Comparative Analysis of Syngeneic and Allogeneic Mixed Lymphocyte Reaction of ''Naturally'' Activated and Resting T Cells

'Naturally' activated (NA) or resting T lymphocytes obtained from the spleen of normal BALB/c mice were compared in their capacity to mount a syngeneic mixed lymphocyte reaction (SMLR). Both T-cell subsets were able to proliferate and secrete IL-3/GM-CSF in SMLR cultures. IL-2 was present in 'resting' T-cell SMLR supernatants, and barely detectable in NA T-cell SMLR supernatants. Both NA and 'resting' T-cell SMLRs were inhibited with anti-class II, anti-CD4, or anti-IL-2R MoAbs. NA T cells exhibited a background proliferative and secretory activity in the absence of syngeneic accessory cells. This autonomous activity was susceptible to anti-CD4, but poorly inhibited with anti-class II MoAbs. Both NA and 'resting' T lymphocytes displayed strong responsiveness to allogeneic stimuli. The analysis of the relative frequency of proliferating cells in the SMLR (BALB/c), or allo-MLR (B10, B10.A, B10.D2) from NA or 'resting' T cells indicated an enrichment for syngeneic reactivity among NA T lymphocytes. The meaning of these results for NA T-cell function and repertoire is discussed.     

SEB活化的人外周血T细胞CD25,CD69的表达

本文用超抗原葡萄球菌肠毒素B(SEB)诱导外周血淋巴细胞增殖。结果显示出微量超抗原能诱导外周血淋巴细胞的增殖,大量的增殖反应发生在SEB刺激后的第5天,增殖的细胞是CD4~+T细胞,它们由刺激前的27％增加到42％。外周血活化的T细胞不依赖外源性IL-2:大量内源性IL-2的存在抑制T细胞的增殖反应。伴随CD4~+T细胞的增殖,CD25和CD69分子表达明显增加。提示SEB能改变外周血T细胞表面的分子表达。     

Stimulation of Peripheral Blood T Cells by an Activated T-Cell Line: a Novel Human Autologous T-T Lymphocyte Reaction

T lymphocyte interactions have generally been described between discrete functional subsets. In our investigation of murine T-cell interactions we described a type of T-T interaction termed the 'Syngeneic T-T Lymphocyte Reaction' in which activated T-cell clones stimulated the proliferation of resting T cells mainly through a mechanism involving cell to cell contact. To investigate whether similar reactions occur in the human immune system we used the human autoreactive T-cell line C.1 to stimulate peripheral T cells. Line C.1 cells, which are not transformed and do not secrete IL2, consistently caused proliferation of purified freshly isolated autologous peripheral human T cells as measured by a [3H]-thymidine incorporation assay. The proliferation was seen in both the CD4 and CD8 subsets and could be inhibited with anti-DR and anti-CD2 antibodies. The stimulation is not due to carryover of classical antigen-presenting cells or to the C.1 line cells acting as antigen-presenting cells. We propose that some activated T cells, probably by expression of a surface molecule, can stimulate resting T cells thereby allowing for antigen-non-specific augmentation of the immune response.     

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4⁺ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4⁺ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4⁺ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.     

Impact of a Low CD34+ Cell Dose on Allogeneic Peripheral Blood Stem Cell Transplantation

Although the CD34⁺ cell dose in allogeneic peripheral blood stem cell transplantation (PBSCT) is considered to be associated with transplantation outcomes, a lower acceptable threshold has not been defined. We retrospectively analyzed 2919 adult patients with hematologic malignancies who underwent related PBSCT in Japan between 2001 and 2014. According to the number of CD34⁺ cells in the graft, we categorized 2494 patients in the standard group (2 to 5 × 10⁶ cells/kg), 377 patient in the low group (1 to 2?×?10⁶ cells/kg), and 48 patients in the very low group (<1?×?10⁶ cells/kg). Compared with the standard group, the low and very low groups showed delayed neutrophil recovery (93.8%, 89.5%, and 78.3%, respectively at day +28; P?<?.001) and platelet recovery (69.3%, 53.0%, and 45.5%, respectively at day +28; P?<?.001). The 2-year overall survival (OS) in the 3 groups was 45.5%, 45.3%, and 29.8%, respectively, with inferior survival in the very low group. However, a higher percentage of high-risk patients may account for the inferior survival in the very low group, and no significant difference in OS was found in a multivariate analysis. There were no differences in relapse, nonrelapse mortality, or the development of graft-versus-host disease among the 3 groups. In conclusion, allogeneic PBSCT with low CD34⁺ cell doses of 1 to 2?×?10⁶ cells/kg gives acceptable results, whereas further investigations are needed to evaluate the effects of lower doses of <1?×?10⁶ cells/kg owing to the smaller number and the higher percentage of patients with adverse prognostic factors in this cohort.     

