Antigen-induced Proliferation Research Articles

Relative Importance of Defined Mycobacterium Tuberculosis Antigens in the T Cell Recognition Repertoire of Latently Infected Individuals not Progressing to Active Disease.

In this study, we have mapped the relative importance of well-defined recombinantly expressed Mycobacterium tuberculosis antigens in the T cell recognition repertoire of latently infected individuals not progressing to active disease. Peripheral blood mononuclear cells from healthy latently infected long term non-progressors were screened for antigen-induced proliferation and Th1 cytokine, Interferon- (IFN-γ) responses. The panel of antigens tested showed a clear spectrum of responsiveness and lead to the identification of a subgroup of frequently recognized antigens (MPT59, CFP7, CFP10, CFP21, TB37.6 /PPE68, ESAT-6, MPT51, and DnaK) with a high cellular response level as measured in both proliferation and IFN-γ assays. Among a subgroup of antigens also screened for responses in tuberculosis patients, CFP21 was identified as differentially recognized in non-progressors. For both cellular assays, we found a positive correlation between responder frequency and magnitude of response. A significant correlation between the level of antigen-specific proliferation and INF-γ secretion was also observed. We have identified a defined set of M. tuberculosis antigens frequently recognized by T cells at a high response level from latently infected long term non-progressors which warrant further investigation for a potential role in immune regulation and protection against progression to active disease.

Anti-Inflammatory Effects of a Novel Nuclear Factor–κ B Inhibitory Derivative Derived from Pyrazolo[3,4-d]Pyrimidine in Three Inflammation Models

Nuclear factor-κB (NF-κB) plays a central role in inflammatory responses, and its physiologic functions are essential for cell survival and proliferation. Currently, drugs targeting NF-κB inhibition have not yet been applied in clinical practice. We investigated the physiologic effect of a novel NF-κB inhibitory compound, 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivative (INH #1), on three inflammatory animal models. The pharmacokinetics were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Acute hepatitis was induced by administrating lipopolysaccharide (LPS) and D-(+)-galactosamine hydrochloride followed by the analysis of survival time and inflammatory mediators. Collagen-induced arthritis (CIA) was induced by immunization with type II collagen (CII), and serum-transfer arthritis (STA) was caused by injecting K/BxN mice serum. Clinical and histologic scores were evaluated in both arthritis models. Immune cell subset analysis, CII-induced interferon-gamma (IFN-γ) production and proliferation, and measurement of anti-CII IgG antibodies were performed in the CIA model. In the acute hepatitis model, INH #1 suppressed tumor necrosis factor-α (TNF-α) production and prevented early death in a dose-dependent manner. INH #1 significantly attenuated arthritis scores and joint inflammation in both arthritis models. Additionally, in the CIA model, dendritic cells (DCs) in the regional lymph nodes were decreased in the treated mice and antigen-induced IFN-γ production and cell proliferation in splenocytes were inhibited, whereas the titers of anti-CII IgG antibodies were comparable regardless of the treatment. Here we revealed that INH #1 exerted anti-inflammatory effects in vivo via inhibition of inflammatory mediators and suppression of cellular immune responses. This compound could be a novel candidate for inhibition of NF-κB in certain inflammatory diseases. SIGNIFICANCE STATEMENT: A novel nuclear factor-κB (NF-κB) inhibitory compound, 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivative (INH #1), which retains physiologically essential NF-κB bioactivity, suppressed inflammation in three different mouse models: the acute hepatitis model, the collagen-induced arthritis model, and the K/BxN serum-transfer arthritis model. These results suggest that this compound could be a novel and potent anti-inflammatory agent.

Hepatocyte integrity depends on c-Jun-controlled proliferation in Schistosoma mansoni infected mice

Schistosomiasis is a parasitic disease affecting more than 250 million people worldwide. The transcription factor c-Jun, which is induced in S. mansoni infection-associated liver disease, can promote hepatocyte survival but can also trigger hepatocellular carcinogenesis. We aimed to analyze the hepatic role of c-Jun following S. mansoni infection. We adopted a hepatocyte-specific c-Jun knockout mouse model (Alb-Cre/c-Jun loxP) and analyzed liver tissue and serum samples by quantitative real-time PCR array, western blotting, immunohistochemistry, hydroxyproline quantification, and functional analyses. Hepatocyte-specific c-Jun knockout (c-JunΔli) was confirmed by immunohistochemistry and western blotting. Infection with S. mansoni induced elevated aminotransferase-serum levels in c-JunΔli mice. Of note, hepatic Cyclin D1 expression was induced in infected c-Junf/f control mice but to a lower extent in c-JunΔli mice. S. mansoni soluble egg antigen-induced proliferation in a human hepatoma cell line was diminished by inhibition of c-Jun signaling. Markers for apoptosis, oxidative stress, ER stress, inflammation, autophagy, DNA-damage, and fibrosis were not altered in S. mansoni infected c-JunΔli mice compared to infected c-Junf/f controls. Enhanced liver damage in c-JunΔli mice suggested a protective role of c-Jun. A reduced Cyclin D1 expression and reduced hepatic regeneration could be the reason. In addition, it seems likely that the trends in pathological changes in c-JunΔli mice cumulatively led to a loss of the protective potential being responsible for the increased hepatocyte damage and loss of regenerative ability.

T Regulatory Cell-Associated Tolerance Induction by High-Dose Immunoglobulins in an HLA-Transgenic Mouse Model of Pemphigus.

Pemphigus vulgaris (PV) is a potentially lethal autoimmune bullous skin disorder caused by IgG autoantibodies against desmoglein 3 (Dsg3) and Dsg1. During the last three decades, high-dose intravenous immunoglobulins (IVIgs) have been applied as an effective and relatively safe treatment regime in severe, therapy-refractory PV. This prompted us to study T- and B- cell polarization by IVIg in a human-Dsg3-dependent mouse model for PV. Using humanized mice transgenic for HLA-DRB1*04:02, which is a highly prevalent haplotype in PV, we employed IVIg in two different experimental approaches: in prevention and quasi-therapeutic settings. Our data show that intraperitoneally applied IVIg was systemically distributed for up to 42 days or longer. IVIg-treated Dsg3-immunized mice exhibited, in contrast to Dsg3-immunized mice without IVIg, significantly less Dsg3-specific IgG, and showed induction of T regulatory cells in lymphatic tissue. Ex vivo splenocyte analysis upon Dsg3-specific stimulation revealed an initial, temporarily reduced antigen-induced cell proliferation, as well as IFN-γ secretion that became less apparent over the course of time. Marginal-zone B cells were initially reduced in the preventive approach but re-expanded over time. In contrast, in the quasi-therapeutic approach, a robust down-regulation in both spleen and lymph nodes was observed. We found a significant down-regulation of the immature transitional 1 (T1) B cells in IVIg-treated mice in the quasi-therapeutic approach, while T2 and T3, representing a healthy stage of B-cell development, appeared to be up-regulated by IVIg. In summary, in two experimental settings employing an active PV mouse model, we demonstrate distinct alterations of T- and B-cell populations upon IVIg treatment, compatible with a tolerance-associated polarization in lymphatic tissue. Our data suggest that the clinical efficacy of IVIg is at least modulated by distinct alterations of T- and B-cell populations compatible with a tolerance-associated polarization in lymphatic tissue.

CD83 expression characterizes precursor exhausted T cell population

T cell exhaustion is a main obstacle against effective cancer immunotherapy. Exhausted T cells include a subpopulation that maintains proliferative capacity, referred to as precursor exhausted T cells (TPEX). While functionally distinct and important for antitumor immunity, TPEX possess some overlapping phenotypic features with the other T-cell subsets within the heterogeneous tumor-infiltrating T-lymphocytes (TIL). Here we explore surface marker profiles unique to TPEX using the tumor models treated by chimeric antigen receptor (CAR)-engineered T cells. We find that CD83 is predominantly expressed in the CCR7+PD1+ intratumoral CAR-T cells compared with the CCR7-PD1+ (terminally differentiated) and CAR-negative (bystander) T cells. The CD83+CCR7+ CAR-T cells exhibit superior antigen-induced proliferation and IL-2 production compared with the CD83- T cells. Moreover, we confirm selective expression of CD83 in the CCR7+PD1+ T-cell population in primary TIL samples. Our findings identify CD83 as a marker to discriminate TPEX from terminally exhausted and bystander TIL.

Targeting arginase-1 exerts antitumor effects in multiple myeloma and mitigates bortezomib-induced cardiotoxicity

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.

Piggybac Transposon Mediated CD19 CAR-T Cells Derived from CD45RA-Positive PBMCs Possess Potent and Sustained Antileukemic Function

Background: The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. We previously demonstrated that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit memory-rich phenotype that is characterized by a high proportion of CD45RA+/CCR7+ T cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotype and antitumor function.Methods: CD45RA+ and CD45RA− PBMCs were isolated by magnetic selection from whole PBMCs, then the CD19-CAR transgene was transduced into these cells using the PB transposon system, as described previously. Transduction efficiency of CD19 CAR transgene was determined 24 hours by flow cytometry after transduction. The phenotype of CD19 CAR-T was evaluated by flow cytometry on day 14. High throughput RNA sequencing was performed to see the T cell activation/exhaustion profile upon antigen stimulation. Sequential killing assays were performed by adding fresh tumor cells into CAR-T cells co-cultured with tumor cells every three days by restoring an effector target ratio of 1:1. To see the durable antitumor efficacy in vivo, we performed in vivo stress test, in which CAR T-cells dosage was lowered to the functional limits, so that these CAR-T cells should be maintained and expanded in vivo, to achieve the antitumor efficacy. We injected 5 x 10 5 of firefly luciferase-labeled CD19+ tumor cells (REH) into NSG mice via tail vein, then these mice were treated with 1 x 10 5 of CD19 RA+ CAR-T, RA− CAR-T, or control CAR-T cells, respectively, at day 6 after the tumor injection.Results: RA+ CAR T cells demonstrated better transient transduction efficiency 24 h after transduction (RA+ CAR-T: 77.5 ± 9.8% vs RA− CAR-T: 39.7 ± 3.8%), and superior expansion capacity after 14 days of culture than RA− CAR-T cells (RA+ CAR-T: 32.5 ± 9.3-fold vs RA− CAR-T: 11.1 ± 5.4-fold). RA+ CAR-T cells exhibited dominant CD8 expression (RA+ CAR-T: 84.0 ± 3.4% vs RA− CAR-T: 34.1 ± 10.6%), less expression of exhaustion marker PD-1 (RA+ CAR-T: 3.1 ± 2.5% vs RA− CAR-T: 19.2 ± 6.4%) and T cell senescence marker CD57 (RA+ CAR-T: 6.8 ± 3.6% vs RA− CAR-T: 20.2 ± 6.9%), and enrichment of naïve/stem cell memory fraction (CAR+/CD45RA+CCR7+ fraction; RA+ CAR-T: 71.9 ± 9.7% vs RA− CAR-T: 8.0 ± 5.3%), which were associated with longevity of CAR-T cells. Transcriptome analysis revealed that RA+ CAR-T cells exhibited the enrichment of naïve/memory phenotype and less expression of canonical exhaustion markers, and these exhaustion profiles even maintained after the antigen stimulation. RA+ CAR-T cells demonstrated sustained killing activity even after multiple tumor rechallenges in vitro, without inducing exhaustion marker expression of PD-1. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, in our study, the expression of CAR molecule on the cell surface following antigen stimulation in RA+ CAR was controlled at a relatively lower level that in RA− CAR-T cells. RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells than RA− CAR-T cells in in vivo stress test (Fig.1A-C). On day15, bone marrow studies in RA+ CAR group exhibited abundant human CD3 positive T cells with less expression of PD-1, and relatively smaller amount of REH cells than RA− CAR group (Fig.1D). Furthermore, in two of long-lived mice in RA+ CAR group, human CD3 positive T cells were expanded even day 50 after treatment as confirmed by sequential bone marrow studies (Fig.1E), which indicated the antigen-induced proliferation and long-term functionality of RA+ CAR-T cells in vivo.Conclusion: Our results suggest that PB-mediated RA+ CAR-T cells exhibit memory-rich phenotype and superior antitumor function, thereby indicating the usefulness of CD45RA+ PBMC as a starting material of PB-CAR-T cells. [Display omitted] DisclosuresYagyu: AGC Inc.: Research Funding. Nagao: AGC Inc.: Current Employment. Kubota: AGC Inc.: Current Employment. Shimizu: AGC Inc.: Current Employment. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

Initial Results of Ibrutinib Plus Venetoclax in Relapsed, Refractory CLL (Bloodwise TAP CLARITY Study): High Rates of Overall Response, Complete Remission and MRD Eradication after 6 Months of Combination Therapy

Initial Results of Ibrutinib Plus Venetoclax in Relapsed, Refractory CLL (Bloodwise TAP CLARITY Study): High Rates of Overall Response, Complete Remission and MRD Eradication after 6 Months of Combination Therapy

Regulating T Cell Population Alleviates SLE by Inhibiting mTORC1/C2 in MRL/lpr Mice.

It’s well known that the mammalian target of rapamycin (mTOR) exerts a critical role in the regulator of immune cells and is associated with T cells dysfunction in patients with systemic lupus erythematosus (SLE). Antigen-induced T-cell proliferation via mTORC1 suppressed by Rapamycin has been used to improve SLE primarily. Previously it has showed that INK128, a highly potent, specific orally inhibitor of mTORC1 and mTORC2, significantly attenuates SLE in pristine-induced lupus mice. Herein we compared the cure effects of INK128 and rapamycin on lupus mice. We treated MRL/lpr mice with INK128 or rapamycin at 12 weeks-age. The effect of the two inhibitors on the lupus mice was determined by immunohistochemistry. The effect of the two inhibitors on T cell populations was investigated by flow cytometry. The mTOR signaling was measured by Western Blot. INK128 remarkably alleviated SLE by reducing splenomegaly, renal inflammation and damage, and resuming T-cell dysfunction. The more effective of INK128 on SLE than rapamycin. INK128 effectively suppressed mTORC1 and mTORC2 activity in T cells, but rapamycin just suppressed mTORC1 activity. Thus, our results show that INK128 is can effectively alleviate SLE and be used as one of the potential clinical therapeutic candidates for SLE.

Quantifying Antigen-Specific T-Cells by Assessing Their Antigen-Induced Proliferation.

A critical property of T cells when activated by their cognate antigen-MHC complex is the initiation of cell cycle activity and clonal expansion. In this chapter, we describe how the proliferation of T cells can be assessed on the single cell level by flow cytometry and how this can be used to identify and potentially isolate antigen-reactive T cells.

Inhibition of arginase modulates T-cell response in the tumor microenvironment of lung carcinoma

ABSTRACT Immunotherapy has demonstrated significant activity in a broad range of cancer types, but still the majority of patients receiving it do not maintain durable therapeutic responses. Amino acid metabolism has been proposed to be involved in the regulation of immune response. Here, we investigated in detail the role of arginase 1 (Arg1) in the modulation of antitumor immune response against poorly immunogenic Lewis lung carcinoma. We observed that tumor progression is associated with an incremental increase in the number of Arg1+ myeloid cells that accumulate in the tumor microenvironment and cause systemic depletion of ʟ-arginine. In advanced tumors, the systemic concentrations of ʟ-arginine are decreased to levels that impair the proliferation of antigen-specific T-cells. Systemic or myeloid-specific Arg1 deletion improves antigen-induced proliferation of adoptively transferred T-cells and leads to inhibition of tumor growth. Arginase inhibitor was demonstrated to modestly inhibit tumor growth when used alone, and to potentiate antitumor effects of anti-PD-1 monoclonal antibodies and STING agonist. The effectiveness of the combination immunotherapy was insufficient to induce complete antitumor responses, but was significantly better than treatment with the checkpoint inhibitor alone. Together, these results indicate that arginase inhibition alone is of modest therapeutic benefit in poorly immunogenic tumors; however, in combination with other treatment strategies it may significantly improve survival outcomes.

Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3−/−) the spleen’s histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3−/− mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3−/− and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3−/− mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3−/− mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1β, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3−/− mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Abstract B67: Development of MyD88 L265P mutation-specific TCR gene therapy for treatment of B-cell lymphoma and leukemia

Abstract Lymphoproliferative malignancies form a major group of cancers and are responsible for nearly 10% of all cancer deaths in developed countries. Adoptive transfer of engineered T cells has been shown to be a promising treatment approach; hence, screening and evaluation of cellular antigens for T-cell therapy with higher specificity and efficacy is a rapidly progressing area of research. Cancer-specific antigens derived by somatic mutations acquired during tumor development, so called “neoantigens,” represent a very advantageous target repertoire. Here we aimed to develop T-cell receptors (TCRs) targeting recurrent neoantigens in lymphoma and leukemia, for a truly cancer-specific immunotherapy. With this purpose, we have generated T-cell lines from multiple healthy donors targeting one of the most common driver mutations found in B-cell lymphomas: a missense mutation on adaptor protein MyD88 changing leucine at position 265 to proline (L265P). T cell lines generated by autologous in vitro priming were reactive selectively against the predicted mutant epitope restricted to HLA-B7, but not against the corresponding wild-type peptide. TCR alpha and beta nucleotide sequences from these T-cell lines were successfully cloned into a retroviral vector and expressed on T cells of healthy donors for further characterization. All cloned TCRs have shown mutation-specific and HLA-restricted reactivity with varying functional avidity. TCR-transduced T cells were able to selectively recognize and kill engineered target cells expressing MYD88 mutation, proving that the mutant epitope can be naturally processed by human proteasome and presented on HLA-B7. In order to further evaluate the therapeutic potency, we tested TCR-transduced T cells with non-Hodgkin lymphoma cell lines naturally harboring MyD88 L265P, and again observed HLA-restricted and mutation-specific reactivity and cytotoxicity, accompanied by antigen-induced proliferation. Taken together, our data suggest that mutation-specific TCRs can be used to target cancer cells with MyD88 L265P, and hold promise for immunotherapy of B-cell malignancies. Therefore, an application to the European Patent Office for variable region sequences of the TCRs has been made (EP19152801.7) in collaboration with Charité Technology Transfer Office. Preclinical safety screening of the TCRs and in vivo efficacy assessment in mice are in progress. Citation Format: Özcan Çinar, Antonia Busse, Antonio Pezzutto. Development of MyD88 L265P mutation-specific TCR gene therapy for treatment of B-cell lymphoma and leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B67.

Extracellular ST6Gal-1 calibrates B cell IgG production by cell non-autonomous extrinsic sialylation

Abstract Cell surface glycans are well-positioned to modify receptor interactions and their downstream biological outcomes. Although canonical glycosylation occurs intracellularly, glycosyltransferases also exist in the blood and can catalyze extracellular reactions with platelet-derived sugars. We have recently reported that such extrinsic glycosylation by the sialyltransferase ST6Gal-1 enhances survival and BCR signaling in immature B cells. Here, we report that mice lacking extrinsic ST6Gal-1 had reduced total and antigen-specific IgG after bone marrow transplantation, despite unaltered IgG half-life and reconstitution of mature B cells and plasma cells. Rather, this deficiency was accompanied by reduced IgG+ splenic B cells and impaired per-cell IgG secretion after BCR stimulation. In vitro, extrinsic sialylation of immature B cells with recombinant ST6Gal-1 enhanced expression of IgM, IgD, and CD86, and potentiated IgG production upon stimulation. Surprisingly, this was not associated with increased IgG germline transcripts or isotype switching, but more robust antigen-induced proliferation, generating more IgG-producing cells. In vivo, hydrodynamic transfection with St6gal1 DNA resulted in an increase in total serum IgG. Finally, infusion of recombinant ST6Gal-1 enlarged splenic follicles and the follicular B cell population, highlighting therapeutic potential in modifying B cell development. Currently, vaccine adjuvants rely on mechanical or inflammatory properties to trigger an immune response. Our results suggest the manipulation of a bloodborne sialyltransferase may be a novel B cell-targeted, biological strategy to boost vaccine-induced and overall IgG production.

Sildenafil does not affect the proliferation of human lymphocytes in the in vitro transplant model.

Sildenafil is used in the treatment of erectile dysfunction and pulmonary arterial hypertension. Numerous studies revealed beneficial effects of its use in renal, liver, heart and bone marrow transplant recipients. Some reports suggested that the drug modulates the function of the immune system, however, its influence on antigen-induced proliferation of lymphocytes remains unknown. Thus, the aim of the study was to investigate the effects of sildenafil on human peripheral blood mononuclear cells (PBMCs) proliferation in a mixed lymphocyte reaction. It was demonstrated that the drug did not affect auto- and alloantigen-induced proliferation of PBMCs and showed no cytotoxic effect.

Increasing CD28-FOXP3+CD8+ Treg and Senescent CD8+NK2GA+Eomes+ NK-like T Cells in Peripheral Blood of Patients with Multiple Myeloma

Immune dysfunction in patients with multiple myeloma (MM) includes TGF-β induced dendritic cell dysfunction, regulatory T cell (Tregs)/Th17 cell imbalance, accumulation of Tregs and myeloid derived suppressor cells. These tumor-induced dysfunctions may contribute to immune escape and even suppress immune cells introduced in adoptive cellular immunotherapy. CD28 independent T cells of the effector memory (TEM) and CD45RA+ effector memory (TEMRA) population were shown to accumulate with age and contribute to the immunosuppressive tumor microenvironment in several solid tumors and hematological cancers. Especially in the CD8+ population, the loss of CD28 is associated with high cytotoxicity and regulatory function while showing high diversity and defective antigen-induced proliferation. In the CD4 subset, regulatory and senescent T cells were studied extensively, while in the CD8 positive subset their heterogeneity is still not clearly defined. Their potential immunosuppressive role and distribution in healthy individuals (HI) as well as patients with multiple myeloma (MM) remains to be observed. Furthermore, a recently characterized CD8+CD28- NK-like T cell subset showing expression of NK-related inhibitory receptors and TCR independent effector function is potentially of interest in the progression of MM. In the present study, we compared the changes of distribution of CD8+CD25+ and CD8+FOXP3+ regulatory T cells (Treg), CD28-CD57+ senescent T cells (Tsen), and CD8+KIR/NK2GA+EOMES+ NK-like T in peripheral blood (PB) between HI and patients with MM by multicolour flow cytometry (Gating strategy shown in Figure 1A). When comparing 35 HIs (Median age is 54) and 14 MM patients (Median age is 52), it was shown that there is no significant change in the proportion of senescent T cells in CD8 (P = 0.2452), TEM/CD8 (P = 0.1686) and TEMRA/CD8 (P = 0.4861) between HIs and the MM group, while both the CD25+FOXP3+ regulatory T cells of the CD4 population (P = 0.0031) and CD28-FOXP3+ regulatory T cells of the CD8 population (P = 0.0014) were shown to increase. There is no significant difference in the percentages of KIR/NK2GA+EOMES+ in the CD8 T cell and TEMRA/CD8 T cell population between HIs and the MM group. Remarkably, although there was no overall increase in senescent T cell in MM patients, senescent CD8+NK2GA+EOMES+ NK-like T cells increased in MM patients in comparison to HIs (P = 0.0068) (Figure 1B). In conclusion, the increase of regulatory T cells of both the CD4 and CD8 population as well as the increase of senescent NK-like T cells in the CD8 population potentially contributes to cancer progression through creation of suppressive microenvironments. Moreover, we found that regulatory CD8 T cells and CD8 NK-like T cells only contribute to a small part of the overall CD28- senescent T cell pool. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ibrutinib Plus Venetoclax in Relapsed/Refractory CLL: Results of the Bloodwise TAP Clarity Study

Ibrutinib Plus Venetoclax in Relapsed/Refractory CLL: Results of the Bloodwise TAP Clarity Study

APOE GENE POLYMORPHISM: THE IMPACT OF APOE4 ALLELE ON SYSTEMIC INFLAMMATION AND ITS ROLE IN THE PATHOGENESIS OF ALZHEIMER’S DISEASE

