Emergence Of CD4 Research Articles

The Correlation between Peripheral Blood Index and Immune Cell Expansion in Vietnamese Elderly Lung Cancer Patients.

(1) Background: The dysfunction and reduced proliferation of peripheral CD8+ T cells and natural killer (NK) cells have been observed in both aging and cancer patients, thereby challenging the adoption of immune cell therapy in these subjects. In this study, we evaluated the growth of these lymphocytes in elderly cancer patients and the correlation of peripheral blood (PB) indices to their expansion. (2) Method: This retrospective study included 15 lung cancer patients who underwent autologous NK cell and CD8+ T cell therapy between January 2016 and December 2019 and 10 healthy individuals. (3) Results: On average, CD8+ T lymphocytes and NK cells were able to be expanded about 500 times from the PB of elderly lung cancer subjects. Particularly, 95% of the expanded NK cells highly expressed the CD56 marker. The expansion of CD8+ T cells was inversely associated with the CD4+:CD8+ ratio and the frequency of PB-CD4+ T cells in PB. Likewise, the expansion of NK cells was inversely correlated with the frequency of PB-lymphocytes and the number of PB-CD8+ T cells. The growth of CD8+ T cells and NK cells was also inversely correlated with the percentage and number of PB-NK cells. (4) Conclusion: PB indices are intrinsically tied to immune cell health and could be leveraged to determine CD8 T and NK cell proliferation capacity for immune therapies in lung cancer patients.

Hepatitis B virus-specific CD4 T cell responses differentiate functional cure from chronic surface antigen+ infection

With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control. We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining, and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly exvivo. ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection. Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses. Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell).

Abstract 5523: LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model

Abstract Objective: Although immunotherapies, e.g., anti-PD-(L)1 has achieved great success in treating cancers, a large portion of patients still cannot benefit from it, due largely to primary refractory or acquired resistance leading to unsatisfied ORR or relapse. TNFR2 is a receptor for TNF-α, expressed highly selective in immune cells, and some tumor cells, transducing survival and immunostimulatory signal to cells expressing this receptor. Targeting TNFR2 may modulate this signaling, therefore a humanized anti-TNFR2 antibody, LBL-019, was developed for monotherapy or in combination with other anti-tumor therapies. Methods: LBL-019 is a monoclonal antibody of IgG1 subtype. The binding affinity to TNFR2 was determined by Fortebio. The epitope of TNFR2 was identified by differential binding to a serial human/mouse chimeric TNFR2 proteins. The binding of TNF-α to either TNFR2 protein or TNFR2 expressed in cells was examined by using ELISA and flow cytometry. The in vitro activity of LBL-019 was measured using several in vitro assays, e.g., cell proliferation and cytokine release in T cells, ADCC and CDC assays. The anti-tumor activity of LBL-019 was investigated in a model of human TNFR2 transgenic mice (Shanghai Model Organisms) implanted with MC38-OVA cells. Results: The binding affinity of LBL-019 was measured as 5.32 nM to human and 3.62 nM to cynomolgus TNFR2. It was found that TNFR2 was expressed in immune cells including T cells and monocytes examined in human PBMC, which was upregulated when T cells were activated. TNFR2 was also found highly expressed in some tumor cell lines and tumor TILs (from in vivo studies described below). The epitope of TNFR2 that LBL-019 binds was identified and mapped out CRD1 domain. The binding of TNF-α with either TNFR2 protein or TNFR2 expressed in immune cells was blocked by LBL-019. The growth of CD8+ T cells and CD4+ T cells was significantly enhanced, when incubated with LBL-019, along with elevated IFN-γ release. In a model of human TNFR2 transgenic mice implanted with MC38-OVA cells, LBL-019 was shown significant tumor growth inhibition (TGI) in a dose dependent manner as compared with vehicle treated mice (p<0.01). It appeared that the antibody with wild type Fc showed a better anti-tumor activity than that with silenced Fc, suggesting that Fc mediated NK killing of tumor cells or Treg may have contributed in part to the observed anti-tumor efficacy in this model. In addition, LBL-019 was also exhibited a synergy of anti-tumor activity, when dosed with anti-PD-1 (TGI reaching 79.51%). Conclusions: LBL-019, a humanized anti-TNFR2 antibody, shows great anti-tumor efficacy in a mouse tumor model, likely due to the mechanisms involving enhanced proliferation of Teff, elevated IFN-γ release, and possibly killing of tumor cells and Treg. The data support further development of LBL-019 as a novel therapeutic antibody for cancer, and clinical trial is expected to launch early 2022. Citation Format: Huan Lin, Xiao Huang, Yurong Qin, Yujia Dang, Peng Zhang, Jianming Sun, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model [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 5523.

