Cell Subset Populations Research Articles

Abstract 4305: Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice

Abstract Cancer is a complex disease that involves both malignant cells and the surrounding microenvironment. The tumor microenvironment (TME) is comprised of various components, including cancer-associated fibroblasts, immune cells, blood vessels, and extracellular matrix elements, all of which interact with cancer cells. It is increasingly recognized that the components of the TME are a critical determinant of therapeutic response and resistance, and as such targeting the TME has emerged as a promising strategy in cancer therapy. Syngeneic models are necessary to study the TME, and more specifically the tumor infiltrating immune cell populations, as these models allow us to monitor various aspects of the immune response. Another important aspect to consider is tumor heterogeneity, which can cause issues during analysis due to immune cell populations not being evenly distributed throughout the tumor. The decision of whether to analyze whole tumors or halved tumors when monitoring changes in immune cell populations is an important consideration. When considering multiple endpoints from a single tumor sample, halved tumors are oftentimes considered as we remain mindful of the 3Rs (replacement, reduction and refinement) in animal research. As such, it is important to understand the effects of splitting samples and how it can affect the data that is generated. To determine the effects of analyzing whole and halved tumor samples on immune cell subsets, studies were designed to subcutaneously implant either CT26.WT or 4T1-Luc2-1A4 cells into female BALB/cAnNHsd mice and harvest tumor samples after the completion of treatment. Mice were treated with isotype control (Clone MPC-11) or anti-mCTLA-4 (Clone 9D9). At the time of sample collection, tumors were kept whole intact or halved and analyzed via flow cytometry to quantify lymphoid and myeloid immune cell subsets within tumor samples. The data revealed differences in immune cell subset populations for both isotype vs. anti-CTLA-4 treated tumors and whole vs. halved tumors. Changes in the lymphoid cell compartment were observed for whole vs. halved tumors of ± ~25-100% and ± ~0-40% for isotype treated and of ± ~0-30% and ± ~25-2600% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively. Changes in the myeloid cell compartment of ± ~5-45% and ± ~10-65% for isotype treated and of ± ~15-100% and ± ~0-70% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively, were also observed. Additionally, differences were observed between the two tumor halves, not just between whole and halved tumors. Taken together, the data support that splitting tumor samples can negatively impact the analysis of the immune cell populations. Tumors can be split if required to perform multiple analyses, however, this should be done with caution, as it can result in trend differences and increases variability within the dataset. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Sheri Barnes, David Draper, Scott Wise. Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4305.

P-017 Impact of yoga intervention on immunosenescence, fertility potential and quality of life in men with Rheumatoid arthritis

Abstract Study question Can a yoga intervention bring about alterations in immune aging, fertility potential and quality of life in men with Rheumatoid arthritis? Summary answer Yoga not only reduces disease severity, minimizes usage of drugs but also reduces the rate of immune aging and cellular aging. What is known already The complex mechanism of Rheumatoid arthritis with infertility in men involves interactions between endocrine, immune, and reproductive systems. Association of autoimmunity with dysregulated androgen (hypogonadism) levels may cause transient infertility in men. Furthermore, usage of disease-modifying antirheumatic drugs (DMARDs) like cyclophosphamide, methotrexate, sulphasalazine etc. may result into decreased quantity and quality of sperm, mitochondrial dysfunctions, reduced fertility potential and ultimately permanent infertility. These drugs can cross blood-testis-barrier and induce changes in sperm impairing spermatogenesis. Complementary and alternative medicine like yoga reduces seminal oxidative stress and its consequences like DNA fragmentation in sperm nuclear/mitochondrial genome. Study design, size, duration Fifty males with RA were enrolled in this 12-week prospective, open-label, single-arm exploratory study, designed to explore the impact of yoga intervention on immunosenescence, fertility potential and quality of life (QoL). Participants/materials, setting, methods The participants were evaluated for pre (day 0) and post (12th week) levels of inflammatory markers: IL-6, IL-17A, TGF-β and TNF-α. T cell sub-set populations along with their aged T cell profiles for immunosenescence as well as telomere length and telomerase activity for cellular aging were studied. Seminal reactive oxygen species (ROS), DNA fragmentation index (DFI) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were estimated as per WHO 2010 guidelines. QoL was measured by WHOQOL-BREF Scale. Main results and the role of chance There was significant reduction in mean levels of TNF-α, IL-6, IL-17A and elevation in TGF-β after 12 weeks. A significant reduction was seen in seminal ROS levels, 8-OHdG and DFI as well. There was a significant decline in aged T cell populations after yoga. There was no change seen in the telomere length but there was an improvement in the telomerase activity. The participants showed significant improvements in QoL after 12 weeks. Limitations, reasons for caution Compliance of patients for Yoga intervention was poor, hence we enrolled large number of patients to achieve the desirable sample size. Wider implications of the findings Adoption of yoga intervention holds the key to maintaining immune homeostasis, reduction of inflammation, reduction in the rate of cellular aging, improved overall QoL, and its associated consequences associated with physical, mental, and reproductive health. Trial registration number N/A

Changes of Cell Adhesion Molecules and T Cell Subset Populations in Acute Myeloid Leukemia Patients Undergoing Intravenous Administration of Cytarabine Supplemented with Idarubicin

Objective. The present study aimed at investigating the efficacy and safety of intravenous administration of cytarabine supplemented with idarubicin in treating acute myeloid leukemia (AML) patients undergoing first attack and its effects on serum levels of cell adhesion molecules, cytokines in response to inflammation, and T cell subset populations in acute myeloid leukemia (AML) patients undergoing first attack. Methods. A total of 88 AML patients eligible for inclusion and exclusion criteria participated in the study and were randomly assigned into the control group (n = 44) in which the patients received intravenous administration of cytarabine and daunorubicin and the study group (n = 44) in which the patients received intravenous administration of cytarabine and idarubicin. Clinical response, incidence of adverse reactions, and quality of life 3 months after therapy were evaluated. Soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), IL-10, and IL-35 were measured by ELISA methods. Phenotypic characteristics of T cell subsets including CD4+, CD8+, CD4+IL-10 Tregs, and CD4+CD25+CD127−Foxp3+ Tregs were analyzed by flow cytometry. Results. The clinical response rate of the study group was better than that of the control group (65.91% vs. 45.45%) ( P < 0.05 ). After treatment, the study group revealed significantly lower levels of sICAM-1, sVCAM-1, IL-10, and IL-35, a lower proportion of Tregs, a higher rate of CD4+/CD8+ T cells, along with increased scores of the Karnofsky Performance Scale (KPS) compared with the control group ( P < 0.05 ). The incidence rate of adverse reactions in the study group was lower than that in the control group (34.09% vs. 61.36%) ( P < 0.05 ). Conclusion. These findings demonstrate that intravenous administration of cytarabine supplemented with idarubicin can improve the immune function and quality of life of AML patients, and this combination drug therapy is effective and safe for AML.

Lactobacillus acidophilus Supplementation Exerts a Synergistic Effect on Tacrolimus Efficacy by Modulating Th17/Treg Balance in Lupus-Prone Mice via the SIGNR3 Pathway.

ObjectiveTacrolimus (Tac) is an immunosuppressant used in the treatment of systemic lupus erythematosus (SLE); however, it induces T cell subset imbalances by reducing regulatory T (Treg) cells. Lactobacillus acidophilus (LA) is reported to have therapeutic efficacy in immune-mediated diseases via T cell regulation.MethodsThis study investigated whether a combination therapy of LA and Tac improves the therapeutic efficacy of Tac by modulating T cell subset populations in an animal model of SLE. Eight-week-old MRL/lpr mice were orally administered with 5 mg/kg of Tac and/or 50 mg/kg of LA daily for 8 weeks. Cecal microbiota compositions, serum autoantibodies levels, the degree of proteinuria, histological changes in the kidney, and populations of various T cell subsets in the spleen were analyzed.ResultsMice presented with significant gut dysbiosis, which were subsequently recovered by the combination treatment of Tac and LA. Double negative T cells in the peripheral blood and spleens of MRL/lpr mice were significantly decreased by the combination therapy. The combination treatment reduced serum levels of anti-dsDNA antibodies and Immunoglobulin G2a, and renal pathology scores were also markedly alleviated. The combination therapy induced Treg cells and decreased T helper 17 (Th17) cells both in vitro and in vivo. In vitro treatment with LA induced the production of indoleamine-2,3-dioxygenase, programmed death-ligand 1, and interleukin-10 via the specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 receptor signals.ConclusionThe present findings indicate that LA augments the therapeutic effect of Tac and modulates Th17/Treg balance in a murine model of SLE.

