CD137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adoptive immunotherapy

The efficient isolation and ex vivo expansion of antigen-specific T cells are crucial for successful adoptive immunotherapy against uncontrollable infections and cancers. Several methods have been reported for this purpose, for example, employing MHC-multimeric complexes, interferon-gamma secretion, and antibodies specific for molecules expressed on T-cell surfaces, including CD25, CD69, CD107a, CD137, and CD154. Of the latter, CD137 has been shown to be one of the most promising targets since it is only expressed on CD8⁺ T cells early after encountering antigen, while being almost undetectable on resting cells. However, detailed comparisons between CD137-based and other methods have not yet been conducted. In this study, we therefore compared three approaches (with CD137, CD107a, and tetramers) using HLA-A24-restricted CMV pp65 and EBV BRLF1 epitopes as model antigens. We found that the CD137-based isolation of antigen-stimulated CD8⁺ T cells was comparable to tetramer-based sorting in terms of purity and superior to the other two methods in terms of subsequent cell expansion. The method was less applicable to CD4⁺ T cells since their CD137 upregulation is not sufficiently high. Collectively, this approach is most likely to be optimal among the methods tested for the isolation and expansion of antigen-specific CD8⁺ cells. K.W. and S.T. are employees of Medical Biological Laboratories Co., Ltd. S.S. is a representative executive of T Cell Technologies, Inc. Y.A. has received financial support through collaboration with Medical Biological Laboratories Co., Ltd.     

Fas/APO-1 (CD95)-mediated cytotoxicity is responsible for the apoptotic cell death of leukaemic cells induced by interleukin-2-activated T cells

Apoptotic cell death is induced by the cross-linking of Fas/APO-1 receptor (CD95) in acute myelogenous leukaemia (AML) cells. Since CD95 ligand (CD95L) is expressed on interleukin-2 (IL-2)-activated T cells, we investigated the involvement of CD95-CD95L pathway in T cell-mediated cytotoxicity against AML cells. Activated CD8⁺ T cells could efficiently kill a parental CD95-sensitive AML cell line, MML-1 and, to a lesser extent, a CD95-resistant clone, MML-1R. Neither MML-1 nor MML-1R cells were killed by activated CD4⁺ T cells. The blocking monoclonal antibody (MoAb) against CD95, ZB4, caused a significant inhibition of T-cell-mediated cytotoxicity against MML-1 cells, but not against MML-1R cells. Interestingly, MML-1 cells underwent the classic nuclear morphologic changes and DNA fragmentation characteristic of apoptosis when cultured with activated T cells. Enumeration of apoptotic and necrotic nuclei showed that both apoptosis and necrosis were induced in MML-1 cells, whereas necrosis was exclusively observed in MML-1R cells. Apoptosis of MML-1 cells was completely blocked in the presence of ZB4 MoAb. Similarly, blocking by ZB4 MoAb significantly inhibited T-cell-mediated lysis of fresh AML cells in a CD95-sensitive group, but not in a CD95-refractory group. In addition CD8⁺ T cells expressed CD95L mRNA more abundantly than CD4⁺ T cells upon activation by IL-2. These findings suggest that T-cell-mediated cytotoxicity against AML cells requires participation of CD95-CD95L pathway for cytotoxic signal transduction leading to target apoptosis.     

Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti–cytotoxic T-lymphocyte–associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti–DEC-205 (dendritic and epithelial cells, 205 kDa)–HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival.Promoting a robust CD8 T-cell response is vital for the generation of an effective antitumor immune response. However, immunosuppressive mechanisms used by the tumor result in the exhaustion of tumor-infiltrating lymphocytes (TIL), leading to cytotoxic T-cell anergy and dysfunction. To overcome this dysfunction, investigators have had considerable success using immune checkpoint inhibitors, such as aCTLA-4 (cytotoxic T-lymphocyte–associated protein 4) mAbs, to promote T-cell function. CTLA-4, a negative regulatory molecule on the surface of T cells, is indispensable for preventing the expansion and activation of autoreactive T cells. Inhibition of this surface receptor using antagonist aCTLA-4 mAb–augmented effector CD4 and CD8 T-cell responses and inhibited or reduced the suppressive function of Treg cells (1–5). However, only a subset of patients treated with aCTLA-4 mAb exhibit an objective clinical response (6).Checkpoint blockade targets T-cell coinhibitory molecules, but other strategies targeting costimulatory molecules, such as the TNF receptor family member OX40 (CD134), have demonstrated success in promoting tumor regression in a wide variety of preclinical models. Treatment with an agonist aOX40 mAb directly stimulated CD4 and CD8 T cells and induced their expansion, differentiation, and up-regulation of prosurvival molecules (7–12). Moreover, OX40 ligation promoted the generation of long-lived memory CD8 T cells and enhanced their function. Recent data indicate that combined aOX40/aCTLA-4 therapy induced a robust effector CD4 and CD8 T-cell response necessary for tumor regression and significantly improved the survival of tumor-bearing hosts relative to therapy with either agent alone (13). Despite this success, the response to combination aOX40/aCTLA-4 treatment was greatly diminished in more established tumors. This therapy may be unable to overcome T-cell anergy in more established immunosuppressive tumor microenvironments, possibly because it is ineffective at specifically targeting and expanding tumor-reactive T cells and relies on limited or defective endogenous priming by dendritic cells (14). Currently, clinically tested vaccines targeting cross-presenting dendritic cells [i.e., anti–DEC-205 (dendritic and epithelial cells, 205 kDa) mAb conjugated to tumor antigens] have demonstrated promise by priming a more robust cytotoxic T-cell response (15–19). The possibility remains that increased Th2-cytokine production by CD4 T cells following combination therapy may reduce its therapeutic efficacy, because inhibition of IL-4 with aOX40/aCTLA-4 treatment significantly improved survival (13). Others have shown that a dominant Th2 cytokine response reduced the efficacy of treatment, whereas a Th1-skewed immune response resulted in more favorable outcomes (20–23).We hypothesized that dendritic cell-targeted vaccination against a tumor-associated antigen in conjunction with combination aOX40/aCTLA-4 mAb immunotherapy would be sufficient to promote a cytotoxic antitumor T-cell response, redirect a Th2 bias in CD4 T cells, and improve survival in mice with established tumors. Here, we report that vaccination using anti–DEC-205/HER2 (human epidermal growth factor receptor 2) mAb along with combination aOX40/aCTLA-4 mAbs significantly expanded effector CD8 T cells, resulting in a more favorable Th1 cytokine profile and inducing a critical accumulation of effector T cells in the tumor, with increased tumor-free survival relative to either therapy alone. Moreover, combination therapy with vaccination reversed T-cell anergy and promoted a robust effector T-cell response to a tumor-associated antigen in a spontaneous adenocarcinoma model.     

Co-expression of CD30 ligand and interleukin 4 (IL-4) receptors by acute myeloid leukaemia blasts is associated with the expansion of IL-4-producing CD30+ normal T cells

CD30 ligand (CD30L), but not its cognate receptor CD30, is frequently expressed on acute myeloid leukaemia (AML) blasts. In the present study, we found that leukaemic blasts presenting surface CD30L displayed a characteristic cytokine-receptor pattern that makes them ideal targets for those cytokines usually produced by Th2-type cell subsets. In particular, even though a broad distribution of Th2 cytokine receptors by AML blasts was shown, we demonstrated the almost exclusive expression of interleukin 4 (IL-4) receptor (R), in the absence of its cognate cytokine, by CD30L+ AML. Furthermore, a number of Th2-associated markers, including CD30, IL-4 and GATA-3, were expressed by residual T cells derived from CD30L+ AML but not from CD30L- AML, in which the presence of the Th1-associated marker LAG-3 was documented in some cases. The production of IL-4 in the absence of interferon gamma (IFN-gamma) was also detected in CD3+/CD30+ T cells from CD30L+ AML. These results, along with the shift toward IL-4-producing specific T-cell clones observed in CD30L+ AML samples by enzyme-linked Immunospot (ELISpot) assay, were consistent with the hypothesis of a Th2 polarization taking place in T cells from CD30L+ AML. The notion that IL-4 was able to enhance in vitro proliferation of CD30L+/IL-4R+ purified leukaemic blasts suggests that the selective interaction of IL-4-producing CD30+ T cells with CD30L+ leukaemic progenitors may have a role in the progression of this particular AML subset.     