Validation of CD4+ CD57+PD1+ T Cells in Bronchoalveolar Lavage as a Biomarker of Lung Allograft Dysfunction

<h3>Purpose</h3> Our analysis on serial bronchoalveolar lavage (BAL) cells obtained from 25 lung transplant recipients (LTRs) using mass cytometry (MC) technology revealed a strong association between the frequency of CD4+ CD57+ PD1+ cells and subsequent lung allograft dysfunction (LAD). Here we present validation of this cell subset as a biomarker of incident LAD in an additional 25 LTRs. <h3>Methods</h3> MC was performed using a panel of 37 heavy metal-tagged Abs against surface markers and intracellular cytokines. This technique was applied to BAL cells from 50 consecutive LTRs at their 3-, 6- and 9-month post-transplant surveillance bronchoscopies. LTRs were randomly assigned into two groups (Fig 1A). Semi-supervised identification algorithm on the first group of LTRs identified CD4+ CD57+ PD1+ cells as the highest discriminatory cluster between stable and LAD patients. We then tested best-fit threshold for frequency of the cell subset and applied it on the second group of LTRs for validation. <h3>Results</h3> Longitudinal analysis of the first randomized group of 25 LTRs demonstrated a higher frequency of CD4+ CD57+ PD1+ cells separating LAD (11.41±4.89%) from stable patients (4.43±2.11%; Fig 1B). Using a receiver operating characteristic curve, we identified a frequency of 7.8% as the best-fit threshold of the cell subset (Fig 1C). Survival analysis showed CD4+ CD57+ PD1+ T cells at 7.8% can discriminate stable from LAD patients in the first group of LTRs (Fig 1D). We then tested the performance of this threshold in the second group of LTRs. We found that in these additional 25 LTRs, those with BAL CD4+ CD57+ PD1+ cells above the threshold had a significantly lower freedom from incident LAD (p=0.05, Fig 1E). <h3>Conclusion</h3> Our data validated that emergence of CD4+ CD57+ PD1+ T cells precedes LAD in a small cohort of LTRs. Further validation in larger cohorts, and molecular and functional studies on this cell population are underway and may lead to improved prediction of LAD and a better understanding of lung transplant immunobiology.

New early prognostic criteria for pregnancy outcome in women with recurrent miscarriage.

To determine the new criteria for predicting the outcome of pregnancy in women with habitual abortion based on features of differentiation of naive T cells and memory cells in a population of T-helper (CD4+) and cytotoxic T lymphocytes (CD8+). The study involved 61 women with threatened and habitual abortion in the first trimester of gestation. Depending on the outcome of pregnancy was allocated to 3 groups: I went to 39 women whose pregnancy ended in timely delivery; in II - 10 women whose pregnancies ended in premature birth; in III - 11 patients in whom there was a spontaneous miscarriage. Using three-color flow cytometry as peripheral venous blood in populations of CD8+ and CD4+ determined by the content Tn, Tcm, Tem and Temra cells. Statistical analysis was carried out in the program «MicrosoftOffice 2010», «Statistica for Windows 6.0» and MedCalc». When conducting a retrospective assessment, it was found that in the group of patients whose pregnancy ended in preterm delivery, the percentage of CD4+ Tem memory cells was significantly higher and CD4+ Tn lower than in the subgroup with timely delivery (p = 0.013 and p = 0.025, respectively ) In patients with early spontaneous miscarriage, the level of CD8+ Tn significantly decreased against the background of the growth of CD8+ Tem memory cells compared with the same parameters in patients with timely delivery (p = 0.040 and p = 0.014, respectively). Prediction of spontaneous abortion is possible up to CD4+ Tn equal to 34.2% or less (sensitivity - 100.0%, specificity - 56.4%, accuracy - 63.8%), premature birth - if the CD4+ Tn equal to 35, 2% or less (sensitivity - 66.7%, specificity - 74.4% accuracy - 72.9%). Thus, the new criteria will allow additional time to identify risk and assign adequate treatment aimed at prolonging the desired pregnancy.

Validation of CD4+ CD57+ PD1+ T Cells in Bronchoalveolar Lavage as a Biomarker of Lung Allograft Dysfunction

Our analysis on serial bronchoalveolar lavage (BAL) cells from 25 lung transplant recipients (LTRs) using cutting-edge mass cytometry (MC) technology revealed a strong association between the frequency of CD4+ CD57+ PD1+ cells and subsequent lung allograft dysfunction (LAD). Here we present validation of this cell subset as a biomarker of incident LAD in an additional 25 LTRs. MC was performed using a panel of 37 heavy metal-tagged antibodies against surface markers and intracellular cytokines. This technique was applied to BAL cells from 50 consecutive LTRs at their 3-, 6- and 9-month post-transplant surveillance bronchoscopies. LTRs were randomly assigned into two groups (Fig 1A). Semi-supervised identification algorithm on the first group of LTRs identified CD4+ CD57+ PD1+ cells as the highest discriminatory cluster between stable and LAD patients. We then tested best-fit threshold for frequency of the cell subset and applied it on the second group of LTRs for validation. Longitudinal analysis of the first randomized group of 25 LTRs demonstrated a higher frequency of CD4+ CD57+ PD1+ cells separating LAD (11.41±4.89%) from stable patients (4.43±2.11%; Fig 1B). Using a receiver operating characteristic curve, we identified a frequency of 7.8% as the best-fit threshold of the cell subset (Fig 1C). Survival analysis showed CD4+ CD57+ PD1+ T cells at 7.8% can discriminate stable from LAD patients in the first group of LTRs (Fig 1D). We then tested the performance of this threshold in the second group of LTRs and found those with BAL CD4+ CD57+ PD1+ cells above the threshold had significantly lower freedom from incident LAD (p=0.05, Fig 1E). Our data validated that emergence of CD4+ CD57+ PD1+ T cells precedes allograft dysfunction in a small cohort of LTRs. Further validation in larger cohorts, and molecular and functional studies on this cell population are underway and may lead to improved prediction of LAD and a better understanding of lung transplant immunobiology.

Emergence of a Specific Intrapulmonary CD4+ T Cell Subset Prior to the Onset of Lung Allograft Dysfunction

Purpose We have applied cutting-edge mass cytometry (MC) technology to serial bronchoalveolar lavage (BAL) cells obtained from lung transplant recipients (LTRs) and have used it to compare the cellular composition of the BAL compartment in patients who remain stable or go on to develop lung allograft dysfunction (LAD), defined here as a drop, from any cause, in the forced expiratory volume in 1 second of 20% from the best post-transplant baseline value. Methods We have collected and cryopreserved BAL cells from 50 consecutive LTRs at their 3-, 6- and 9-month post-transplant surveillance bronchoscopies. Cryopreserved samples were then recovered and labeled with a panel of 37 heavy metal-tagged antibodies against surface markers and intracellular cytokines before MC. We applied t-distributed stochastic neighbor embedding (tSNE; Fig1 A) which assists with better visualization of multi-dimensinal analysis to identify both common and rare cell populations in the BAL. We further applied an algorithm called cluster identification, characterization and regression (CITRUS; Fig1 B) to allow unsupervised identification of differentially represented BAL cellular subsets between stable and LAD patients. Results Longitudinal analysis of the first 25 patients demonstrated different cell subsets and cytokine-producing cells, with substantial intra- and inter-patient variation. Our data identified an elevation in the frequency of CD4+ CD57+ PD-1+ cells separates LAD (18.41±4.89%) from stable patients (4.43±2.11%; Fig1 C). Emergence of CD57+ PD-1+ events among CD4+ T cells preceded LAD by up to several weeks (Fig1 D), suggesting that these cells may contribute to LAD development rather than simply correlating with it. Conclusion Our data suggest that emergence of CD4+ CD57+ PD-1+ T cells may predict and/or contribute to allograft dysfunction. Molecular and functional studies on this cell population are underway to obtain a better understanding of their role in lung transplant immunobiology.

Emergence of CD4+ and CD8+ Polyfunctional T Cell Responses Against Immunodominant Lytic and Latent EBV Antigens in Children With Primary EBV Infection

Long term carriers were shown to generate robust polyfunctional T cell (PFC) responses against lytic and latent antigens of Epstein-Barr virus (EBV). However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM]) and asymptomatic (AS) EBV infection. Evaluation of IFN-γ secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-γ, TNF-α, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs) as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions) concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6–12 months. In conclusion, EBV antigen-specific CD4+ and CD8+ PFC responses emerge during the first year of primary EBV infection, with greatest responses toward immunodominant epitopes in both lytic and latent proteins, correlating to steady decline in PBMC and plasma viral loads.

Changes of the Immune Status in Patients with Purulent Peritonitis

The analysis of cellular and humoral immunity indicators in the blood of patients with purulent peritonitis (PP) with the determination of their diagnostic value while determining the stage of peritonitis was performed. The levels of CD3, CD4, CD8, CD4 / CD8, CD19, CD16, CD95, CD25, IgA, IgM, IgG during hospitalization of 126 patients with PP was investigated. Monitoring of immune responsiveness stated regular changes in cellular and humoral immunity caused by the influence of cytokine profile, describing immunosuppression, the extent of which depends on the length and stage of PP. The progressive deepening of PP stages (in reactive and toxic stage) led to an increase in white blood cell count, CD95, due to the influence of dose-dependent increase in IL-6, CD19, IgM, G, and NK-cells (CD16) and CD25, the dynamics of which is closely related and dependent of IL-2.This was accompanied by a progressive lymphocytopenia, a decrease in CD3, CD4, which at constant or slightly increasing concentration led to decrease in CD8 IRI (CD4 / CD8) caused by the impact of immunosuppressive IL-6, stepwise reduction of IgA. Severe secondary immunodeficiency was observed in patients with end-stage PP and lymphocytopenia progressed along with the growth of white blood cell and marker CD95 with disruption of T-lymphocytes subsets (CD4 decrease at a constant or insignificant growth of CD8) with critical IRI indicators. The decrease in CD25, CD16 and CD19 dysfunction with sharp inhibition of the antibodies synthesis – decrease in IgM and G, A concentrations was also determined.

Abstract 4093: TGF-β insensitivity promotes the proliferation of human CD8+ T cells in metastatic castration resistant prostate cancer (MCRPC) patients

Abstract Introduction: Tumor derived TGF-β is a potent immunosuppressant molecule which promotes prostate cancer (CaP) progression. We previously reported that tumor derived TGF-β can suppress the growth of human CD8+ T cells. Here we report a preclinical study on the antitumor response and growth of human CD8+ T cells which were rendered insensitive to TGF-β. Methods: Whole blood (50 ml/each pt) was collected from three human pts who previously had MCRPC and docetaxel chemotherapy. CD8+ T cells were isolated using the EasySep CD8+ T cells enrichment kit, and then cultured in the presence of clinical grade IL-2 (100unit/ml, Prometheus Labs) and CD-3 Biotin/CD28/Anti-Biotin beads (1:1, Miltenyi Biotec). We developed an TβRIIDN-TK-IRES-anti-PSMA chimeric T cell receptor construct (TβRIIDN-TK-IRES-PZ-1) using an anti-PSMA IgTCR(ζ) gene (provided by Dr. Sadelain) and a dominant negative TGF-β type II receptor (TβRIIDN) and Thymidine kinase (TK). Infection with a retroviral vector encoding this construct could induce T cells to be PSMA reactive, TGF-β insensitive, and undergo TK inducible cell suicide. The purification of CD8 T cells was confirmed by immunofluorescence assay. The transfection of TβRIIDN-TK-IRES-PZ-1 into the packaging cell line GP2-293 was performed by using lipofectamine 2000, and the retrovirus particles were infected into CD8+ T cells. Three days after infection, the expression of TK, anti-PSMA IgTCR(ζ) in CD8+ T cells were evaluated by PCR and RT-PCR. The growth of the CD8+ T cells were evaluated by cell counter every 3 days, and [3H] Thymidine incorporation assay was used to evaluate cell growth under the treatment of TGF-β (10ng/ml, 48hours). Results: Highly purified CD8+ T cells were isolated from each pt (CD3+: 94.5%; CD8+: 98.6%). Group 1: CD8+ T cells infected with TβRIIDN-TK-IRES-PZ-1 strongly express anti-PSMA IgTCR(ζ) gene and TK gene compared with the uninfected control group (Group 2). On the 7th day after transfection, the infected cells in Group 1 increased 4 fold (from 0.045x106/ml to 0.18x106/ml); however, Group 2 only increased &amp;lt;2 fold (from 0.064x106/ml to 0.12x106/ml). On the 17th day, the cells in Group 1 were expanded 11.5 fold (from 0.675x106 to 7.8x106) and Group 2 only 3.43 fold (from 0.96x106 to 3.3x106). Thymidine incorporation assay demonstrated that under the treatment of TGF-β, there was no significant change in Group 1, while the growth of cells in Group 2 were suppressed by 46.7%. Conclusion: We have reported that tumor derived TGF-β suppresses functioning and proliferation of CD8+ T cells, which results in immune escape and progression of CaP. Our results indicate that CD8+ T cells rendered insensitive to TGF-β can enhance the proliferation of CD8+ T cells in human samples obtained from pts. In addition, it is possible that in combination with an anti-PSMA IgTCR(ζ) and TK inducible gene, this construct may offer a novel immunotherapy for CaP and men with advanced disease. Citation Format: Qiang Zhang, Brian Helfand, Ann V. LeFever, Chung Lee, Timothy Michael Kuzel. TGF-β insensitivity promotes the proliferation of human CD8+ T cells in metastatic castration resistant prostate cancer (MCRPC) patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4093. doi:10.1158/1538-7445.AM2014-4093

Expansion of a CD8+PD-1+ Replicative Senescence Phenotype in Early Stage CLL Patients Is Associated with Inverted CD4:CD8 Ratios and Disease Progression

Patients with chronic lymphocytic leukemia (CLL) display immune deficiency that is most obvious in advanced stage disease. Here we investigated whether this immune dysfunction plays a pathologic role in the progression of early stage disease patients. We carried out eight-color immunophenotyping analysis in a cohort of 110 untreated early stage CLL patients and 22 age-matched healthy donors and correlated our findings with clinical outcome data. We found a significant reduction in naive CD4(+) and CD8(+) T cells in CLL patients. Only the CD4(+) subset showed significantly increased effector memory cells (T(EM) and T(EMRA)) in the whole cohort (P = 0.004 and P = 0.04, respectively). However, patients with inverted CD4:CD8 ratios (52 of 110) showed preferential expansion of the CD8 compartment, with a skewing of CD8(+) T(EMRA) (P = 0.03) coupled with increased percentage of CD57(+)CD28(-)CD27(-) T cells (P = 0.008) and PD-1 positivity (P = 0.027), consistent with a replicative senescence phenotype. Furthermore, inverted CD4:CD8 ratios were associated with shorter lymphocyte doubling time (P = 0.03), shorter time to first treatment (P = 0.03), and reduced progression-free survival (P = 0.005). Our data show that the emergence of CD8(+)PD-1(+) replicative senescence phenotype in early stage CLL patients is associated with more aggressive clinical disease. Importantly, these findings were independent of tumor cell prognostic markers and could not be accounted for by patient age, changes in regulatory T-cell frequency, or cytomegalovirus serostatus (n = 217).

Antiretroviral therapy in Ukraine

Purpose of the study : The study aimed to assess results of ART in Ukraine. Methods: National official cohort data from August 2004 to December 2010 were analysed. We included all individuals starting their first ART in this period. Results: As of 01/01/2012, the ART receives 26,720 patients. Among those receiving ART, adults (15+) 91.5% (24,452 patients), of which 12,696 men (51.9%), 11,756 women (48.1%). The share of active IDUs among persons receiving ART is 8.3%. ART regimen in 1st line receives 24,807 patients (92.8%), regimen of the 2nd line - 1836 patients (6.9%), regimen 3rd line - 77 patients (0.3%). Total for the treatment using 64 combinations of antiretroviral drugs that are based on: 1 NNIZT + 2 NIZT - 17 combinations; 1 IP + 2 NIZT - 19 combinations, 3 NRTIs - 6 combinations; integrase inhibitor - 9 combinations; 3 NIZT + 1 IP - 6 combinations, 1 NRTI + 1 NRTI + 1 IP - 3 combinations, other - 4. The pooled data analysis of the cohort showed that after 12 months of treatment, 82.9% of patients who started ART cohorts continued to receive it (the minimum rate of survival), 8.1% of people - died in the 1 year after treatment, 9.0% - interrupted ART. Maximum survival at 12 months (number of persons in the cohort who remained alive) was 91.9%. After 12 months of treatment regimen in 1st line continued to receive 81.1% of the total number of patients who started ART in the cohort. 6 years after initiation of ART schemes are one of the 1st line 83.8% of the total number of patients who continue to receive therapy, the scheme of the 2nd line gets 15.6% of patients, the scheme 3rd line - 0.6% of patients. As a result of monitoring the functional status in 2004-2005, patients with limited physical activity and/or are not capable of self-care accounted for about 64% of all patients prescribed ART, in 2010 there were 50%. There is a clear correlation between the growth of CD4 and duration receiving ART. There is a clear downward trend in the percentage of patients with a level of less than 350 CD4 cells/ml. If at the beginning of treatment the percentage of such patients of the cohort was 88%, then 12 months therapy - 43% after 7 years - 8%. In a cohort of patients receiving ART 7 years, 95% demonstrated high adherence to ART. Conclusion: These generalized figures, in particular the survival, retention on ART, indicate positive trends in the provision of care to patients, and at the same time, emphasize the importance of ensuring access to treatment for all who need.

Treatment of collagen‐induced arthritis by Natura‐α via regulation of Th‐1/Th‐17 responses

Cytokines and CD4(+) Th cells play a crucial role in the pathogenesis of rheumatoid arthritis. Among the Th populations, Th-1 and Th-17 have been described as pathogenic in collagen-induced arthritis (CIA) whereas Th-2 and Treg were found to have protective effects. The objective of this study was to examine the affect of Natura-alpha, a newly developed cytokine regulator, on CIA and on Th cell development. Natura-alpha treatment was administered before or during arthritis induction. Anti-type II collagen antibodies and cytokine expression were evaluated by ELISA. Emergence of CD4(+)CD25(+)Foxp3(+) T cells was assessed by flow cytometry. Th-17 differentiation of naive CD4 T cells was assessed in cultures with anti-CD3 and anti-CD28. We showed that Natura-alpha both prevented and treated CIA. We further demonstrated that in vivo treatment with Natura-alpha inhibited IL-17 production and anti-type II collagen IgG development. We showed in vitro, using an APC-free system, that Natura-alpha acted directly on differentiating T cells and inhibiting the formation of Th-1 and Th-17 cells but did not affect Th-2 cells. Since Natura-alpha inhibits a large spectrum of important pathogenic factors in CIA, it may provide a new and powerful approach to the treatment of rheumatoid arthritis and other inflammatory diseases.

Inhibitory Effects of Cyclosporine on Human Regulatory T Cells In Vitro

Background Inevitable hepatitis C virus (HCV) recurrence after liver transplantation is a major barrier to the survival of a transplanted liver. It may be promoted by immunosuppression and the emergence of CD4 +CD25 + regulatory T cells (Treg). Treg cells can mediate the induction and maintenance of immunological self-tolerance as well as transplant tolerance. We investigated the effects of cyclosporine (CsA), a widely used immunosuppressive agent, on human CD4 +CD25 + Treg cells. Methods Human CD4 +CD25 + cells isolated from healthy donors were cultured in the presence of 40 or 400 ng/mL CsA. The suppressive activity of Treg was assessed in mixed leukocyte reactions (MLR) using CD25 + and autologous activated peripheral blood mononuclear cells (PBMC). Phenotype analysis (flow cytometric, Q-PCR) and cytokine production (ELISA) of Treg cells were then performed on cultures. Results CsA (40 or 400 ng/mL) inhibited the proliferative capacity of PBMC and CD4 +CD25 + Treg in a dose-dependent manner. Interestingly, addition of 40 ng/mL CsA in MLR impaired the suppressive activity of CD4 +CD25 + cells, whereas a higher dose of CsA had no effect on Treg function. It appears that a therapeutic dose of CsA (40 ng/mL) did not change the phenotype of CD4 +CD25 + T cells, but altered Treg activity by switching the regulatory to an inflammatory cytokine profile. Conclusion CsA significantly impaired the function of CD4 +CD25 + Treg cells by inducing interleukin-2 (IL-2) and interferon-γ (IFN-γ) secretion. The present studies suggested that CsA may block the induction of immune tolerance and decrease the risk of hepatitis C recurrence.

Effects of interferon-gamma and tumour necrosis factor-alpha on the development of cytotoxic T lymphocytes in autologous mixed lymphocyte tumour cultures with human melanoma

We have studied the influence of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the development of cytotoxic T lymphocytes (CTL) against melanoma in mixed lymphocyte tumour cultures (MLTC). In these MLTC, TNF-alpha at 10(4) U/ml increased the expansion of the CTL up to 10(4)-fold over recombinant IL-2 (rIL-2) alone. IFN-gamma at 10(4) U/ml and combinations of TNF-alpha plus IFN-gamma at 10(2)-10(3) U/ml promoted the proliferation more variably. MLTC generated with rIL-2 showed a predominance of CD8+ cells, while 2 weeks of culture in the presence of IFN-gamma at 10(4) U/ml, or with IFN-gamma and TNF alpha at 1 x 10(2)-10(3) U/ml, favoured the emergence of CD4+ cell populations. The cytotoxic activity of the lymphocytes generated in these MLTC showed a consistent decline of K562 cytotoxic activity following exposure to the combination of IFN-gamma and TNF-alpha. Despite the altered T cell subset distribution with different combinations of cytokines, no consistent alteration in the specific anti-tumour cytotoxicity against melanoma was detected. These results suggest that TNF-alpha and IFN-gamma influence the activation, phenotypic, and functional outcome of MLTC-generated CTL, and may account for the phenotypic variations observed in T cell populations generated in vitro.

A Novel Cellular Pathway of SCL T-Leukemogenesis Revealed by a Conditional Transgenic Mouse Model.

The acquired activation of stem cell leukemia (SCL) during T-lymphopoiesis is a common event in T-cell acute lymphoblastic leukemia. Here, we generated a novel tamoxifen-inducible transgenic mouse model (lck-ERT2-SCL) to study the cellular targets of acquired SCL activation during T-cell development. Upon tamoxifen treatment we observed the thymic emergence of abnormal, non-cycling CD8+TCRβlow and immature CD4+CD8+ (double-positive, DP) cells displaying increased viability. Unexpectedly, fetal thymic organ culture analysis of lck-ERT2-SCL thymi revealed the development of DP cells before the emergence of CD8+TCRβlow cells, which implied the derivation of CD8+TCRβlow cells from DPs rather than immature CD8 single-positive (SP) thymocytes. Interestingly, histone deacetylase (HDAC) inhibition with trichostatin A (TSA) had a divergent effect on SCL perturbed thymopoiesis: TSA increased T-cell receptor surface expression within DP and CD8 SP cells however did not alter the CD8 shifted CD4/CD8-ratio. Furthermore, we studied the expression of NOTCH1 in SCL induced TCRβlow thymocytes. Strikingly, we found that SCL induced NOTCH1-upregulation in DP TCRβlow cells. We therefore conclude that SCL promotes the emergence of abnormal CD8+TCRβlow cells by an only partially HDAC dependent mechanism from DP TCRβlow cells. Moreover, SCL induced DP TCRβlow cells are characterized by upregulated NOTCH1, which in turn might promote the effect of acquired NOTCH1 mutations during T-leukemogenesis.

T cells stimulated by CD40L positive leukemic blasts-pulsed dendritic cells meet optimal functional requirements for adoptive T-cell therapy

Adoptive T-cell immunotherapy may provide complementary therapy for childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In this study, we have analyzed the functional characteristics of anti-BCP-ALL effector T cells generated by co-culturing T lymphocytes and dendritic cells (DC) from allogeneic human stem cell transplantation (HSCT) donors. After 21-day co-culture with DC pulsed with CD40L+ apoptotic BCP-ALL blasts, T cells presented with both effector and central memory phenotype, and showed high and specific cytotoxic activity against leukemic cells (average lysis = 77%), mostly mediated by CD8+ T cells. Noticeably, growth of CD4 T cells was maintained (45% of total cells), which actively produced Th1 cytokines (IFN-gamma, TNF-alpha, IL-2), but not IL-4, IL-5 and IL-10. Anti-BCP-ALL T cells expressed CD49d and CXCR4 (implicated in the recruitment to bone marrow), and CD62L and CCR7 (involved in the migration to lymphoid organs). In accordance with this profile, T cells significantly migrated in response to the chemokines CXCL12 and CCL19. In conclusion, stimulation of T cells with CD40L+BCP-ALL cells-loaded DC not only elicited the generation of potent and specific anti-leukemic cytotoxic effectors, but also the differentiation of specific and functional Th-1 CD4 lymphocytes. These effectors are fully equipped to reach leukemia-infiltrated tissues and have characteristics to support and orchestrate the anti-tumor immune-response.