Infectious Complications of Daratumumab-Containing Therapy for Multiple Myeloma

IntroductionOver the past several years, new drug therapies have transformed the treatment approach in multiple myeloma. Amongst these, daratumumab (a monoclonal antibody directed against CD38) has been a clear breakthrough in relapsed myeloma and is currently being studied in frontline regimens. It is known that multiple myeloma patients are inherently immunosuppressed by functional hypogammaglobulinemia and decreased T-cell counts, but the impact of anti-CD38 monoclonal antibodies on the remaining innate immunity and infection rates is unclear. In this study, we measured infection rates and immune cell subset populations in multiple myeloma patients treated with daratumumab-containing regimens.MethodsWe conducted a retrospective analysis of multiple myeloma patients who received daratumumab at our institution from October 2015 to December 2016. Patients were divided into four groups based on administration regimen of daratumumab: (1) single agent with dexamethasone; (2) double therapy with another agent (proteasome inhibitor (PI), immune-modulating drug (IMiD), mitogen-activated protein kinase (MEK) inhibitor or mAB) (3) triple therapy with IMiD ± PI ± mAB or cyclophosphamide; (4) combination therapy with high-dose chemotherapy. We evaluated the incidence of infection, hospitalization, and 90-day survival.ResultsWe studied a total of 170 subjects, 108 (64%) were male and 62 (36%) female. The median age was 67 years (range: 40-87).They received a total of 340 different daratumumab regimens: single therapy (n=99, 29%), double therapy (n=171, 50%), triple therapy (n=52, 15%) and combination therapy (n=18, 5%).An infection event occurred in 124 of the regimens (36.5%). The causative agent was found in 114 cases (92%): 79 viral (69%) and 53 bacterial (46%). Our study found that patients who developed an infection had statistically significant lower nadir ANC (1007 ± 1034 vs 1459 ± 1248), t(256) = 3.047, p = 0.003; and lower nadir ALC (529 ± 544 vs 769 ± 588), t(332) = 3.698, p < 0.001 compared to patients who didn't develop an infection. There were no differences in the absolute CD4 and absolute CD 56 between patients who developed viral vs bacterial infections.Patients who developed a viral infection had statistically significant lower nadir ALC (529 ± 569 vs 717 ± 583), t(332) = 2.108, p = 0.036 compared to patients who didn't develop a viral infection.A hospitalization event occurred in 82 cases (24%). The reason for the hospital admission was an infection process in 38 (51%) of the cases. No statistically significant differences in the rates of hospitalization were seen in the different daratumumab regimen groups.The overall 90-day overall survival for this cohort was 96 % (n=327). In patients who didn't achieve 90-d survival, the cause of death was related to an infection in 62% (n=8).ConclusionMultiple myeloma patients treated with daratumumab-containing regimens had a relatively high rate of infectious complications (36.5%), which were identifiable and most often viral. Infections were associated with significantly lower nadir ANC and ALC, and viral infections were associated with a significantly lower ALC than in patients who did not develop viral infections. No differences were appreciated in absolute CD4 or absolute CD56 in viral vs. bacterial infections, or in infected vs. non-infected patients. Further studies are needed to elucidate the entire immunomodulatory effects of daratumumab therapy in patients with multiple myeloma. Increased awareness of its associated risk of infection is important to permit clinical recognition and appropriate anti-infective measures. DisclosuresDavies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria.

The Effect of Hyperbaric Oxygen Therapy on Human Adipose-Derived Stem Cells.

Adipose-derived stem cells are considered as candidate cells for regenerative plastic surgery. Measures to influence cellular properties and thereby direct their regenerative potential remain elusive. Hyperbaric oxygen therapy-the exposure to 100% oxygen at an increased atmospheric pressure-has been propagated as a noninvasive treatment for a multitude of indications and presents a potential option to condition cells for tissue-engineering purposes. The present study evaluates the effect of hyperbaric oxygen therapy on human adipose-derived stem cells. Human adipose-derived stem cells from healthy donors were treated with hyperbaric oxygen therapy at 2 and 3 atm. Viability before and after each hyperbaric oxygen therapy, proliferation, expression of surface markers and protein contents of transforming growth factor (TGF)-β, tumor necrosis factor-α, hepatocyte growth factor, and epithelial growth factor in the supernatants of treated adipose-derived stem cells were measured. Lastly, adipogenic, osteogenic, and chondrogenic differentiation with and without use of differentiation-inducing media (i.e., autodifferentiation) was examined. Hyperbaric oxygen therapy with 3 atm increased viability, proliferation, and CD34 expression and reduced the CD31/CD34/CD45 adipose-derived stem cell subset and endothelial progenitor cell population. TGF-β levels were significantly decreased after two hyperbaric oxygen therapy sessions in the 2-atm group and decreased after three hyperbaric oxygen therapy sessions in the 3-atm group. Hepatocyte growth factor secretion remained unaltered in all groups. Although the osteogenic and chondrogenic differentiation were not influenced, adipogenic differentiation and autodifferentiation were significantly enhanced, with osteogenic autodifferentiation significantly alleviated by hyperbaric oxygen therapy with 3 atm. Hyperbaric oxygen therapy with 3 atm increases viability and proliferation of adipose-derived stem cells, alters marker expression and subpopulations, decreases TGF-β secretion, and skews adipose-derived stem cells toward adipogenic differentiation. Therapeutic, V.