慢性疲劳综合征患者外周血淋巴细胞亚群及CD25^＋调节性T细胞表达的研究

研究慢性疲劳综合征（CFS）患者外周血淋巴细胞亚群及CD25＋调节性T细胞的表达情况。使用流式细胞仪检测84例CFS患者（CFS组）、50例健康体检者（健康对照组）外周血淋巴细胞亚群（T细胞、CD4＋T细胞、CD8＋T细胞、B细胞、NK细胞）及CD25＋调节性T细胞的表达情况。结果显示,CFS组与健康对照组的T细胞、CD8＋T细胞百分率以及CD4＋/CD8＋比值无显著差别（P〉0.05）;而CFS组NK细胞、CD4＋T细胞及CD25＋调节性T细胞百分率显著增高,B细胞百分率显著降低（P〈0.05）。结论：慢性疲劳综合征患者外周血淋巴细胞各亚群比例异常,提示其免疫功能失衡,而CD25＋T调节性细胞可能在该病进程中有重要意义。     

Antibody Dependent Cell Mediated Cytotoxicity and Phagocytosis of Senescent Erythrocytes by Autologous Peripheral Blood Mononuclear Cells

Human red blood cells (RBCs) have a life span of 120 days in circulation, after which they are removed primarily by resident macrophages. Autoimmune antibodies are commonly found on effete RBCs and appear to contribute to their removal from the circulation. In this article, we focused on senescent erythrocytes and studied their removal, in comparison to young RBCs, in two RBC-depletion in vitro assays: antibody dependent cell mediated cytotoxicity (ADCC) and erythrophagocytosis. The results were determined prior to and following the addition of anti-D antibodies to the systems. Old (O-RBC) and young (Y-RBC) erythrocytes were separated by differential centrifugation. When incubated with autologous peripheral blood mononuclear cells as attacking cells, both anti-D treated O-RBCs and anti-D treated Y-RBCs were phagocytized and underwent contact lysis. However, O-RBCs had a significantly higher tendency to be phagocytized ( p =0.05) and a higher predisposition to undergo lysis ( p =0.043) than did Y-RBCs. When incubated with attacking cells without anti-D antibodies, O-RBCs were phagocytized while Y-RBCs were not phagocytized at all ( p =0.046). No contact lysis of either source of target cells occurred when incubated with attacking cells alone without anti-D sera. These in vitro results suggest that ADCC may serve as an additional pathway of elimination of senescent erythrocyte in addition to the classical phagocytosis pathway.     

Peripheral Blood Mononuclear Cells from Women with Recurrent Abortion Exhibit an Aberrant Reaction to Release Cytokines upon the Direct Contact of Human Leukocyte Antigen-G-Expressing Cells

PROBLEM: In search for pathogenesis of recurrent abortion, we examined whether lymphocytes/macrophages from women with recurrent abortion exhibited an aberrant ability to release cytokines upon the direct contact of human leukocyte antigen (HLA)-G. METHOD OF STUDY: The amounts of cytokines released from peripheral blood mononuclear cells (PBMCs) from women with recurrent abortion were compared with those from normal multiparous women or normal nulligravidous women when cocultured with or without HLA-G-expressing target cells. RESULTS: When cocultured with HLA-G-expressing target cells, the amount of interleukin-1β released from PBMCs was increased in recurrent aborters whereas it decreased in both normal multiparous and nulligravidous women. The amount of interleukin-3 released from PBMCs did not differ with or without HLA-G-expressing cells in recurrent aborters, whereas it increased in the presence of HLA-G-expressing cells in normal controls. The amount of tumor necrosis factor-α released from PBMCs was decreased in the presence of HLA-G-expressing cells in both recurrent aborters and normal controls. CONCLUSION: The aberrant reaction of maternal lymphocytes/macrophages in releasing cytokines upon the contact of HLA-G expressed on trophoblasts may impact negatively on trophoblastic growth, which may be pathogenic in recurrent abortion.     

Examination of Peripheral Blood Mononuclear Cells and Sera from Thai Adults Naturally Infected with Malaria in Assays of Blastogenic Responsiveness to Mitogenic Lectins and Allogeneic Cell Surface Antigens