In vitro culture of skin‐homing T lymphocytes from inflammatory skin diseases

We, in this study, describe how T lymphocytes in a skin biopsy can proliferate in vitro for up to 3 months by using T-cell growth factors - interleukin-2 (IL-2) and IL-4 yielding approximately 100-160 million T lymphocytes within 1 month. We established cell lines from three tuberculin skin tests, four positive patch tests, 15 of 16 biopsies from atopic dermatitis (AD), 15 of 19 biopsies from mycosis fungoides (MF), 12 of 24 biopsies from psoriasis vulgaris, which was significantly less than AD (P < 0.05), and with a reduced cumulative number of lymphocytes (P < 0.05). Omitting IL-2 and IL-4 led to immediate halt of proliferation. Blood mononuclear cells from patients and biopsies from healthy persons never gave cell lines. All cells were T lymphocytes expressing CD45RO+, HLA-DR+ and CD150. The CD7 expression was significantly increased in cell lines from AD (P < 0.05). T-cell receptor beta-chain studies by using reverse transcription-polymerase chain reaction showed that all T lymphocytes had access to the skin compartment. Single-stranded conformational analysis showed clonally expanded T cells numbering between 40 and 60 clones. After approximately 2 months of growth, the mean CD4+ : CD8+ ratio was for AD 1.20, MF 0.65 and psoriasis 0.85. Patients with AD treated with cyclosporin-A had almost no growth of CD8+ cells in vitro. Our findings indicate a changed homeostasis among skin-homing lymphocytes for in vitro culture. Our culture system of skin-homing T lymphocytes leads to a prominent cellular expansion allowing for a range of studies of in vivo activated skin T lymphocytes.

Neonatal Autoimmune Disease: Influence of CD4+ CD25+ Regulatory T Cells

Although previous studies have emphasized the tolerogenic property of murine neonatal immune system, recent studies indicate that neonatal mice are prone to autoimmune disease. This chapter will summarize the evidence for neonatal propensity to autoimmune ovarian disease (AOD) and describe the new finding that autoantibody can trigger a T cell–dependent autoimmune disease in neonatal but not adult mice. Based on depletion or addition of the CD4+CD25+ T cells, disease resistance of older mice is explicable by the emergence of CD4+CD25+ regulatory T-cell function after day 5, whereas disease susceptibility is associated with resistance to regulation by CD4+CD25+ T cells.

Monoassociation of SCID mice with helicobacter muridarum, but not four other enterics, provokes IBD upon receipt of T cells

Recently, a number of animal models for different aspects of inflammatory bowel disease (IBD) have been developed. The aim of this study was to use one of these to determine whether particular, ostensibly innocuous, intestinal bacteria could provoke or exacerbate IBD. Conventionally reared C.B17 SCID mice were compared with germ-free and gnotobiotic mice, monoassociated with 1 of 5 intestinal bacteria, after transfer of CD45RB(high) CD4(+) T cells from conventionally reared congenic BALB/c mice. Recipient mice were monitored over 7-12 weeks for clinical signs of IBD, and tissues were analyzed by histology/flow cytometry for abnormal inflammation and CD4(+) T cell outgrowth. Neither germ-free mice nor mice monoassociated with segmented filamentous bacteria, Ochrobactrum anthropi, a nonpathogenic mutant of Listeria monocytogenes, or Morganella morganii developed any signs of IBD. In contrast, mice monoassociated with Helicobacter muridarum displayed an accelerated development of IBD in 5-6 weeks compared with 8-12 weeks observed in conventionally reared mice. The outgrowth of CD4(+) T cells in spleen and large intestine of H. muridarum monoassociated mice, as well as in conventionally reared mice was significantly higher than that in the other monoassociated mice. Among the intestinal bacteria tested, H. muridarum can serve as a provocateur of IBD in this model.