Number Of Cytotoxic T Lymphocytes Research Articles

Systemic chemokine-modulatory regimen combined with neoadjuvant chemotherapy in patients with triple-negative breast cancer

BackgroundHigher cytotoxic T lymphocyte (CTL) numbers in the tumor microenvironment (TME) predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) and positive long-term outcomes in triple-negative breast cancer (TNBC). pCR...

Immune response in neuroinflammatory caused by mild traumatic brain injury (experimental study)

The study of the organism immune response in mild traumatic brain injury (mTBI) is an important stage in the formation of a scientific understanding of this type of brain damage. The purpose of the study: to research of the systemic immune response parameters in the experimental modeling of mTBI. The cellular composition and phenotype of immune cell subpopulations were assessed, and the content of pro- and anti-inflammatory cytokines in the mTBI and blood serum of injured rats was determined. A high content of leukocytes and interleukin 6 (IL 6) in the blood was found 2 hours after the injury. The number of cytotoxic T-lymphocytes and B-lymphocytes increased with a decrease of the neutrophils, monocytes, eosinophils and T helpers number one day after mTBI. During the indicated periods of observation, an increase in the content of cytokines TNFα and IL 6 in the cerebrospinal fluid and blood serum was observed, and the indicators of anti-inflammatory IL 10 increased 7–14 days after mTBI. On the 14th day after injury, the level of stab neutrophils remained low, while the number of CD45, CD3, CD20, and CD8 positive lymphocytes continued to increase, which indicates the progression of the inflammatory response. Thus, after the application of mTBI, an inflammatory reaction develops, accompanied by the manifestation of the activity of immune components. Cytokines are registered in the peripheral circulation and cerebrospinal fluid, the number of B-lymphocytes and cytotoxic T-lymphocytes increases.

Evaluation of the effectiveness of detoxification in the immunorehabilitation of cancer patients

Purpose. Evaluation of the effectiveness of detoxification using enterosorbents in the immunorehabilitation of cancer patients.
 Materials and methods. 87 patients with metastatic forms of solid tumors of various localizations with metastatic lesions were examined. The patients were divided into 2 groups: study group (41 patients who received detoxification therapy in addition to standard therapy) and comparison group (46 patients who received only standard therapy). The effectiveness of therapy with these drugs was evaluated after one month by assessing clinical and immunological parameters. The assessment of the state of cellular immunity was carried out by flow cytometry.
 Results. Disturbances in the function of the immune system in the form of insufficiency of the cellular effector link of immunity were detected in all examined patients before the start of therapy. The state of the cellular link of immunity was characterized by an increase in the number of transitional and non-classical subsets of monocytes, an increase in the content of T-, B- and NKT-cells, but with a decrease in NK-cells. An increase in the ratio between the number of T-helpers (CD3+CD4+) and the number of cytotoxic T-cells (CD3+CD8+) was observed in the examined patients. Restoration of cell subsets to the level of control values was found in the examined patients after detoxification therapy as part of immunorehabilitation measures. Significant changes in the number of cells in the blood were absent in the comparison group after the therapy which indicated the absence of a complete relief of the systemic inflammatory response.
 Conclusion. Chronic toxicosis with the development of a systemic inflammatory reaction on the background of the immune system dysfunctions were found in cancer patients. The use of enterosorbents in complex immunorehabilitation therapy is substantiated and confirmed by the relief of asthenovegetative and dyspeptic syndromes, normalization of the immune system which makes it possible to recommend detoxification therapy during immunorehabilitation measures.

Systemic infusion of TLR3-ligand and IFN-α in patients with breast cancer reprograms local tumor microenvironments for selective CTL influx

BackgroundPresence of cytotoxic T lymphocytes (CTL) in the tumor microenvironment (TME) predicts the effectiveness of cancer immunotherapies. The ability of toll-like receptor 3 (TLR3) ligands, interferons (IFNs) and COX2 inhibitors...

Cytotoxic T Lymphocytes Control Growth of B16 Tumor Cells in Collagen-Fibrin Gels by Cytolytic and Non-Lytic Mechanisms.

Cytotoxic T lymphocytes (CTLs) are important in controlling some viral infections, and therapies involving the transfer of large numbers of cancer-specific CTLs have been successfully used to treat several types of cancers in humans. While the molecular mechanisms of how CTLs kill their targets are relatively well understood, we still lack a solid quantitative understanding of the kinetics and efficiency by which CTLs kill their targets in vivo. Collagen-fibrin-gel-based assays provide a tissue-like environment for the migration of CTLs, making them an attractive system to study T cell cytotoxicity in in vivo-like conditions. Budhu.et al. systematically varied the number of peptide (SIINFEKL)-pulsed B16 melanoma cells and SIINFEKL-specific CTLs (OT-1) and measured the remaining targets at different times after target and CTL co-inoculation into collagen-fibrin gels. The authors proposed that their data were consistent with a simple model in which tumors grow exponentially and are killed by CTLs at a per capita rate proportional to the CTL density in the gel. By fitting several alternative mathematical models to these data, we found that this simple "exponential-growth-mass-action-killing" model did not precisely describe the data. However, determining the best-fit model proved difficult because the best-performing model was dependent on the specific dataset chosen for the analysis. When considering all data that include biologically realistic CTL concentrations (E≤107cell/mL), the model in which tumors grow exponentially and CTLs suppress tumor's growth non-lytically and kill tumors according to the mass-action law (SiGMA model) fit the data with the best quality. A novel power analysis suggested that longer experiments (∼3-4 days) with four measurements of B16 tumor cell concentrations for a range of CTL concentrations would best allow discriminating between alternative models. Taken together, our results suggested that the interactions between tumors and CTLs in collagen-fibrin gels are more complex than a simple exponential-growth-mass-action killing model and provide support for the hypothesis that CTLs' impact on tumors may go beyond direct cytotoxicity.

Abstract 5492: Determining effects of the CD206 positive M2 macrophage depleting engineered exosome in combination with anti PD1 therapy in breast tumor

Abstract Introduction: Triple negative breast cancer (TNBC) subtype of breast cancer, which lacks molecular markers such as HER2 and estrogen/progesterone receptors, has limited treatment options. Exosomes are nano-sized (30-150 nm) spherical vesicles, derived from the endosomal system for intercellular communications, can be engineered to express targeting peptides to target specifically CD206 positive M2 macrophage. Investigators have used either synthetic nanoparticles or fusion protein to deliver Fc-IgG2b to initiate Antibody dependent cellular cytotoxicity (ADCC) but reports are showing a lack of ADCC following tagging with gold nanoparticles. To overcome this, in our lab, we developed engineered exosomes in non-tumorous cells, HEK293, to carry therapeutic Fc-mIgG2b to enhance ADCC and to carry peptide to deplete CD206+ M2 macrophages. We investigated the effectiveness of engineered therapeutic exosomes to target and deplete immunosuppressive CD206+ M2 macrophages in TNBC in combination with anti PD-1. Methods: In this study, we developed syngeneic metastatic TBNC model (luciferase + 4T1 cells in Balb/c) by implantation of 50K 4T1-luci cells in fat pad. On day 1 of tumor implantation, animals were treated with vehicle, engineered exosomes, anti-PD1 plus engineered exosomes, 2 dose/week for 3 weeks. They were euthanized 1 day after the last dose of treatment. Single-cell suspension was made from the primary TME, lung, and spleen. Using flow cytometry surfaces markers were investigated to determine different T-cell and myeloid cell populations. Results: We observed very surprising results following three weeks of therapies. Although the number of cytotoxic T-cells was significantly higher in the primary and metastatic TME in exosome-treated animals, there was also an increased number of proliferative T reg cells. The number of CD8+ cells was decreased following concurrent therapy with an anti-PD1 antibody. Most interestingly, lung tissue macrophages (CD11c) showed significantly increased PD-L1 expression following exosomes and anti PD1 with engineered exosomes therapies. Conclusion: This study demonstrates that treatment with engineered therapeutic exosome targeting M2 macrophages with anti PD-1 could be utilized effectively. This study will also make a greater impact in exosome engineering based targeted therapy and will open new arena to target and deplete pathogenic cell in different tumors using engineered exosome. Citation Format: Mahrima Parvin, Mohammad H. Rashid, Ahmet Alptekin, Ali S. Arbab. Determining effects of the CD206 positive M2 macrophage depleting engineered exosome in combination with anti PD1 therapy in breast tumor. [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 5492.

Kinetic measurements of human CD8+ T cell cytotoxic activity in a 384-well plate format.

The elimination of infected or cancerous cells by CD8+ cytotoxic T lymphocytes (CTL) is a crucial effector mechanism of the immune system. Upon antigen recognition, CTL stop migrating, establish a tight contact with target cells and deliver cytotoxic molecules such as perforin and granzymes that lead to target cell apoptosis. The ability of CTL to control a population of infected cells or a tumor depends on multiple parameters, such as the relative numbers of CTL and target cells, the intrinsic cytotoxic activity of CTL, the intrinsic resistance of target cells and the repertoire of immune checkpoints tuning cytotoxic activity at the CTL:target cell interface. In this context, in vitro assays to precisely measure CTL:target cell interactions and cytotoxic activity over time are required to monitor natural or therapeutic responses. We here present an image-based method that allows recording of positions and survival of CTL and target cells over time in a high-throughput format. The protocol relies on the staining of CTL and target cells with fluorescent dyes and the automated imaging of cells deposited in wells of a 384-well plate with an automated cell imaging device. We discuss potential applications offered by the kinetic assessment of CTL cytotoxic activity in a high-throughput format.

Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

Mechanism of action of DSP-7888 (adegramotide/nelatimotide) Emulsion, a peptide-based therapeutic cancer vaccine with the potential to turn up the heat on non-immunoreactive tumors

BackgroundWilms’ tumor 1 (WT1) is highly expressed in various solid tumors and hematologic malignancies. DSP-7888 (adegramotide/nelatimotide) Emulsion is an investigational therapeutic cancer vaccine comprising three synthetic epitopes derived from WT1. We evaluated the mechanism of action of DSP-7888 Emulsion, which is hypothesized to induce WT1-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs).MethodsThe ability of nelatimotide and adegramotide to induce WT1-specific CD8+ T cells and CD4+ T cells was assessed in human peripheral blood mononuclear cells (PBMCs). The ability of DSP-7888 Emulsion to induce WT1-specific CTLs in vivo was assessed using human leukocyte antigen-I (HLA-I) transgenic mice. To assess how adegramotide, the helper peptide in DSP-7888 Emulsion, enhances WT1-specific CTLs, HLA-I transgenic mice were administered DSP-7888 or nelatimotide-only Emulsion. Interferon-gamma secretion under antigen stimulation by splenocytes co-cultured with or without tumor cells was then quantified. The effects of combination treatment with DSP-7888 Emulsion and an anti–programmed cell death protein 1 (PD-1) antibody on tumor volume and the frequency of tumor-infiltrating WT1-specific T cells were assessed in HLA-I transgenic mice implanted with WT1 antigen-positive tumors.ResultsThe peptides in DSP-7888 Emulsion were shown to induce WT1-specific CTLs and HTLs in both human PBMCs and HLA-I transgenic mice. Unlike splenocytes from nelatimotide-only Emulsion-treated mice, splenocytes from DSP-7888 Emulsion-treated mice exhibited high levels of interferon-gamma secretion, including when co-cultured with tumor cells; interferon-gamma secretion was further enhanced by concomitant treatment with anti-PD-1. HLA-I transgenic mice administered DSP-7888 Emulsion plus anti-PD-1 experienced significantly greater reductions in tumor size than mice treated with either agent alone. This reduction in tumor volume was accompanied by increased numbers of tumor-infiltrating WT1-specific CTLs.ConclusionsDSP-7888 Emulsion can promote both cytotoxic and helper T-cell-mediated immune responses against WT1-positive tumors. Adegramotide enhances CTL numbers, and the CTLs induced by treatment with both nelatimotide and adegramotide are capable of functioning within the immunosuppressive tumor microenvironment. The ability of anti-PD-1 to enhance the antitumor activity of DSP-7888 Emulsion in mice implanted with WT1-positive tumors suggests the potential for synergy.

Oncolytic adenovirus with MUC16-BiTE shows enhanced antitumor immune response by reversing the tumor microenvironment in PDX model of ovarian cancer

ABSTRACT The improved survival rate of ovarian cancer (OC) is related to the action of infiltrating cytotoxic T lymphocytes (CTLs). Recently, oncolytic adenoviruses (OAds) have emerged as a key player in treating solid tumors; however, the immunosuppressive tumor microenvironment (TME) and the body-mediated antiviral immune response limit their therapeutic effect. In this study, we tested the hypothesis that bispecific T-cell engagers (BiTEs) could activate and redirect CTLs to increase the anti-tumor effect of OAds. We modified the parental OAd to express a MUC16-targeting BiTE antibody (OAd-MUC16-BiTE), which retained its oncolytic properties and replication ability in vitro. This BiTE secreted from infected tumor cells into the microenvironment binds to MUC16 on target cells and cross‐links them to CD3 on T cells, leading to activation, proliferation, and toxicity of T cells against MUC16+ tumor cells. In cell coculture assays, OAd-MUC16-BiTE–mediated oncolysis enhanced T-cell–mediated tumor cell killing and bystander effect. In ex vivo tumor cultures freshly derived from OC patients, OAd-MUC16-BiTE overcame the suppressed immune TME, achieving stronger toxicity than the parental virus. Moreover, in the cell-derived xenograft and patient-derived xenograft model, OAd-MUC16-BiTE showed stronger antitumor activity and increased the number of CTLs, compared with the parental virus. Further, we demonstrated that the OAd-MUC16-BiTE-mediated anti-tumor activity is related to the reversal of the TME and improved MHC I antigen presentation. Overall, our results show how arming OAds with BiTE can overcome limitations in oncolytic virotherapy, yielding a potent therapy that is ready for clinical assessment.