Evaluation of natural killer and lymphokine-activated killer (LAK) cell activity in vivo in patients treated with high-dose interleukin-2 and adoptive transfer of autologous LAK cells

Summary This study investigated the development of peripheral blood natural killer (NK) and lymphokineactivated killer (LAK) cell activity in vivo in cancer patients treated with high doses of recombinant interleukin-2 and autologous LAK cell infusion. It was found that interleukin-2 administration by bolus injection resulted in an early but transient rise of NK and LAK cell activity in vivo during the first 5–9 days of treatment (postpriming period), whereas continuous infusion of interleukin-2 caused an increase in both cytotoxic activities in the second phase of the treatment, concomitant with infusions of autologous LAK cells. Elevated NK but not LAK cell activity in vivo in the continuous-infusion patients persisted up to 180 days after completion of therapy. In both bolus and continuous interleukin-2 protocols, augmented NK cell activity in vivo appeared to be correlated with a beneficial response to therapy.Supported by the US-Poland Cancer Program (NCI) and by NCI Contract NO1-CM-73705     

血脂康胶囊对老年颈动脉粥样硬化患者CIMT、血脂、CD40/CD40L、MMP-9的影响

目的 观察老年颈动脉粥样硬化(CAS)患者行血脂康胶囊治疗前后的颈动脉内膜中层厚度(CIMT)、血脂、白细胞分化抗原40及其配体(CD40/CD40L)和基质金属蛋白酶9(MMP-9)变化,探讨血脂康胶囊对患者上述指标的影响.方法 选择CAS患者60例(GAS组),其中合并脑梗死(CI)34例;健康体检者20例(对照组).CAS组用血脂康胶囊治疗24周,治疗前后行颈CIMT检测,采用ELISA法检测血清CD40/CD40L和MMP-9,并行血脂检测.对照组在查体时检测上述指标.结果 与对照组比较,CAS组CIMT及血清CD40、CD40L、MMP-9明显升高(P＜0.05或＜0.01);CAS合并CI患者的CIMT及血清CD40、CD40L、MMP-9较无CI患者明显升高,CAS组治疗前后的CIMT及血清CD40、CD40L、MMP-9比较有统计学差异(P均＜0.05).结论 血脂康胶囊可通过调节血脂水平,降低CAS患者的CIMT及血清CD40、CD40L、MMP-9,从而延缓CAS进展,减少CI发生率.     

CD154, a marker of antigen-specific stimulation of CD4 T cells, is associated with response to treatment in patients with chronic HCV infection

Summary. CD4 T‐cell function is crucial for the eradication of HCV, and insufficient function is observed in chronic carriers. The monitoring of T‐cell responses is complicated by the scarcity of antigen‐specific T cells and the relative inefficiency of virus‐specific T cells to produce effector cytokines. CD154 is a marker of activation expressed on T cells induced through their T‐cell receptor. We analysed CD4 T‐cell responses in 72 patients with chronic or resolved HCV infection (23 treatment naïve, 49 treatment experienced, including 16 who had achieved a sustained response). In an additional prospective protocol, 20 of the chronically infected patients were analysed before and after 8–12 weeks of combination therapy with peg‐interferon‐α and ribavirin. T‐cell responses were measured by detecting the expression of CD154 and Th1 cytokines after stimulation with recombinant HCV proteins and were correlated with pretreatment status and outcome of therapy. Broader T‐cell responses were observed in treatment naïve than in experienced patients, while the outcome of a preceding therapy regimen did not influence T‐cell responses. In the prospective cohort, an on‐treatment increase in CD154+ cytokine? T‐cell activity was associated with response to treatment, while a decrease was observed in nonresponders. Stronger antigen‐independent activity of CD154+ cytokine+ T cells was observed in responders than in nonresponders. Our data indicate that CD154 as a marker of activation of CD4 T cells is a suitable tool for the analysis of T‐cell responses in patients with HCV infection.     

B cells CD19+ in patients with B‐cell chronic lymphocytic leukaemia and autoimmune haemolytic anaemia

The study presents results of B and T lymphocytes population analysis in patients with chronic lymphocytic leukaemia B cells and autoimmune haemolytic anaemia (CLL‐B + AIHA). We evaluated the following groups of patients: (1) with newly recognized CLL‐B and co‐existent AIHA (untreated), (2) after short‐term treatment with corticosteroids, (3) after treatment with chemotherapy and corticosteroids. The control groups were made of patients with CLL‐B without AIHA. The populations of lymphocytes and determination of cells immunophenotype were performed by means of flow cytometry. The analysed data were obtained from 25 patients. The untreated patients with CLL‐B + AIHA presented significantly more numerous population of neoplastic cells CD19+ CD5+ in comparison with patients without AIHA. The patients with AIHA showed a reduced percentage of B CD19+ CD22+ cells in comparison with those without AIHA. Untreated patients with AIHA or after a short‐term corticosteroid treatment showed a higher ratio of the number of CD19+ CD5+ cells to the number of T CD4+ and T CD8+ lymphocytes than CLL‐B patients without AIHA. It can be presumed that the differences found may be related to the pathogenesis of the autoimmune haemolysis syndrome in patients with CLL‐B.     

Susceptibility to opportunistic infections in HIV-infected patients with increased CD4 T-cell counts on antiretroviral therapy may be predicted by markers of dysfunctional effector memory CD4 T cells and B cells

OBJECTIVES: HIV-infected patients responding to combination antiretroviral therapy (ART) after experiencing severe immunodeficiency may exhibit persistent immune defects and occasionally experience opportunistic infections (OIs) despite increased CD4 T-cell counts. The investigation of immune defects in such patients was examined in this study. METHODS: CD4 effector memory T-cell (T(em)-cell) function [assessed by blood cytomegalovirus (CMV) interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) counts] and B-cell dysregulation [assessed by serum immunoglobulin A (IgA) and IgE levels] were examined in 27 patients with increased CD4 T-cell counts after receiving ART for over 2 years. Two of these patients and one other had developed OIs on ART and are described in detail. RESULTS: Serum levels of IgA and IgE were higher than reference intervals (P<0.001) and CMV IFN-gamma ELISPOT counts were lower than those in non-HIV-infected controls (P<0.001) in the HIV-infected patients. Low CMV IFN-gamma ELISPOT counts were associated with high IgA levels (r=-0.5, P=0.01, Spearman's correlation test) and segregated with high IgE levels (P=0.06, Fisher's test). CMV IFN-gamma ELISPOT counts and serum IgA and IgE levels did not change significantly over a median time of 35 (range 8-60) months after the first measurement, whereas CD4 T-cell counts increased. All three patients who experienced OIs had repeatedly low CMV IFN-gamma ELISPOT counts and increased serum levels of IgA and/or IgE. CONCLUSION: Low CD4 T(em)-cell function and B-cell dysregulation are immune defects that may persist independently of changes in the CD4 T-cell count in HIV-1-infected patients responding to ART and are associated with an increased risk of developing an OI.     

Induction of CD4+CD8+ double positive T cells and increase in CD5+ B cells in efferent lymph in sheep infected with Trypanosoma evansi

The effects of Trypanosoma evansi on efferent lymphocyte phenotypes draining from a lymph node primed with Pasteurella haemolytica vaccine were studied in sheep. The prefemoral efferent lymphatic ducts of the infected sheep along with those of two uninfected sheep were surgically cannulated. Lymph was collected and lymphocytes recovered from it analysed by two-colour indirect immunofluorescence staining and cytofluoremetry in a fluorescence activated cell analyser (FACSCAN). The study showed the appearance and persistence of T. evansi in the efferent lymph for a long period of time and the appearance of CD4⁺CD8⁺ (double positive, DP) T lymphocytes in the efferent lymph of infected animals. The infection also resulted in increases in CD5⁺ B cells in the prefemoral efferent lymph. In addition, there were decreases in the output of conventional B cells, CD5⁺ and CD4⁺ T cell subsets but large increases in CD8⁺ cells followed by terminal depletion of all cell subsets. In contrast, inoculation of sheep with pasteurella vaccine antigen alone produced little alterations in the proportions, but large increases in the numbers of all T cell subsets except that of CD8⁺ cells which also showed little variation; and there was a concurrent increase in the numbers and proportions of efferent B cells. In addition, the abnormal expression of DP and CD5⁺ B cells did not occur in the uninfected vaccinated sheep. It is concluded that these abnormal changes in the kinetics of efferent lymphocyte phenotypes are likely to play a role in the genesis of the generalized immunosuppression seen in trypanosome-infected hosts.     

Increased regulatory T cells and impaired functions of circulating CD8 T lymphocytes is associated with viral persistence in Hepatitis B virus‐positive newborns

Hepatitis B Virus (HBV) infection in infancy or early childhood leads to high rate of persistent infection (25–90%). The immunological basis of high rate of viral persistence in vertically acquired HBV infections is not completely understood. CD8 T cells play a pivotal role in clearing the Hepatitis B virus infection in adults. Herein, we sought to delineate the role of T cells in viral persistence in HBsAg+ve newborns . At birth peripheral and cord blood of HBsAg+ve (N = 12), HBsAg‐ve (N = 10) and healthy newborns (HC: N = 15) were evaluated for T‐cell frequency and functionality by flow cytometry. No significant differences were observed in the frequency of CD8 and CD4 T cells in all the three groups. However, significantly higher frequency of FoxP3 expressing regulatory T cells were observed in HBsAg+ve (63.79%) compared with HBsAg‐ve (28.12%) and HC (11.06%) (P < 0.05). Moreover, HBsAg+ve newborns showed functional defect in CD8 T cells by decreased IFN‐γ production and lower CD107A expression (cytotoxic capacity) compared with HBsAg‐ve and HC, which positively correlated with decreased TCRζ‐chain expression CD8 T cells (r² > 0.93, P < 0.05). Despite equal frequency of CD8 T cells in all the three groups, CD8 T cells in HBsAg+ve newborns are dysfunctional. An expansion of regulatory T cells and impaired TCR signalling may represent the immune tolerant state of the adaptive immune system in response to chronic HBV infection.     

Possible involvement of allogeneic antigens recognised by donor-derived CD4 cytotoxic T cells in selective GVL effects after stem cell transplantation of patients with haematological malignancy

Cytotoxic T lymphocyte (CTL) lines specific for allogeneic antigens were generated by in vitro stimulation of donor-derived peripheral blood mononuclear cells obtained from patients who received human leucocyte antigen (HLA)-matched allogeneic haematopoietic stem cell transplantation (HSCT). One of the allogeneic antigen-specific CD4+ CTL lines, CTL-A, generated from a patient with T cell acute lymphoblastic leukaemia, recognised HLA-DPB1*0501-positive Epstein-Barr virus-immortalised human B cell line (EBV-B cells), phytohaemagglutinin blasts and leukaemia cells, but not interferon-gamma (IFN-gamma) treated HLA-DPB1*0501-positive fibroblasts, indicating that this CD4+ T-cell line recognised a minor histocompatibility antigen (mHa) that is preferentially expressed in haematopoietic cells in an HLA-DPB1*0501-restricted manner. The other CD4+ CTL line, CTL-B, generated from a patient with chronic myeloid leukaemia, recognised mismatched HLA-DQB1*0303 on EBV-B cells and phytohaemagglutinin (PHA) blasts. Interestingly, this CTL line did not recognise IFN-gamma-treated recipient's skin fibroblasts, as HLA-DQ was merely upregulated even after IFN-gamma stimulation in non-haematopoietic cells including fibroblasts, endothelial cells and hepatocytes. These results suggest that these CD4 positive CTLs, specific for mismatch HLA-DQ and mHa that are preferentially expressed on haematopoietic cells, may play an important role in induction of selective graft-versus-leukaemia effect without development of graft-versus-host disease after allogeneic HSCT.     

Selection and transplantation of autologous CD34+ B-lineage negative cells in advanced-phase multiple myeloma patients: a pilot study

The feasibility of sequential positive and negative selection of mobilized CD34+ B-lineage negative cells to achieve tumour-free autografts in multiple myeloma (MM) patients was evaluated. Peripheral blood stem cells (PBSC) of 14 patients with advanced disease were mobilized. CD34+ cells were enriched in 12 of the patients by the avidin-biotin immunoabsorption technique. Subsequently, CD10+, CD19+, CD20+ and CD56+ cells (B-lin cells) were removed by immunomagnetic depletion. Minimal residual disease (MRD) was detected by flow cytometry and PCR-based molecular analysis of the patient specific IgH complementary-determining region III (CDRIII). Positive selection of stem cells produced a median recovery of 54.7% of the initial content of CD34+ cells (median purity 71.9%). Negative depletion of B-lineage cells reduced the number of CD34+ cells to 33.3% of the baseline value (median purity 72.7%). However, long-term culture assays showed the recovery of >60% of primitive haemopoietic progenitor cells after depletion of the B-lineage-positive cells. All evaluable patients had detectable disease in PBSC collections. The first step of positive selection of CD34+ cells resulted in >2 logs of tumour cell purging. However, molecular assessment showed the persistence of the disease in 6/7 cases. Immunofluorescence analysis demonstrated 1 additional log of B-cell purging by negative depletion. More importantly, molecular evaluation of IgH CDRIII region showed the disappearance of myeloma cells in 6/7 patients. 12 patients received a median of 3.9 x 106 CD34+ B-lin- cells/kg after conditioning with high-dose melphalan and showed a rapid reconstitution of haemopoiesis. These results were similar to two similar cohorts of patients who received either unmanipulated PBSC or positively selected CD34+ cells after the same conditioning regimen. Severe extrahaematological toxicity was limited to mucositis; no late infections were observed. We concluded that autotransplantation of purified CD34+ B-lin- cells was associated with a rapid and sustained recovery of haemopoiesis and low peritransplant morbidity. Sequential positive and negative enrichment of stem cells reduced tumour cell contamination in B-cell malignancies below the lower limit of detection of molecular analysis.     

Generation and administration of HA-1-specific T-cell lines for the treatment of patients with relapsed leukemia after allogeneic stem cell transplantation: a pilot study

Since HA-1-specific T cells have been shown to make a significant contribution to the clinical responses in patients with relapsed leukemia, we investigated the feasibility of adoptive transfer of in vitro induced HA-1-specific CD8 positive T cells to patients with relapsed leukemia after allogeneic stem cell transplantation. The in vitro generation of clinical grade HA-1-specific T-cell lines from HA-1 negative donors was seen to be feasible and 3 patients were treated with HA-1-specific T-cell lines. No toxicity after infusion was observed. Although in one patient, during a period of stable disease, HA-1-specific T cells could be detected in the peripheral blood and bone marrow, these patients had no clear clinical response.     

Selective ex vivo expansion of cytomegalovirus-specific CD4+ and CD8+ T lymphocytes using dendritic cells pulsed with a human leucocyte antigen A*0201-restricted peptide

Adoptive transfer of ex vivo-generated cytomegalovirus (CMV)-specific T lymphocytes may be effective in preventing CMV disease in allogeneic haematopoietic stem cell transplantation (HSCT) recipients. We developed a procedure for expansion of CMV-specific T lymphocytes based on the antigen-presenting function of donor dendritic cells (DCs), pulsed with a human leucocyte antigen A*0201-restricted pp65 nonamer peptide. CMV-specific T lymphocytes were identified following induction of interferon gamma (IFN-gamma) secretion prompted by peptide exposure. Both CD8+ and CD4+ CMV-specific T lymphocytes were selectively produced in these cultures and showed CMV-restricted cytotoxicity. The simultaneous and selective expansion of CD4+ and CD8+ CMV-specific lymphocytes might be instrumental for more efficient in vivo function of infused CMV-specific lymphocytes.     

胰岛素抵抗的慢性丙型肝炎患者外周血CD4+CD25+调节性T细胞的数量及其功能

目的 了解胰岛素抵抗的慢性丙型肝炎患者外周血CD4 +CD25+调节性T细胞(Treg)数量和功能的变化.方法 筛选40例HLA-A2+慢性丙型肝炎患者(其中20例合并胰岛素抵抗),流式细胞仪检测患者CD4+CD25+Treg细胞占外周血中CD4+T细胞的频率,液闪计数仪检测对HCV特异性CD8+T细胞增殖的抑制作用,ELISA法检测IFN-y水平.统计学处理采用t检验.结果 胰岛素抵抗的慢性丙型肝炎患者外周血CD4 +CD25+ Treg细胞占CD4+T细胞的(9.5±1.9)％,明显低于慢性丙型肝炎患者的(11.2±2.2)％(t=2.615,P＜0.05).胰岛素抵抗指数(HOMA-IR)≥4患者的CD4+CD25+ Treg细胞比例为(9.0±1.8)％,明显低于HOMA-IR＜4患者的(10.8±2.3)％(t=2.413,P＜0.05).胰岛素抵抗的慢性丙型肝炎患者CD4+CD25+ Treg细胞和去除Treg的外周血单个核细胞(PBMC)共培养上清液中IFN-y为(4 050±580) pg/mL,明显高于慢性丙型肝炎患者的(2 005±330)pg/mL(t=13.705,P＜0.01).HOMA-IR≥4患者IFN-y为(5 682±986)pg/mL,明显高于HOMA-IR＜4患者的(2 819±660) pg/mL(t=7.630,P＜0.01).结论 随着胰岛素抵抗程度加重,慢性丙型肝炎患者外周血CD4+ CD25+ Treg细胞频率减低,对HCV特异性CD8+T细胞增殖的抑制作用减弱.     

Low percentages of regulatory T cells in common variable immunodeficiency (CVID) patients with autoimmune diseases and its association with increased numbers of CD4+CD45RO+ T and CD21low B cells

BackgroundCommon variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies defined by marked reductions in serum IgG, IgA and/or IgM levels and recurrent bacterial infections. Some patients are associated with defects in T cells and regulatory T cells (Tregs), resulting in recurrent viral infections and early-onset autoimmune disease.MethodsWe analyzed whether there is an association between Tregs cells (CD4+CD25+CD127^low and CD4+CD25+FoxP3+); memory T cells (CD4+CD45RO+); memory B cells (CD19+CD27-IgD-); and CD21^low B cells (CD19+CD38^lowCD21^low); as well as autoimmune manifestations in 36 patients with CVID (25 women and 11 men, mean age 24 years), all by flow cytometry.ResultsFourteen patients presented with autoimmune diseases (AI) (39%), including 11 with autoimmune thrombocytopenia (ITP) (31%); two with vitiligo (6%); one with systemic lupus erythematosus (LES) (3%); and one with multiple sclerosis (MS) (3%). CVID patients with AI had a reduced proportion of Tregs (both CD4+CD25+CD127^low and FoxP3+ cells) compared with healthy controls. CVID patients with AI had expanded CD21^low B cell populations compared with patients who did not have AI. A correlation between increased CD4+CD45RO T cell populations and reduced Tregs was also observed.ConclusionsOur results showed that 39% of patients with CVID had AI and reduced Tregs populations. Research in this area might provide noteworthy data to better understand immune dysfunction and dysregulation related to CVID.