approach will enable spatial and functional studies of the interface between the tumor and the immune system and provide necessary information about signaling pathway activation in situ, the latest representing a novel state-of-the-art in tissue diagnostics. Citation Format: Hampus Elofsson, Agata Zieba Wicher, Carolina Oses Sepulveda, Tony Ullman, Maria-Giuseppina Procopio, Alix Faillétaz, Diego G. Dupouy, Charlotte Stadler. Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system. [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 4336.

Abstract 6424: Immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes in ovarian clear cell carcinoma relative to high-grade serous ovarian carcinoma

Abstract Recent clinical trials have demonstrated the potential efficacy of immune checkpoint inhibitors (ICIs) for ovarian clear cell carcinoma (OCCC). However, little is known about the immune characteristics of OCCC. In this study, we aimed to investigate the distinct immune characteristics of tumor-infiltrating lymphocytes (TILs) in OCCC to high-grade serous ovarian carcinoma (HGSOC). We isolated peripheral blood mononuclear cells (PBMCs) and TILs from patients with epithelial ovarian cancer. The expression of immune checkpoint receptors and T-cell transcription factors of TILs in OCCC (n=26) and HGSOC patients (n=44) were accessed using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-CTLA-4, and their proliferation was assessed to predict response to ICIs. CD8 TILs and tumor-infiltrating regulatory T cells (Tregs) exhibited higher expression of immune checkpoint receptors, including PD-1, CTLA-4, and 4-1BB, compared to PBMCs. Tregs were more infiltrated in advanced/recurrent stage than early stage OCCC. However, immune characteristics of CD8 TILs and Tregs were not significantly different regardless of stage. When we compared the characteristics of TILs in OCCC (n=26) with those in HGSOC (n=44), the expression of PD-1 on CD8 TILs was significantly lower in OCCC than in HGSOC. The frequencies of PD-1highCD8 TILs and PD-1+TOX+CD8 TILs were also lower in OCCC than HGSOC. In ex vivo assay (n=19), 9 cases responded to anti-PD-1 in terms of enhanced proliferation capacity of CD8 TILs. In addition, when anti-CTLA-4 was added to anti-PD-1, an additional increase in proliferation of CD8 TILs was observed in patients who responded to anti-PD-1. Responders to anti-PD-1 had significantly lower frequencies of effector Tregs in the TILs than non-responders. Overall, CD8 TIL in OCCC was found to be less exhausted than HGSOC in terms of PD-1 and TOX expressions. Tumor-infiltrating Tregs affected on reinvigoration ability of CD8 TILs upon ICIs treatment in OCCC. This study provides the rationale and evidence for establishing the optimal strategies for ICIs treatment in patients with OCCC. Citation Format: Junsik Park, Yong Jae Lee, Jung Chul Kim, Jung-Yun Lee. Immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes in ovarian clear cell carcinoma relative to high-grade serous ovarian carcinoma. [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 6424.

A NOVEL BISPECIFIC CD8 TREG MODULATOR TARGETING CYTOLYTIC CD8 REGULATORY T CELLS REDUCES PATHOGENIC CD4 T CELLS AND INFLAMMATION IN TRANSLATIONAL MODELS OF INTESTINAL AUTOIMMUNE AND INFLAMMATORY DISEASE

We have characterized a novel CD8 Treg network in autoimmune patient peripheral blood and tissues, in which a subset of cytolytic CD8 Treg eliminate pathogenic CD4 T cells, reducing inflammation and ameliorating disease in response to pathogenic CD4 T cell activation.

Human small intestine contains 2 functionally distinct regulatory T-cell subsets

Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited. We performed a detailed characterization of Foxp3+ CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions. Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed. SI Foxp3+ CD4 T cells were CD45RA-CD127-CTLA-4+ and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3+ Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold. The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease.