Abstract 6122: Different characteristics of tumor immune microenvironment between LUSC and LUAD

Abstract Background: Patients with lung squamous cell carcinoma (LUSC) have higher tumor mutation burden (TMB) and PD-L1 expression than those with lung adenocarcinoma (LUAD). Several studies showed that tumor immune microenvironment (TIME), mainly the tumor-infiltrating immune cells, may affect the efficacy of immunotherapy. However, the comparison of tumor immune microenvironment between LUAD and LUSC is little known. Methods: 14 patients with LUSC and 21 patients with LUAD were enrolled in the retrospective analysis of multiple immunofluorescence staining to assess the number of immune cells. Genomic profiling of DNA was performed on formalin-fixed paraffin-embedded tumor samples from 3752 LUAD and 459 LUSC patients by NGS with 733 cancer-related genes panel. TMB is defined as the total counts of single nucleotide variants and indels in the exon region. Results: Overall, as the most abundant tumor-infiltrating immune cells in both groups, CD3+ cells are more in LUAD [median (number/mm2 in LUSC vs in LUAD): 312 vs 918, p = 0.02]. However, there were no significant differences in CD8+ T cells (median: 48 vs 36, p = 0.74), indicating higher infiltration of T lymphocytes rather than cytotoxic T lymphocytes (CTLs) in LUAD than that in LUSC. Unlike in tumor, LUAD patients had both more T lymphocytes (p < 0.001) and CTLs (p < 0.01) in stroma. Besides, LUSC was correlated with fewer M2 tumor-associated macrophages (TAMs) and PD-1+ immune cells (p < 0.001; p = 0.02), while no significant differences were found in tumor-infiltrating FOXP3+ cells (p = 0.41) and B lymphocytes (p = 0.61). In addition, natural killer cells were the fewest among the tumor-infiltrating lymphocytes detected in both groups (median: 2 vs 13). Similar to the previous studies, TMB level was higher in LUSC (median: 8.94 vs 3.36, p < 0.0001). Although LUSC patients had a larger proportion of positive PD-L1 expression (45% vs 30.5%, p < 0.0001) and strong PD-L1 expression in tumor cells (20.9% vs 10.7%, p < 0.0001), the number of CTLs and TAMs expressing PD-L1 is significantly lower than that in LUAD ( p = 0.04; p = 0.0001). Conclusion: LUAD had more immune invasion, while LUSC was related to higher TMB and PD-L1 expression. It suggested that the mechanisms affecting the benefits of immunotherapy may be different. The immune microenvironment of tumor and stroma should be considered simultaneously to explore the mechanism of immunotherapy and the respective thresholds should be selected according to the types of immune cells. Citation Format: Na Li, Mengna Hu, Ding Zhang, Xiaoyu Ji. Different characteristics of tumor immune microenvironment between LUSC and LUAD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6122.

The impact of tumor-infiltrating lymphocytes on tumor features and pathological characteristics in breast cancer patients: the role of cytotoxic T lymphocytes and regulatory T cells.

Though tumor-infiltrating lymphocytes (TILs) have a predictive impact in cancer patients, their association with presentation and prognosis in breast cancer is less consistent. This study aimed to assess the level of infiltrating cytotoxic T lymphocytes (CTLs) and regulatory T lymphocytes (Tregs) and their association with the clinicopathological features of breast cancer. Tissue samples from female patients (n=153) diagnosed with primary invasive breast cancer were stained with CD8 (a CTL marker) and Foxp3 (a Treg marker) using immunohistochemistry. CTLs were distributed between tumor bed and stroma whereas Treg cells were mainly located in the stroma. The level of intratumoral CTLs correlated positively with Tregs in both tumor and stroma (rho=0.312, p<0.001 and rho=0.176, p=0.031; respectively). Stromal CTLs correlated positively with stromal Tregs (rho=0.319, p=0.005). Tumor size correlated inversely with the number of Treg cells in the tumor bed (rho= - 0.179, p=0.028). Tregs were associated with lymphovascular invasion status in the tumor bed (p=0.042). The ratio of intratumoral CTLs to Tregs was associated with estrogen receptor positivity and luminal subtype (p=0.029 and p=0.045, respectively). The median number of CTLs was significantly lower in patients using aspirin or antihypertensive medications compared to nonusers (p=0.024 and p=0.03, respectively). TILs were distributed differently in tumor tissues of breast cancer patients. CTLs infiltrates were found in both tumor bed and stroma while Tregs were dominant in the stroma. TILs were also distinctly associated with tumor features. The impact of TILs on prognosis and treatment outcomes in Jordanian breast cancer patients needs further investigation.

The uterine natural killer cell, cytotoxic T lymphocyte, and granulysin levels are elevated in the endometrium of women with nonstructural abnormal uterine bleeding

To examine the expression of uterine natural killer (uNK) cells and cytotoxic T lymphocytes (CTLs) in endometrial biopsies from reproductive-age women with and without nonstructural abnormal uterine bleeding (AUB) and evaluate the expression of granulysin within these cell populations and potential modulation of matrix metalloproteinase (MMP) expression. Experimental study, retrospective design. Academic research laboratory. Patients with nonstructural AUB with no other gynecological pathologies and control patients without AUB. None. Immunohistochemical analysis of granulysin, CD56 (uNK cell marker), and CD8 (CTL marker) expression as well as granulysin messenger ribonucleic acid (mRNA) expression levels in endometrial biopsy samples. Assessment of granulysin regulation of human endometrial stromal cell MMP-1 and MMP-3 mRNA expression. The numbers of uNK cells and CTLs were significantly greater in endometrial biopsy tissue from women with AUB than those from controls. In accord with the increased expression of uNK cells and CTLs, granulysin expression was significantly greater in endometrial biopsies from patients with AUB than in from controls and colocalized to both cell types but not endometrial stromal or epithelialcells. The increased granulysin protein expression was associated with the increased granulysin mRNA expression in adjacent serial sections from these same samples. The treatment of the human endometrial stromal cell line t-HESC with granulysin resulted in a significant increase in MMP-1 and MMP-3 mRNA expression. In the current study, immunohistochemistry showed an increased expression of uNK cells, CTLs, and granulysin among subjects with AUB compared with that of subjects without AUB, leading to conclusions that disturbances in the balance of immune cells and an increase in granulysin expression may have implications in the pathophysiology of AUB and include enhanced MMP-1 and MMP-3 expression.

Аsessment of relationships of cytotoxic Т-lymphocyte disturbances with other compartments of the immune system in post-COVID patients

Assessment of the state of the cells of the immune system in post-COVID patients currently remains little studied. The consequences of the influence of the virus itself, as well as drugs received by patients in the treatment of infection, on various body systems, including the immune system, contribute to disorders leading to post-COVID syndrome. The aim of the study was to study the level of cytotoxic T cells and features of immune system disorders in patients who had undergone SARS-CoV-2 infection. Materials and methods of research: 78 patients were examined 6 months after suffering COVID-19; studied 25 parameters of the blood system (general blood test), 50 parameters of the immune system, including T-lymphocytes, B-lymphocytes and their functional markers, NK, TNK cells, phagocytic part of the immune system, and humoral factors, including general and specific immunoglobulins and complement fragments. Results and discussion: when analyzing the results obtained, it was found that 44.8% of the examined individuals had a 1.7-fold decrease in the number of cytotoxic T-cells. This phenomenon was accompanied by a decrease in the level of T-lymphocytes in general, TNK-lymphocytes, some growth of T-helpers, an increase in the ratio of CD4/CD8 cells, levels of B-lymphocytes, due to B-total and B1, B2-lymphocytes of non-memory cells related to plasma cells (or otherwise the most actively producing immunoglobulins B cells). All these data indicate that in such patients, an immune response is formed to a greater extent according to the TH2 type (increased production of antibodies), and not according to the TH1 type (formation of cellular immune responses). In addition, in such patients, a violation of the TH1 response is observed and the likelihood of developing pathological processes associated with an inadequate response to viral antigens (including re-infection with the SARS-CoV-2 virus, exacerbations of chronic infections) increases. It is possible that the disorders we have identified in such patients may contribute to the occurrence or exacerbation of allergic and autoimmune processes in case of impaired functioning (hyperactivation) of the B-cell link of the immune system. All this requires further more detailed research, both in terms of further clinical observations of these patients, and the development of methods for correcting the identified disorders of the immune system.

P–238 Impaired oocyte fertilization is associated with a proinflammatory M1 phenotype of macrophages and the activation of the NLRC4 inflammasome

Abstract Study question Are inflammatory signatures in the blood of clinically asymptomatic patients predictive for oocyte fertilization success in ART treatment? Summary answer The proinflammatory M1 phenotype of macrophages combined with increasing numbers of cytotoxic T-cells and an upregulated inflammasome NLRC4 is associated with impaired oocyte fertilization. What is known already Oocyte fertilization is an indispensable step towards embryogenesis and reproductive success. Patients with acute or chronic inflammation are at risk of reduced fertilization rates and failure of assisted reproductive technology (ART) treatment. Inflammasomes are intracellular multiprotein complexes that mediate inflammatory tissue remodeling. They can be activated by endogenous and exogenous stimuli and result in maturation of the cytokines IL–1βand IL–18. Appropriate inflammasome activation serves for pathogen defense and tissue damage repair, while aberrant activation of inflammasomes can enhance uncontrolled tissue damage. Study design, size, duration This a prospective study including 39 patients stratified by fertilization rate (cut off 66%) of mature M II oocytes after ICSI procedure, compares 20 patients with high (mean 84,8%, range 67 –100%) versus 19 patients with low (mean 29.4%, range 0–65%) rates. Participants/materials, setting, methods We performed FACS analysis of immune cell composition (leukocytes, neutrophils, monocytes, macrophage types M1 and M2, cytotoxic T-cells, T-helper cells) and utilized RT-qPCR analysis of three inflammasomes (AIM 2, NLRP3 and NLRC4) and their down-streaming proteins caspase 1 and IL–1ß. We focused on an initial and a late state during controlled ovarian stimulation (COS) procedure. Main results and the role of chance This study reports a cellular and molecular inflammatory signature associated with reduced oocyte fertilization rates after ICSI treatment in clinically asymptomatic patients. On a cellular level proinflammatory M1 macrophages were significantly elevated in the low fertilization group (p &amp;lt; 0.01), additionally the ratio of proinflammatory M1 macrophages to anti-inflammatory M2 phenotype was inversely associated with oocyte fertilization rate (p &amp;lt; 0.001). Cytotoxic T cells (p &amp;lt; 0.05) and the inflammasome NLRC4 (p &amp;lt; 0.01) revealed identical patterns of association with fertilization rates in the group comparison. In line with this pattern, there was a significant upregulation of Caspase 1 with a consecutive increase in IL–1ß activity in women with low fertilization rates (p &amp;lt; 0.05). No significant association was shown for the other tested immune cells (leukocytes, neutrophils, monocytes, T-helper cells) and inflammasomes (NLRC3, AIM2). Limitations, reasons for caution The preliminary results in this small study indicate an activation of the inflammasome NLRC4. This, however, has to be assessed on the protein level in a larger group of patients. Wider implications of the findings: Systemic macrophage augmentation of M1 phenotype and cytotoxic T cell presence in combination with upregulated inflammasome NLRC4 are associated with impaired oocyte fertilization. The predictive information of the identified immunologic signature could indicate targeted treatment to down regulate systemic inflammation and the use of oocyte activators in the ART laboratory. Trial registration number none

Republican registry of primary immune deficiencies in the chuvash republic and description of postvaccinal immunity disorders in a pregnant patient with common variable immune deficiency

In recent years, primary immunodeficiencies have turned from the class of rare diseases to the category of more common disorders which may be encountered by doctors of any clinical discipline. The first case of primary immunodeficiency disorder (PID) in Chuvashia was detected in 1993. Since that time, the Department of Internal Diseases with the Course of Clinical Immunology at the I. Ulyanov Chuvash State University registered all the cases of PID diagnosed in the region, introducing them into the Republican Registry of PID. The study was aimed for searching epidemiological indexes, clinical and laboratory manifestations of PID in Chuvash region. The study was based on the patient data obtained by retrospective analysis of 85 case histories of PID patients, treated at different departments of the Republican Clinical Hospital, and the City Chuvash Pediatric Clinical Hospital of Public Health Ministry in 2000-2019, as well as on 49 outpatient records of the patients included into the Regional PID Registry. Various forms of PIDs were diagnosed according to the criteria developed by the European Society for Immunodeficiency and the Pan-American Group on Immunodeficiency (1999). The results of this study showed that the incidence of PID in the Chuivash Region is 3.4:100,000. The incidence of common variable immune deficiency (CVID), the most common form of PID in the region, was 1.58 per 100,000 population. The average age at the time of CVID diagnosis in Chuvash patients was 30.4±16.1 years, and the age of CVID debut was 11.3±15.0 years. The delay in proper diagnosis from the moment of clinical manifestation of CVID was, on average, 17.9 years in the region. At the time of CVID diagnosis, the patients showed marked decrease in the levels of 3 or 2 immunoglobulin classes (IgG and IgA), and T-helper cell contents (CD3+CD4+) in peripheral blood. Prevalence of selective IgA deficiency with сlinical symptoms was 0.83 per 100,000 population of the region, and the incidence of the asymptomatic form of this PID was 1 : 167. In patients with selective IgA deficiency, there were also disorders in the T cell system manifesting as decreased relative number of cytotoxic T-cells as well as elevated IgG and IgM levels. The age of diagnosis of X-linked agammaglobulinemia in the region was 3.5±3.0 years. In addition to disturbances of humoral adaptive immunity in children with this disease, a decrease in absolute T cell numbers was detected. In conclusion, the article describes disturbances of postvaccinal immunity in a pregnant patient with CVID, with asymptomatic clinical course, thus leading to false interpretation of the serological markers of TORCH infections and wrong strategy of pregnancy management.

DOSE-DEPENDENT CHARACTER OF DISTURBANCE OF HEMATOPOIETIC AND IMMUNE SYSTEMS FUNCTION, PRODUCTION OF SOME HORMONES IN EXPERIMENTAL URANIUM ACETATE DIHYDRATE EXPOSURE

The paper presents the results of an experimental study of the dose-dependent nature of functional changes in the body systems under chronic administration of uranyl acetate dihydrate in doses of 0.5 and 5.0 mg/kg per element for 18 weeks. The study was performed on 45 male outbred rats. It has been shown that uranyl acetate dihydrate in a dose of 0.5 mg/kg had no significant effect on hematological parameters. At the same time, activation of bactericidal activity of neutrophils, a decrease in the immunoregulatory index, and an increase in the blood concentration of tumor necrosis factor (TNF-α) have been revealed. The toxicant administered to rats in a dose of 5 mg/kg led to a decrease in the absolute number of erythrocytes, hemoglobin, hematocrit, platelets, the release of myelocytes into the blood, basophilia, monocytosis, the appearance of leukolysis cells and plasmatization of lymphocytes. On the part of the immune system, an increase in the biocidal capacity of neutrophilic granulocytes, TNF-α production, an increase in the number of CD8+ cells, and a reduction in the CD4+/CD8+ ratio have been found. Uranyl acetate dihydrate had a dose-dependent effect only on the number of cytotoxic T-lymphocytes, T-cells with the CD4+CD8+ phenotype, on the immunoregulatory index, and on the level of TNF-α. Hyperglycemia and glucosuria were also dose-dependent. An increase in glucose in the blood and urine indicated a violation of carbohydrate metabolism and kidney function. There was a decrease in the concentration of thyroxine, testosterone and an increase in the level of insulin. Uranyl acetate dihydrate led to the development of insulin resistance. The level of hormones did not depend on the dose of the toxicant administered to the animals.

DOSE-DEPENDENT CHARACTER OF DISTURBANCE OF HEMATOPOIETIC AND IMMUNE SYSTEMS FUNCTION, PRODUCTION OF SOME HORMONES IN EXPERIMENTAL URANIUM ACETATE DIHYDRATE EXPOSURE

The paper presents the results of an experimental study of the dose-dependent nature of functional changes in the body systems under chronic administration of uranyl acetate dihydrate in doses of 0.5 and 5.0 mg/kg per element for 18 weeks. The study was performed on 45 male outbred rats. It has been shown that uranyl acetate dihydrate in a dose of 0.5 mg/kg had no significant effect on hematological parameters. At the same time, activation of bactericidal activity of neutrophils, a decrease in the immunoregulatory index, and an increase in the blood concentration of tumor necrosis factor (TNF-α) have been revealed. The toxicant administered to rats in a dose of 5 mg/kg led to a decrease in the absolute number of erythrocytes, hemoglobin, hematocrit, platelets, the release of myelocytes into the blood, basophilia, monocytosis, the appearance of leukolysis cells and plasmatization of lymphocytes. On the part of the immune system, an increase in the biocidal capacity of neutrophilic granulocytes, TNF-α production, an increase in the number of CD8+ cells, and a reduction in the CD4+/CD8+ ratio have been found. Uranyl acetate dihydrate had a dose-dependent effect only on the number of cytotoxic T-lymphocytes, T-cells with the CD4+CD8+ phenotype, on the immunoregulatory index, and on the level of TNF-α. Hyperglycemia and glucosuria were also dose-dependent. An increase in glucose in the blood and urine indicated a violation of carbohydrate metabolism and kidney function. There was a decrease in the concentration of thyroxine, testosterone and an increase in the level of insulin. Uranyl acetate dihydrate led to the development of insulin resistance. The level of hormones did not depend on the dose of the toxicant administered to the animals.

Abstract PD11-09: Vadis trial: Phase II trial of nelipepimut-s peptide vaccine in women with DCIS of the breast