We have previously observed that Thai adults who are infected with malaria have a loss of peripheral blood T cells, and that patient sera contain lymphocytotoxic antibodies. In the present study, we examined peripheral blood mononuclear cells from Thai adults naturally infected with Plasmodium falciparum and Plasmodium vivax for the capacity to undergo blastogenesis in response to phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic cell surface antigens in a one-way mixed leukocyte reaction. In addition, sera from actively infected patients were examined with regard to suppressive capabilities toward normal lymphocyte blastogenesis by using the same assays. We found that patient mononuclear cells exhibited normal reactivity to phytohemagglutinin, concanavalin A, and pokeweed mitogen when compared with controls. However, peripheral blood mononuclear cells from patients had a decreased stimulatory capacity in the allogeneic mixed leukocyte reaction, and P. vivax, but not P. falciparum, lymphocytes exhibited decreased responsiveness in the mixed leukocyte reaction. Furthermore, sera from patients with active malaria induced decreased responsiveness by normal mononuclear cells to phytohemagglutinin and concanavalin A, but not pokeweed mitogen; pooled P. falciparum sera caused decreased responsiveness to allogeneic cell surface antigens in the mixed leukocyte reaction. These studies indicate that despite the lost of circulating T cells during the course of infection with malaria, blastogenic responsiveness remains intact, and that sera from patients with malaria are capable of exerting negative immunoregulatory effects.     

Induction of Natural Killer Cell Activity and Allocytotoxicity in Human Peripheral Blood Lymphocytes after Mixed Lymphocyte Culture

The K-562 tumour cell is a highly susceptible target for natural killer (NK) cell lysis by lymphocytes of human peripheral blood. We have studied the antigenic relationship between the recognition sites for lysis of lymphoid and various tumour target cells by cytolytic T lymphocytes (CTL) and NK cells induced in mixed lymphocyte cultures (MLC). The characteristics of these two effector cell types have also been investigated. It was demonstrated that fresh NK cells lose their NK lytic activity when cultured alone. Cell lines not susceptible to lysis by fresh NK cells are lysed by MLC-induced NK cells. There is no antigenic relationship between the recognition sites for the alloreactive T lymphocytes and MLC-generated NK cells expressed on the lymphoid target cells and the tumour target cells, respectively. The MLC-generated alloreactive T cells and NK cells are not identical. The MLC-generated NK cells are different from the fresh NK cells present in the peripheral blood.     

Profile of Toll-Like Receptors on Peripheral Blood Cells in Relation to Acute Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

Toll-like receptors (TLRs) play a key role in the cross-talk between the innate and adaptive immune systems. Previous studies investigating associations between certain TLRs and acute graft-versus-host disease (aGVHD) have reported contrasting results, and no studies relating aGVHD to the expression and function of all human TLRs together have been published to date. We prospectively evaluated the expression of 9 TLRs on T lymphocytes and monocytes by flow cytometry in relation to aGVHD in 34 patients. Induction of TNF-α, IL-4, IFN-γ, and monocyte chemotactic protein 1 on TLR activation was assessed by ELISA on cell supernatants. Nineteen patients developed aGVHD, at a median time of 28 days (range, 20-50 days) after transplantation. A 2-step multivariate analysis was performed using principal component analysis and multifactor analysis of variance. The levels of TLR-5 expression on monocytes and T lymphocytes were positively correlated to aGVHD (P = .01), whereas levels of TLR-1 and -9 were negative predictors (P = .03 and .01, respectively). This profile of TLR-1, -5, and -9 can promote an overall immunostimulatory/proinflammatory response. If our findings are confirmed by further studies, this TLR profile could be a useful biomarker of aGVHD.     

In Vitro Production of Monoclonal and Polyclonal Immunoglobulins by Peripheral Blood Mononuclear Cells in Human Plasma Cell Myeloma

The in vitro monoclonal and polyclonal immunoglobulin (Ig) production of peripheral blood mononuclcar cells was studied in human multiple myeloma (four IgG myelomas, one IgA myeloma) and in one patient with benign monoclonal gammopathy. Using an enzyme-linked immunosorbent assay with anti-class-specific antisera and antisera against idiotypic structures on the myeloma protein, it was possible to quantilate separately monoclonal and polyctonal Ig of the same class in cell culture supernatants. After stimulation with pokcweed mitogen (PWM) patients' cells produced lower amounts of polyclonal Ig than cells from healthy adults. In contrast, production of monoclonal Ig could not be enhanced by PWM. Moreover, the kinetics of monoclonal Ig production was different from that of polyclonal Ig. Myeloma cells contained large amounts of monoclonal Ig while their content of polyclonal Ig was low. A rapid release of preformed monoclonal Ig during the first day of culture was not inhibited by puromycin. A later phase of release was partly suppressed by puromycin and was probably caused by active protein synthesis.     

GM-CSF诱导外周血单核细胞的分化及其对混合淋巴细胞反应的影响

作者对 Ｇ Ｍ Ｃ Ｓ Ｆ 作用下的外周血单核细胞（ Ｐ Ｍ Ｃ） 的形态特征及免疫表型进行了测定, 并观察了其对混合淋巴细胞反应（ Ｍ Ｌ Ｒ） 的影响。结果显示, Ｇ Ