Peripheral Blood CD8 Lymphocytes Research Articles

Membrane (CD8⁺PD-1⁺ and CD4+PD-1⁺) and soluble (sPD-1 and sPD-L1) forms of immune checkpoints in melanoma, breast cancer, and oral mucosal cancer patients: A observational study

Background. The PD-1/PD-L1 pathway plays an important role in tumor evasion from immunological surveillance. In addition to the membrane forms of PD-1 and PD-L1, there are soluble variants: sPD-1 and sPD-L1. Both membrane and soluble forms have immunoregulatory properties and can affect the function and number of different immune cell populations.
 Aim. To study the relationship between the initial level of CD8⁺PD-1⁺ and CD4+PD-1⁺ lymphocytes and soluble forms of sPD-1 and sPD-L1 with the percentage of the main effector and regulatory populations of peripheral blood (PB) lymphocytes and tumor-infiltrating lymphocytes.
 Materials and methods. The study included melanoma, breast cancer and the oral mucosa cancer patients. The percentage of cell populations of PB lymphocytes and tumor-infiltrating lymphocytes was determined by flow cytometry before treatment. The concentrations of sPD-1 and sPD-L1 proteins were studied in blood serum using enzyme immunoassay.
 Results. The relationship of the level of CD8⁺PD-1⁺ cells with certain populations of CD8-lymphocytes in PB and tumor tissue was found. In the PB of melanoma patients with CD8⁺CD11b⁺CD28⁺ and CD8⁺CD11b⁻CD28⁻ T cells, in breast cancer patients with a population of CD8⁺CD11b⁺CD28⁺ lymphocytes. In the tumor tissue of all patients there was a positive correlation with a population of regulatory CD8⁺CD11b⁻CD28⁻ T cells. The immunoregulatory properties of sPD-1 and sPD-L1 were confirmed. Both sPD-1 and sPD-L1 levels were positively correlated with the number of suppressor CD8⁺CD11b⁻CD28⁻ T cells and negatively with the level of CD8 lymphocytes, CD8⁺CD11b⁺CD28⁺ cytotoxic/memory T cells, B cells and activated CD25 lymphocytes.
 Conclusion. The results of the study can make a certain contribution to the study of the prognostic significance of membrane and soluble forms of PD-1 and PD-L1, taking into account the peculiarities of their relationship with suppressor and effector populations of lymphocytes of systemic and local immunity.

PD36 ‐ Evaluation of intracellular cytokines IL‐2, IFNγ, IL‐4 and IL‐5 in children with atopic dermatitis and correlations with other immunological and epidemiological parameters

Atopic dermatitis is a chronic inflammatory disorder of the skin, charachterised by a TH2 skewed cytokine profile, usually elevated total serum IgE and can be co coexpressed with other atopic clinical manifestations. The atopic skin is prone to bacterial colonization and that has an effect on immunological parameters. Atopic Dermatitis has been correlated with IgG hypoglobulinaemia, low serum IgA and downregulation of the CD8 lymphocytes in peripheral blood.

Detection of Circulating Antigen-Specific CD8+ Lymphocytes against Leukemia Associated Antigens WT1, PR1 and Survivin Using HLA 0201 Pentamers After Allogeneic Stem Cell Transplantation.

Abstract 5115 INTRODUCTIONAllogeneic stem cell transplantation (allo-SCT) is a potentially curative treatment option for patients with hematological disorders. Alloreactive donor-derived T lymphocytes exert a beneficial graft-versus-leukemia (GVL) effect through the recognition of leukemia-restricted (or preferentially expressed) antigens as Wilms tumor protein (WT1), survivin (SURV) or proteinase (PR1). Currently research in transplant immunology focuses in enhancing GVL while preventing the deleterious graft-versus-host disease (GVHD) that could be achieved by manipulating donor-derived antigen-specific T-populations. In this study we tested the presence of peripheral blood leukemia-associated antigen-specific CD8+ T-lymphocytes during post allo-SCT follow-up. PATIENTS AND METHODSForty-three consecutive HLA*0201 patients (homo or heterozygotous) undergoing conventional myeloablative (n=24) or non-myeloablative (n=19) allo-SCT as treatment of hematological disorders were included. Allogeneic donor was an HLA-identical sibling in 26 cases (60.5%) and unrelated in 17 cases (39.5%). Hematopoietic stem cell source included mobilized peripheral blood (n=20), bone marrow (n=18) and umbilical cord blood (n=5). As GVHD prophylaxis regimens Cyclosporine plus Methotrexate (n=20) or Cyclosporine plus Mofetil micofenolate (n=23) were employed. In addition, 22 patients received rabbit antithymocyte globulin at 6-8mg/kg. At last follow-up four patients had relapsed 9-14 months after allo-SCT. We sought for leukemia-antigen specific CD8 lymphocytes in peripheral blood samples drawn within a median of 7 months (range 2-38) when lymphocyte recovery had occurred and complete donor chimerism was achieved. We used four color multiparametric flow cytometry in a FACSCanto II acquiring at least 5 ×105 viable (Propidium Iodide low) lymphoid gated events, stained with MnAbs: CD8-FITC and CD3PE/APC MnAb. To identify leukemia-antigen specific CD8 lymphocytes we used class I HLA pentamers 0201 APC or PE conjugated (Proimmune, London, UK) against the following nonapeptides: Proteinase 1: VLQELNVTV (169-177) WT1: RMFPNAPYL (126-134) and SURV: ELTLGEFLKL (95-104). As positive staining control we used CMV pp65: NLVPMVATV (495-503) and as negative controls we used irrelevant nonapeptide and peripheral blood samples from patients lacking HLA* 0201 genotype. RESULTSDetection of donor-derived CD8+ lymphocytes against CMV pp65 occurred in 61% of recruited patients with a median percentage of 0.1% (range 0.03-13 over CD3+CD8+ events). Likewise, it was possible to detect CD8+ lymphocytes specific for PR1, WT1 and SURV in 65.2%, 47.8% y 39.1% of recruited patients respectively. Median percentage of PR1 and WT1 leukemia-antigen specific lymphocytes was 0.1% (range 0.04-1% over CD3+CD8+ events) and for WT1 0.1% (range: 0.01-0.2%). Detection of leukemia-antigen specific CD8+ lymphocytes was not significantly associated with clinical variables such as conditioning regimen (conventional or non-myeloablative), Disease status at transplant, donor type (sibling or unrelated), ATG use or HLA-disparity degree. The presence of WT1 specific CD8+ lymphocytes was significantly more frequent in patients undergoing allo-SCT for lymphoid hematological malignancy (p=0.04). By contrast, the presence of circulating anti-PR1 specific CD8+ lymphocytes was not more frequently found in patients undergoing allo-SCT for myeloid malignancies. Of note, none of the four patients who eventually relapsed harbored circulating leukemia-specific CD8+ lymphocytes. CONCLUSIONSMultiparametric flow cytometry is a useful tool to detect and quantify rare donor-derived CD8+ lymphocytes specific for leukemia-associated antigens as PR1, WT1 or SURV. The presence of these populations in peripheral blood is not associated to conventional clinical variables and in our series anti-WT1 CD8+ lymphocytes were more frequently detected in patients receiving allo-SCT for lymphoid malingnacies. By contrast, larger series are needed to assess if the lack of these leukemia-associated antigen-specific CD8 lymphocytes in peripheral blood could identify patients in a higher risk of relapse. Financial supportThis study was supported by a grant of Conserjeria de Salud, Junta de Andalucia 2006/0355. J. Serrano López is a post-doc fellow from Fundación Española de Hematología y Hemoterapia. DisclosuresNo relevant conflicts of interest to declare.

191 Anti-FasL treatment preserves SIV-specific memory and slows progression to AIDS in rhesus macaques

Previously we reported an increased survival advantage in SIV-infected rhesus macaques after injection with a FasL-blocking monoclonal antibody (Salvato et al., Clin Dev Immunol. 2007: 93462). In the present study we analyzed the effect of such FasL blocking on immune parameters and disease progression in a cohort of SIV-infected rhesus macaques. Upon infection, various immune parameters in the blood developed highly significant differences between animals given anti-FasL RNOK203 antibody and those given an isotype control antibody. The anti-FasL treated animals had a clear preservation of central memory CD4 lymphocytes starting from acute infection through at least set point viremia. The total memory CD4 lymphocytes in these animals were also significantly higher throughout the 25-week study period. Anti-FasL treatment resulted in significantly higher percentages of memory CD8 lymphocytes in peripheral blood. Besides these changes in the memory lymphocyte compartment, the treated animals had a higher response to infection as evidenced by increased turnover of CD4 and CD8 lymphocytes expressing Ki67. Importantly, the antigen-specific cellular immune responses were significantly higher at 4, 6 and 17 weeks after infection in anti-FasL treated animals. Preservation of memory lymphocytes and cellular immunity are considered the main correlates of protection in SIV infection. Our study demonstrates that FasL is a major contributor towards depletion of anti-SIV immunity and that blocking this pathway is a beneficial strategy that should be considered for vaccine formulations.

A substype analyse of peripheral blood CD8 lymphocyte in patients with chronic hepatitis virus B

目的 探讨慢性乙型肝炎患者外周血CD8淋巴细胞亚型与其临床状态及HBV复制的关系.方法采用流式细胞技术多色荧光分析法,检测了18名健康体检者和107例慢性乙型肝炎患者外周血CD8淋巴细胞CD45RO、CD45RA和CD28的表达及病毒载量检测.结果慢性病毒携带、慢性活动性肝炎患者CD8淋巴细胞与正常对照比较均无明显差别.慢性活动性肝炎组CD3+CD8+/CD45R0+淋巴细胞百分率为(15.7±3.35)%,明显高于正常对照(P＜0.05),其CD3+CD8+/CD45RA+淋巴细胞百分率为(12.3±4.39)%,明显低于正常对照(P＜0.05).慢性活动性肝炎组CD3+CD8+CD28+细胞百分率为(23.7±3.35)%,明显高于正常对照(P＜0.05),而慢性病毒携带组CD3+CD8+CD28-细胞百分率为(16.3±4.37)%,明显高于正常对照(P＜0.05).高病毒载量与低病毒载量患者的CD3+CD8+/CD45R0+亚型、CD3+CD8+/CD45RA+亚型均无明显差别,CD3+CD8+CD28+亚型和CD3+CD8+CD28-亚型亦无明显差别.结论慢性乙型肝炎患者病情的进展与CD8淋巴细胞总量无关;可能是由于机体免疫功能增强,CD3+CD8+CD45RO+亚型、CD3+CD8+CD28+亚型淋巴细胞数升高,诱发细胞毒性淋巴细胞(CTL)作用的结果.但血清中病毒载量的变化与外周血中CD8淋巴细胞亚型无明显相关性。

Role of peripheral CD8 lymphocytes and soluble IL-2 receptor in predicting the duration of corticosteroid treatment in polymyalgia rheumatica and giant cell arteritis.

To determine if the presence of low percentages of CD8 positive cells or high levels of soluble interleukin-2 receptors (sIL-2R) define a subgroup of patients with more severe polymyalgia rheumatica and giant cell arteritis (PMR/GCA). 38 PMR/GCA patients were followed up prospectively. Serum levels of sIL-2R and peripheral blood CD8 lymphocytes were measured before the start of corticosteroid treatment, after six months of treatment and at the last visit. Phenotypical analysis of lymphocyte subpopulations was performed with a two colour technique, and assay of sIL-2R was performed using an enzyme-linked immunosorbent kit. Forty four healthy people matched for age and gender comprised a healthy control group. The median duration of follow up was 28 months (range 7-65). Corticosteroid treatment lasted a median of 23.5 months (7-65). Eleven patients (29%) were in remission at the end of follow up; 45% of the patients had at least one relapse or recurrence. Compared with controls, patients with active disease had a significantly lower percentage of CD8 cells and significantly increased sIL-2R levels. Erythrocyte sedimentation rate, C reactive protein, and sIL-2R values were significantly less after six months of steroid treatment compared with before treatment. The percentage of CD8 cells remained significantly lower at six months and the end of follow up compared with controls, while sIL-2R levels remained significantly greater. Patients in whom the percentage of CD8 cells at six months was lower than one SD of the mean of normal controls (26%) had a significantly longer duration of corticosteroid treatment, a greater cumulative dose of prednisone and more relapses or recurrences compared with patients in whom the percentage was in the normal range. The duration of treatment and the cumulative dose of prednisone were not influenced by the percentage of CD8 cells before treatment therapy or by the levels of sIL-2R after six months of treatment. A reduced percentage of CD8 cells after six months of treatment may be a useful outcome parameter which would identify a group of PMR/GCA patients likely to experience more severe disease, defined as longer duration of corticosteroid treatment, higher cumulative dose of prednisone, and relapse or recurrence of disease.