T helper 17 and regulatory T-cell profile and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in pediatric patients with beta-thalassemia

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment option for hereditary hemoglobin disorders, such as beta-thalassemia; However, this procedure is not without constraints, mainly engendering complications such as acute graft-versus-host disease (aGvHD), chronic GvHD (cGvHD), and susceptibility to infections. The clinical outcomes of allo-HSCT are highly dependant on the quality and quantity of T-cell subsets reconstitution. Following the allo-HSCT of six pediatric patients afflicted with beta-thalassemia, their mononuclear cells were isolated, and then cultured with a combination of phorbol myristate acetate (PMA)/ionomycin and Brefeldin A. The content of CD4 T-cell subsets, including T helper 17 (Th17) cells and regulatory T cells (Tregs), were determined by specific conjugated-monoclonal antibodies three and six months post-HSCT. An increased frequency of total CD4 T-cells, Tregs and Th17 cells was observed at day 90 and 180 after allo-HSCT, albeit the numbers were still lower than that of our healthy controls. In patients who developed cGvHD, a lower Th17/Treg ratio was observed, owing it to a decreased proportion of Th17 cells. In conclusion, creating balance between Th17 and Treg subsets may prevent acute and chronic GvHD in patients after allo-HSCT.

Probiotic-educated Tregs are more potent than naïve Tregs for immune tolerance in stressed new-born mice.

When new-born mice are subjected to acute maternal separation stress, cow-milk based formula feeding, and brief recurrent hypoxia with cold stress, they develop gut inflammation similar to the phenotype of neonatal necrotizing enterocolitis, characterised by an increase in gut mucosal effector T (Teffs) and reduced Foxp3+ regulatory T (Tregs) cells. The imbalance can be prevented by probiotic Limosilactobacillus reuteri DSM 17938 (LR 17938). We hypothesised that LR 17938 could potentiate a tolerogenic function of Tregs. To analyse whether LR 17938 can educate Tregs to improve their tolerogenic potency during neonatal stress, we isolated T cells (Tregs and Teffs) from 'donor' mice fed with either LR 17938 (107 cfu) or control media. The cells were adoptively transferred (AT) by intraperitoneal injection (5 × 105 cells/mouse) to new-born (d5) recipient mice. Mice were then separated from their dams, fed formula by gavage, and exposed to hypoxia and cold stress (NeoStress) for 4 days. We analysed the percentage of Tregs in CD4+T helper cells in the intestine (INT) and mesenteric lymph nodes (MLN) of recipient mice. We found that: (1) the percentage of Tregs in the INT and MLN following NeoStress were significantly reduced compared to dam-fed unstressed mice; (2) AT of either naïve Tregs or LR-educated Tregs to mice with Neostress increased the percentage of Tregs in the INT and MLN compared to the percentage in NeoStress mice without Treg treatment; however, LR-educated Tregs increased the Tregs significantly more than naïve Tregs; and (3) AT of LR-educated Tregs reduced pro-inflammatory CD44+Foxp3-NonTregs and inflammatory CX3CR1+ dendritic cells in the intestinal mucosa of NeoStress mice. In conclusion, adoptive transfer of Tregs promotes the generation of and/or migration of endogenous Tregs in the intestinal mucosa of recipient mice. Importantly, probiotic-educated Tregs are more potent than naïve Tregs to enhance immune tolerance following neonatal stress.

The Proportion of Recent Thymic Emigrant Lymphocytes in Breastfed and Formula Fed Term Neonates

Recent thymic emigrants (RTEs) represent a distinct T cell subset characterized by a tolerance-prone status. We have recently demonstrated that the proportion of regulatory T cells (Tregs) is nearly two-fold higher in exclusively breastfed compared with exclusively formula-fed neonates. However, it has been unknown whether the type of milk is also associated with the proportion of the RTE cell compartment. Cord blood (CB) and, at three weeks of age, peripheral venous blood samples were collected from 19 healthy-term neonates. A maternal blood sample was also taken. The proportion of RTEs, naïve CD4 cells, naïve RTEs, and Tregs was analyzed by flow cytometry in blood samples. RTE cell proportions were comparable between CB and 3 weeks. At both time points, there was no difference in the proportion of naïve CD4 cells, RTE CD4 cells, and naïve RTE CD4 cells between the feeding groups. The fold change of RTE cells between birth and three weeks of life was highest in mixed-fed babies. Since RTE counts were comparable across the feeding groups at birth, this most likely reflects a postnatal upregulation, to which the dual antigenic exposure to both non-inherited maternal antigens via breastmilk, as well as to other environmental antigens in formula milk, may contribute.