Comparing the functionality of proleukin® and akron interleukin-2 through an analysis of key T cell subsets

Background & Aim Recombinant human interleukin-2 (IL-2) promotes the proliferation of activated T cells and has long been used in vivo to stimulate and maintain the growth of effector T cells and increasingly, to expand T cells in vitro for adoptive cell therapy in patients with cancer. IL-2 was approved by the FDA for treatment of metastatic renal cancer in 1992 and for treatment of metastatic melanoma in 1998. However, its administration in the approved dose has been associated with potentially fatal side effects, including vascular toxicity. There have recently been efforts to improve upon the existing product, marketed as Proleukin® (aldesleukin), through modifications to the molecule, through the development of fusion proteins, and through the development of low-dose formulations. Akron Biotechnology manufactures IL-2 for in vitro use. The product is embedded in several clinical and commercial CAR T therapies. Moreover, the company has sought to register a novel, low-dose formulation of its IL-2 to treat patients suffering from acute and chronic graft versus host disease (GVHD) . The data confirms the functional similarity of Proleukin® and Akron's IL-2, bolstering the product's utility for both in vitro and in vivo uses. Methods, Results & Conclusion Methods CyTOF: antibody panel designed by Dana Farber Cancer Institute. There are three categories of target proteins: surface proteins,intracellular markers and signaling molecules. Different markers were checked after stimulation with Akron IL-2 or Proleukin® (15 minutes) and verified with lymphocyte subset and intrinsic signaling cascade. Results The research focused on three major T cell subset populations (Treg, Tcon and CD8 T cells).Upon stimulation with Akron IL-2 and Proleukin®, CD25, CD122, CD132, CTLA-4, PD-1,PD-L1 markers were checked for T cell subsets, observed via t-SNE plot. The data indicated that the antibody panel setting was accurate and reliable; each marker was expressed in the expected T cell subset. For example: CD25 was expressed exclusively in Tregs. The expression of pSTAT5, pSTAT3, and pSTAT1 was identical in Akron IL-2 and Proleukin®, controlling for exposure and dosage . These results confirm that Akron IL-2 and Proleukin® are identical in their ability to boost adaptive immune response. Conclusions This study demonstrates that Akron's IL-2 is functionally similar to Proleukin®, its utility for adoptive immunotherapies, and potentially for treatment of patients suffering from autoimmune diseases.

Efficacy and tolerability of anti-programmed death-ligand 1 (PD-L1) antibody (Avelumab) treatment in advanced thymoma

BackgroundThymic epithelial tumors are PD-L1–expressing tumors of thymic epithelial origin characterized by varying degrees of lymphocytic infiltration and a predisposition towards development of paraneoplastic autoimmunity. PD-1–targeting antibodies have been evaluated, largely in patients with thymic carcinoma. We sought to evaluate the efficacy and safety of the anti-PD-L1 antibody, avelumab (MSB0010718C), in patients with relapsed, advanced thymic epithelial tumors and conduct correlative immunological studies.MethodsSeven patients with thymoma and one patient with thymic carcinoma were enrolled in a phase I, dose-escalation trial of avelumab (MSB0010718C), and treated with avelumab at doses of 10 mg/kg to 20 mg/kg every 2 weeks until disease progression or development of intolerable side effects. Tissue and blood immunological analyses were conducted.ResultsTwo of seven (29%) patients with thymoma had a confirmed Response Evaluation Criteria in Solid Tumors–defined partial response, two (29%) had an unconfirmed partial response and three patients (two thymoma; one thymic carcinoma) had stable disease (43%). Three of four responses were observed after a single dose of avelumab. All responders developed immune-related adverse events that resolved with immunosuppressive therapy. Only one of four patients without a clinical response developed immune-related adverse events. Responders had a higher absolute lymphocyte count, lower frequencies of B cells, regulatory T cells, conventional dendritic cells, and natural killer cells prior to therapy.ConclusionThese results demonstrate anti-tumor activity of PD-L1 inhibition in patients with relapsed thymoma accompanied by a high frequency of immune-related adverse events. Pre-treatment immune cell subset populations differ between responders and non-responders.Trial registrationClinicalTrials.gov - NCT01772004. Date of registration – January 21, 2013.

Intranasal administration of Lignosus rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) extract attenuates airway inflammation in murine model of allergic asthma.