ApoE is a member of lipoprotein family. It is the most common lipoprotein in the central nervous system (CNS), secreted by astrocytes, microglia, neurons and immunocompetent cells, including lymphocytes, monocytes and macrophages. According to recent data, it has endotheliotropic and immunomodulatory functions, regulating inflammatory activation of mononuclear phagocytes and antigen-induced lymphocyte proliferation. APOE4 allele is a major genetic risk factor of Alzheimer’s disease, with prevalence 3-12 times higher in those who have this allele. Mechanisms that predispose carriers of the allele to earlier clinical presentation of neurodegeneration include changes in lipid metabolism in the CNS, in the buildup of neurotoxic amyloid-beta oligomers, in the clearance of amyloid-beta peptides from the CNS and in regulation of immune response. In this review the functions of ApoE protein in central nervous and immune system and changes in functional activity of the protein in APOE4 carriers are discussed. The impact of APOE4 allele on monocyte phenotype and inflammatory activation of monocytes, on specific cell-mediated immune response to amyloid-beta antigens and on effectiveness of immunomodulatory therapy in patients with Alzheimer’s disease summarized, as well as the possible role of changes in the immune response characteristic for APOE4 carriers in the increased risk of Alzheimer’s disease.

Combined Application of Xymedon Hydrochloride with Rabies Vaccine on the Background of Immunosupression

The effect of xymedon hydrochloride on the immune response of rabbits immunized with the rabies vaccine prepared from strain Shchelkovo-51 under the influence of immunosupression induced by cyclophosphamide has been studied. Xymedon hydrochloride was injected at a dose of 5 mg kg body weight in a volume of 1 cm3 twice a day in combination with the rabies vaccine. In addition, the immune response higher level of 1: 12800 was observed in the immunosupressed animals compared to 1: 6400 in the control animals immunized without the immunostimulant. At 6 months after vaccination, the antibody concentration of 1: 93.75 ± 44.68 remaining below the protective level was recorded, which indicated the weakness of the humoral link in the immune system. The counts of T- lymphocytes, B-lymphocytes, T-helpers, and T-suppressors in the blood of the immunesuppressed rabbits immunized with the rabies vaccine in combination with xymedon hydrochloride increased 3.2-fold, 1.9-fold, 7.7-fold, and 22.0-fold, respectively, when compared with the reference values. Therefore, the protective effect was followed by increasing the antigeninduced and spontaneuous lymphocyte proliferation, the numbers of T-helper cells and T lymphocytes exhibiting the cytotoxic and suppressive activity, and the virus-specific neutralizing antibody titers in cases wherein the Shchelkovo-51 vaccine in combination with xymedon hydrochloride was administered. It should be reasonable to apply the two-step rabies vaccination with the use of the immunostimulant, in particular, xymedon hydrochloride, to ensure the adequate immune response in immunodeficient animals.

Large cell neuroendocrine lung carcinoma induces peripheral T-cell repertoire alterations with predictive and prognostic significance

ObjectivesThis study was performed to evaluate for a potentially important role of T cells in the pathophysiology and treatment sensitivity of large cell neuroendocrine lung carcinoma (LCNEC), an orphan disease with poor prognosis and scarce data to guide novel therapeutic strategies. Materials and methodsWe performed T-cell receptor (TCR) β-chain spectratyping on blood samples of patients treated within the CRAD001KDE37 trial (n = 35) using age-matched current or former (n = 11) and never smokers (n = 10) as controls. The data were analyzed in conjunction with the complete blood counts of the probands as well as the data about response to treatment and overall survival in the clinical trial. Results and conclusionUntreated stage IV LCNEC patients had significant T-cell repertoire alterations (p < 0.001) compared to age-matched smokers. These changes correlated positively with blood lymphocyte counts (r = 0.49, p < 0.01), suggesting antigen-induced T-cell proliferation as the causative mechanism. At the same time, LCNEC patients showed mild lymphopenia (1.54 vs. 2.51/nl in median, p < 0.01), which reveals a second, antigen-independent mechanism of systemic immune dysregulation. More pronounced T-cell repertoire alterations and higher blood lymphocyte counts at diagnosis were associated with a better treatment response by RECIST and with a longer overall survival (441 vs. 157 days in median, p = 0.019). A higher degree of T-cell repertoire normalization after 3 months of therapy also distinguished a patient group with more favourable prognosis (median overall survival 617 vs. 316 days, p = 0.036) independent of radiological response. Thus, LCNEC induces clinically relevant changes of the T-cell repertoire, which are measurable in the blood and could be exploited for prognostic, predictive and therapeutic purposes. Their pathogenesis appears to involve antigen-induced oligoclonal T-cell expansions superimposed on TCR-independent lymphopenia.