Abstract Background: Peptide cancer vaccines may be most effective when used in earlier stage cancers or pre-cancers where systemic and tumor microenvironmental immune suppression are less profound. Nelipepimut-S (NPS) plus granulocyte-macrophage colony-stimulating factor (GM-CSF) is a vaccine comprised of a human leukocyte antigen (HLA) restricted peptide from the extracellular domain of the HER2 protein (E75) combined with GM-CSF. We have completed a randomized phase II trial of preoperative vaccination with NPS+GM-CSF vs. GM-CSF alone with the primary outcome being NPS-specific cytotoxic T lymphocyte (CTL) responses. Methods: HLA-A2 positive, DCIS patients were enrolled and randomized to either NPS+GM-CSF vs GM-CSF alone. The patients received two vaccinations prior to surgery at 2-week intervals. The number of NPS-specific CTL was measured at specified intervals (pre-vaccination, time of surgery, 1 month (+/- 7 days) post-op, and 3 months (+/- 7 days) post-op) using a flow cytometry-based dextramer assay. Differences in NPS-specific CTL responses between the two groups and between baseline pre-vaccination and 1-month post-op were analyzed using either a two-sample t-test or Wilcoxon rank sum test, when appropriate. The incidence and severity of adverse events, graded according to Common Terminology Criteria for Adverse Events (CTCAE) version (v) 4.03, were recorded and compared between treatment groups. Results: 45 patients were registered; 7 withdrew consent, 1 opted for surgery at an external facility, 20 were ineligible due to negative HLA-A2, and 4 failed screening for other reasons, leaving 13 patients enrolled. The 13 patients were randomized (2:1) into treatment groups, with nine patients receiving NPS+GM-CSF and four patients receiving GM-CSF alone. The two groups were well-matched for age; however, the GM-CSF alone group had higher percentages of African American (50% vs. 22%) and Hispanic (25% vs. 11%) patients as compared to the NPS+GM-CSF group. In general, vaccination was well-tolerated with similar treatment-related toxicity profiles in the NPS+GM-CSF vs GM-GSF groups (Grade 1 - 93.3% vs. 89.3%, Grade 2 - 6.7% vs. 10.7%, respectively). The mean NPS-specific CTL% in the NPS+GM-CSF group at 1-month post-op was double that of the GM-CSF alone group (0.10 +/- 0.12% vs. 0.05 +/- 0.08, p=0.70). In addition, between baseline pre-vaccination and 1-month post-op, the NPS+GM-CSF group experienced an 11-fold increase in percentage of NPS-specific CTL (0.01 +/- 0.02% vs. 0.11 +/- 0.12%) as compared to only a 2.25-fold increase of NPS-specific CTL in the GM-CSF alone group (0.04 +/- 0.07% vs. 0.09 +/- 0.15%). Conclusions: NPS+GM-CSF is safe and well-tolerated when given preoperatively to patients with DCIS. In HLA-A2 positive patients with DCIS, a single inoculation with NPS+GM-CSF can induce in vivo immunity and a continued antigen-specific T-cell response one month post-surgery. This data provides support for further testing of NPS+GM-CSF in the neoadjuvant and adjuvant settings in an attempt to prevent invasive recurrence in DCIS. Citation Format: Anne E O'Shea, Guy T Clifton, Na Qiao, Brandy Heckman-Stoddard, Malgorzata Wojtowicz, Eileen Dimond, Isabelle Bedrosian, Diane Weber, Alex Husband, Ricardo Pastorello, Lana Vornik, George Peoples, Elizabeth A Mittendorf. Vadis trial: Phase II trial of nelipepimut-s peptide vaccine in women with DCIS of the breast [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD11-09.

Role of Hypoxia and the Adenosine System in Immune Evasion and Prognosis of Patients with Brain Metastases of Melanoma: A Multiplex Whole Slide Immunofluorescence Study.

Simple SummaryThe introduction of immune-checkpoint inhibitors improved the therapeutic landscape for patients with advanced malignant melanoma. However, many patients, including patients with melanoma brain metastases, do not derive benefit from immune-checkpoint blockade. Hence, biomarkers are needed to identify potential mechanisms of resistance and optimize patient selection. This study aimed to explore the role of hypoxia-mediated immunosuppression within the tumor microenvironment of patients with metastatic melanoma using multiplex immunofluorescence. We analyzed the prognostic relevance of the hypoxia surrogate marker GLUT-1, the adenosine-synthesizing ectoenzymes CD73/CD39, and the infiltration by CD8 positive lymphocytes, and evaluated their spatial interaction within the tumor microenvironment (TME). Finally, we outlined the role of the melanoma immune phenotype for the patient’s prognosis and discussed the importance of tumor hypoxia and the adenosine system in shaping the tumor immune phenotype.Following the introduction of immune checkpoint inhibitors, a substantial prolongation of the overall survival has been achieved for many patients with multiple brain metastases from melanoma. However, heterogeneity between individual tumor responses is incompletely understood. In order to determine the impact of the individual tumor phenotype on the prognosis of melanoma patients, we examined surgical sections from 33 patients who were treated with radiotherapy (whole-brain radiotherapy, WBRT, stereotactic radiotherapy, STX, or both) and Ipilimumab. We analyzed multiplex staining of the hypoxia marker GLUT-1, the adenosine (ADO)-associated enzymes CD73 and CD39, and CD8, a marker of cytotoxic T lymphocytes (CTL) on a single-cell basis using QuPath. Additionally, the MOSAIC interaction analysis algorithm was used to explore the hypothesis that CTL systematically avoid GLUT-1high tumor areas. Our results revealed, that a strong GLUT-1 expression, low numbers of CTL, or exclusion of CTL from the tumor were correlated with significant prognostic detriment. Hypoxic tumors overall have smaller amounts of CTL, and spatial analysis revealed a repellent effect of hypoxia on CTL. In contrast to in vitro studies, specific upregulation of ADO-related enzymes CD73 and CD39 in GLUT-1high tumor regions was never observed. In this study, we could show direct in vivo evidence for hypoxia-mediated immunosuppression in melanoma. Moreover, this study suggests a significant prognostic relevance of the tumor immune phenotype, the strength of CD8 infiltration in the tumor, and the expression of hypoxia marker GLUT-1 on melanoma cells. Last, our results suggest a temporal stability of the microenvironment-mediated immunosuppressive phenotype in melanoma.