Asthma is a chronic inflammatory disorder in the airways that involves the activation of cells and mediators. Lignosus rhinocerotis (Cooke) Ryvardan or Tiger Milk mushroom is a medicinal mushroom that is traditionally used to treat inflammatory diseases including asthma. In this study, the protective effects of intranasal administration of L. rhinocerotis extract (LRE) in ovalbumin (OVA)-induced airway inflammation mouse model were investigated. Mice were sensitized by intraperitoneal (i.p) injection on days 0 and 14, followed by a daily challenge with 1% OVA from days 21 to 27. Following OVA challenge, LRE and dexamethasone were administered via intranasal and i.p. injection respectively. On day 28, the level of serum immunoglobulin (Ig)E, differential cell counts and T-helper (Th) 2 cytokines in bronchoalveolar lavage fluid (BALF) fluid, cell subset population in lung-draining lymph nodes (LNs), leukocytes infiltration and mucus production in the lungs of the animals was measured. Results demonstrated that intranasal administration of LRE significantly suppressed the level of inflammatory cell counts in BALF as well as populations of CD4+ T-cells in lung draining LNs. Apart from that, LRE also significantly reduced the level of Th2 cytokines in BALF and IgE in the serum in OVA-induced asthma. Histological analysis also demonstrated the amelioration of leukocytes infiltration and mucus production in the lungs. Overall, these findings demonstrated the attenuation of airway inflammation in the LRE-treated mice therefore suggesting a promising alternative for the management of allergic airway inflammation.

Profiling Mesenchymal Stem Cell Immunomodulation In Vitro

Abstract Mesenchymal stem cells (MSCs) are multipotent adult stem cells found in a variety of tissues, including bone marrow, adipose, and umbilical cord. Although MSCs have the ability to differentiate into a variety of cell types useful for regenerative medicine, their ability to modulate the immune system has been at the forefront in the clinic. The mechanisms by which MSCs affect the immune system are not fully understood. This makes it difficult to design specific quality control measures for MSCs used for cell therapy as well as to understand what parameters are the most critical for their immunomodulatory abilities. This specific testing is important to determine batch-to-batch variability and to better forecast patient-to-patient efficacy. In this study, we demonstrate a workflow to investigate the effects of MSCs on immune cell subset populations. Human MSCs were co-cultured with human T cell subtypes (Th1, Th2, Th17, and Treg) that have been differentiated utilizing CellXVivo™ Differentiation Kits. Co-culture supernates are then analyzed via Proteome Profiler™ Antibody Arrays to screen for changes in cytokine levels, relative to MSCs cultured in the absence of T-cells. Analytes discovered to change in the screening arrays are then quantitated using Luminex® Assays. We hypothesize that these tools and techniques will allow for a better understanding of the mechanisms contributing to MSC immunomodulation and provide methods for routine quality control testing of MSC populations prior to clinical use.

Elevated levels of CCR6+ T helper 22 cells correlate with skin and renal impairment in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease with a variety of pathological features. Our study investigated the potential role of CCR6+ T cells in organ impairment of SLE patients. We analyzed CCR6+/− T cell subset populations and compared the concentrations of IL-22, IFN-γ, TNF-α, and IL-17A cytokines in 67 patients with newly diagnosed SLE and 26 healthy controls. We found that SLE patients had elevated percentages of CCR6+ T, CCR6+ Th22, Th17, Th17.1, and CCR6− Th2 cell subsets, along with increased concentrations of IL-22, IFN-γ, TNF-α, and IL-17 cytokines. Higher levels of CCR6+ T and CCR6+ Th22 cells, along with plasma IL-22 were observed in SLE patients with sole skin and/or renal impairment. The percentage of Th22 cells also correlated with Revised Cutaneous Lupus Erythematosus Disease Area and Severity Index (RCLASI) and IgG levels, and inversely correlated with C3 levels in SLE patients with sole skin impairment. SLE patients with sole renal impairment showed a correlation between the percentage of Th22 cells and ESR levels. Our data indicated that CCR6+ Th22 cells may contribute to the pathogenesis of new onset SLE patients with skin or renal impairment, and CCR6 may, thus, be a possible therapeutic target for SLE treatment.

Immune System Profiling Following Autologous Stem Cell Transplant for High-Risk Neuroblastoma Predicts Disease-Free Survival

Objective: Neuroblastoma (NB) is the most common extra cranial solid tumor in childhood. It accounts for approximately 7% of pediatric cancer diagnoses each year in the United States yet is responsible for 15% of pediatric cancer deaths annually. Occurring overwhelmingly in patients with high-risk (HR) disease.Measurable immune surveillance correlates with improved overall disease-free survival in patients with a myriad of cancer subtypes, including NB. Current therapeutic strategies in HR-NB incorporate manipulation of the host immune response, including autologous stem cell transplantation (autoSCT), cis-retinoic acid, and chimeric antibody targeting the tumor-associated antigen disialoganglioside (GD2). Understanding how such therapies affect the immune system and its relation to clinical response is of critical importance. We speculate that patients with higher proportions of regulatory T cells (Tregs) and slower recovery of other lymphocyte subsets will have higher rates of progressive disease and/or relapse.Methods: We reviewed the records of all University of Chicago patients between 2009 and 2015 who received autoSCT as part of upfront therapy for HR neuroblastoma. Data included complete blood count and peripheral blood lymphocyte flow cytometric (FACS) subset monitoring. Our analysis aimed to determine the association between lymphocyte subset kinetics following autoSCT with pre-transplant treatment, transplant conditioning, and disease-free/overall survival. Patients who had a follow-up of greater than one-year post-transplantation were included in the analysis.Results: Thirty-four patients (59% male, median age 5.6 years) received autoSCT between 2009 and 2015 for HR neuroblastoma. Seventy-six percent (n = 26) received either carboplatin, etoposide, melphalan (CEM) or busulfan/melphalan (BuMel) for conditioning. Among those patients, twenty-seven percent received MIBG prior to their autoSCT. The other patients received BSO/melphalan or tandem transplantations. Sixty-eight percent of these patients went on to receive chimeric antibody after their autoSCT irrespective of their conditioning regimen. Antibody therapy with IL-2 or GM-CSF was initiated at a median of 101 days (mean 98.9) post-autoSCT. All patients had normal neutrophil counts at 100 days post transplant, while the total absolute lymphocyte count (ALC), CD8+ T cells, NK cells and CD19+ B cells, were below normal levels in the majority of patients, (100%, 89%, 79% and 94% of patients respectively). Patients who received MIBG prior to autoSCT had a higher proportion of CD3+CD4+CD25+ T cells, the subset containing Tregs, among their total CD3+ population at 100 days post-transplant, (79.8% versus 32.8% p-value = 0.02). Following single autoSCT, patients with higher ALC and CD4+ cells had increased disease free survival in evaluations after six months post transplant. (Figure 1) CD8+ cells, NK cells and CD19+ cell counts were not significant in this analysis.Conclusions: Patients with HR NB have differential immune cell recovery following autoSCT that is dependent on the type of therapy received prior to autoSCT. Specifically, patients who received MIBG had markedly higher proportion of CD3+CD4+CD25+ T cell recovery compared to patients not receiving pre-autoSCT MIBG. This is the subset containing Tregs, which may indicate suppression of an anti-NB immune response long-term. Immune reconstitution is globally poor in all patients at the time of chimeric antibody immunotherapy that is initiated around day 100-post autoSCT. There are features of immune reconstitution that appear to correlate with improved outcomes, including elevated ALC, CD8+ and CD4+ cell counts when evaluated at least six months post transplantation. Additional characterization of T cell subsets is currently being performed to quantify the number of Treg cells in the CD3+CD4+CD25+ T cell subset population. Prospective evaluation of both immune cell recovery and functional evaluation of cells is required to confirm these findings, which may have a profound impact on how immune modulatory therapy is scheduled for those patients with high-risk neuroblastoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Probiotic feeding affects T cell populations in blood and lymphoid organs in chickens.

This study was performed to evaluate the effects of Lactobacillus acidophilus bacteria as a probiotic on chicken T cell subset populations in peripheral blood and lymphoid tissues. Thirty chickens were divided into three groups and fed sterilised cow milk, a mixture of milk and L. acidophilus (probiotic), or neither, as the control group. Chickens were euthanised after 14 and 21 days, and whole blood and ileal, bursal, and caecal tonsillar tissues were collected. The populations of T cell subsets, including CD4+, CD8+, and TCR1+ cells, were evaluated by immunohistochemistry and flow cytometry. After 21 days of treatment the percentage of blood CD4+, CD8+, and TCR1+ cells was significantly higher in the probiotic-fed group than in the control group. After 14 days of treatment, a significantly greater number of CD4+ T cells were found in the ileum of probiotic-fed chickens than in chickens from the other two groups. This difference was even greater after 21 days. In addition, after 21 days, a significantly greater number of TCR1+ cells were found in the caecal tonsils of milk-fed chickens than in chickens from the control group. The findings indicate that probiotics may alter the distribution of T cells in the blood and lymphoid tissues in young chickens; however, transient changes in lymphoid tissues indicate that probiotics likely do not permanently affect mucosal immunity.

Abstract 3356: Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker

Abstract Gene expression profiling experiments have demonstrated that the immune microenvironment in follicular lymphoma (FL) plays an important role in controlling the behavior of the disease. FL patients with tumors enriched for T cell-related transcripts have a more favorable prognosis. As T cells have been shown to mediate anti-tumor immunity in other cancers, they may also be critical in controlling the progression of FL. T cells are often identified in and around malignant follicles in FL by immunohistochemistry, but it is not known whether their presence relates to their specificity for antigens expressed by FL cells. Hypothesizing that at least a fraction of T cells in FL are truly lymphoma antigen-specific, we have begun to characterize the T cell receptor (TCR) repertoire of FL tissues using a next generation sequencing platform, and have deeply sequenced T cell receptor (TCR) cDNAs of T cell subset populations present in pretreatment FL biopsy specimens. Regulatory T cell (Treg) TCRs in FL tissues revealed a highly oligoclonal expansion, compared to those in control lymph nodes. Furthermore, an inverse correlation was observed between the diversity of Treg and CD8+ TCRs in FL specimens. Interestingly, a tumor from an FL patient, who has received no anticancer treatment for more than 10 years, was found to have missense mutations in the peptide-binding domain of both HLA class I and class II molecules, which might have presented tumor-specific antigens and enhanced host immune responses. Although further verification is required, our data suggest that the T cell repertoire is skewed and restricted in FL and support the evolving understanding of the microenvironment in this disease. Citation Format: Justin Paul Kline, Xiao Liu, Girish Venkataraman, Jiaying Lin, Kazuma Kiyotani, Sonali M. Smith, Magdeline Montoya, Yusuke Nakamura. Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3356. doi:10.1158/1538-7445.AM2015-3356

Deciphering the adaptive immune system of bats (VET2P.1131)

Abstract Bats are natural reservoirs for several viruses, many of which cause morbidity and high mortality rates in other mammals, including humans but they generally don’t cause disease in bats. In fact, fruit bats have been found to be the principal suspect that initiated the latest 2014 West-Africa Ebola outbreak. How bats cope with such deadly viruses without getting sick remains unknown. Efforts are being increasingly devoted to dissect the bat’s immune system, however, due to the lack of reagents progress has been limited. Our group is focusing on characterizing bats adaptive immunity. The first step consists of generating a systematic knowledge on the various immune cell populations (T and B cells mainly). Subsequently, functional assays will be optimized to study how bats adaptive immunity reacts to viral infections. Screening of human and mouse antibodies against the main components of the bats adaptive immunity revealed poor cross-reactivity to T and B cell main surface markers (CD3, CD4, CD8, CD19). Combining flow cytometry and real-time PCR approaches, we have been able to characterize different T cell subset populations present in bats spleen and blood. Understanding how bats control viral infection can provide important insights into anti-viral strategies that may result in the development of new therapeutics for other mammals

Abstract B294: Antitumor efficacy of AUR-012, a peptide antagonist of the PD1 immune checkpoint pathway, correlates well with the modulation of specific T-cell populations.

Abstract Recent breakthroughs in achieving highly durable clinical responses via immunotherapy to inhibit immune checkpoint proteins including CTLA4 and PD1 have revolutionized the outlook for cancer therapy. However, along with impressive clinical activity (response rate of ∼25% with either anti-CTLA4 or anti-PD1 as single agent, but more than 50% with a combination), immune-related toxicities due to the breaking of immune self-tolerance (25-30% with anti-CTLA4 and up to 15-17% with anti-PD1) are becoming increasingly visible. Sustained inhibition of the signaling as a result of a very long half-life (&amp;gt;15-20 days) and &amp;gt;70% target occupancy for months are likely contributing to severe adverse effects observed in clinical trials with antibodies targeting immune checkpoint proteins. Our efforts are therefore focused towards developing novel immune checkpoint blockers with potent antitumor activity but with a shorter pharmacokinetic profile as a strategy to better manage severe adverse effects. Herein we report our findings from the pharmacodynamic characterization of a novel peptide inhibitor of the PD1 signaling that displays desired pharmacokinetic and efficacy profile. Peptide antagonist AUR-012, constructed with elements from human PD-1 ectodomain, displayed equipotent antagonism towards PD-L1 and PD-L2 with potent activity in rescue of lymphocyte proliferation and effector functions. Rescue of proliferation of immune cell subset populations analyzed in vitro using anti-CD3/anti CD-28 stimulation indicated that the functional capacity of T-cells has been reinvigorated by AUR-012 resulting in a complete rescue of CD4+ and CD8+ T cell proliferation in the presence of PD-L1. Similar effect in the rescue of proliferation was also observed for B-cells and NK cells. Interestingly, the proliferation of CD4+, FOxP3+ T cells was completely abolished with AUR-012 treatment indicating a complete suppression of regulatory T cells. Sustained activation of circulatory immune cells and their ability to secrete IFN-γ up to 72 h indicate that pharmacodynamic effects persist even after the clearance of AUR-012 in animal models, thus supporting a dosing interval of up to 3 days irrespective of the short half-life of the peptide. In preclinical models of melanoma, breast, kidney and colon cancers, AUR-012 showed superior showed superior efficacy compared to therapeutic agents currently used in the clinic in inhibition of both primary tumor growth and metastasis. Additionally, antitumor activity of AUR-012 in a pre-established CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4+ and CD8+ T cells, and a reduction in PD1+ T cells (both CD4+ &amp; CD8+) in tumor and blood. In conclusion, the observed correlation between antitumor activities with the modulation of specific T-cell populations supports the use of immunophenotyping as a potential therapeutic biomarker strategy in the planned clinical studies with AUR-012. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B294. Citation Format: Murali Ramachandra, Pottayil G. Sasikumar, Rajeev K. Shrimali, Sreenivas Adurthi, Raghuveer Ramachandra, Leena K. Satyam, Amit A. Dhudashiya, Dodheri S. Samiulla, K B Sunilkumar. Antitumor efficacy of AUR-012, a peptide antagonist of the PD1 immune checkpoint pathway, correlates well with the modulation of specific T-cell populations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B294.

Altered functional B cell subset populations in patients with chronic fatigue syndrome compared to healthy controls

Chronic fatigue syndrome (CFS) is a heterogeneous disorder of unknown aetiology characterized by disabling fatigue, headaches, sleep disturbance and several other symptoms. The onset of CFS may follow a viral infection or period of stress. Patients with CFS do not have hypogammaglobulinaemia, predisposition to recurrent bacterial infections or symptoms of autoimmunity. To date, defects in B cell numbers or function have not been shown in the literature. However, treatment with anti-B cell therapy using Rituximab has recently shown benefit to CFS patients. We therefore postulated that patients with CFS had a subtle humoral immune dysfunction, and performed extended B cell immunophenotyping. We undertook a detailed characterization of the proportions of the different B cell subsets in 33 patients with CFS fulfilling the Canadian and Fukada criteria for CFS and compared these with 24 age- and gender-matched healthy controls (HC). CFS patients had greater numbers of naive B cells as a percentage of lymphocytes: 6·3 versus 3·9% in HC (P = 0·034), greater numbers of naive B cells as a percentage of B cells: 65 versus 47% in controls (P = 0·003), greater numbers of transitional B cells: 1·8 versus 0·8% in controls (P = 0·025) and reduced numbers of plasmablasts: 0·5 versus 0·9% in controls (P = 0·013). While the cause of these changes is unclear, we speculate whether they may suggest a subtle tendency to autoimmunity.

Inhibitors of the Mammalian Target of Rapamycin and Transplant Tolerance

Certainly, achieving and maintaining donor-specific hyporesposiveness is a main challenge nowadays in organ transplantation to improve long-term graft survival. Immunosuppression seems to be mandatory in the majority of renal transplant patients. However, some specific drugs have shown to have interesting immunomodulatory effects, regardless of their immunosuppressive activity. Sirolimus, an immunosuppressive agent with a distinctive action mechanism has shown to be able to directly influence the main two cell subset population in charge of controlling alloimmune responses: regulatory T cells and dendritic cells. Here, we discuss and analyze the main mechanisms by which sirolimus may modulate the alloimmune response, thus facilitating a protolerogenic state in renal transplantation.

RAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45(+) leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye.

CD5+ B cells with the features of subepithelial B cells found in human tonsils

This study describes a CD5+ B cell that differs from the majority of the CD5+ B cells from human tonsils. This cell, isolated from in vivo activated B cells, expressed activation markers and featured a CD23-, IgMhigh, IgDlow surface phenotype, responded to T cell-independent type-2 antigens in vitro, and was detected in the subepithelial (SE) areas, the tonsil equivalent of the splenic marginal zone (MZ). Most of the cells utilized unmutated Ig VH genes, although cells with mutated genes also were found, a finding confirmed by single-cell studies. Mutated sequences were more frequent in suspensions enriched for CD27+ cells. Repeated VDJ gene sequences were observed in different molecular clones from the same cell suspension, suggesting in situ expansion. These CD5+ B cells seem to share features with previously characterized tonsil CD5- SE B cells and differ from the majority of tonsil CD5+ B cells, which have the surface phenotype of follicular mantle B cells, lack activation markers, do not respond to T cell-independent antigens, and utilize unmutated VH genes. These data are discussed considering the present views on the origin of B cell subset populations and the relationships between MZ and B1 cells.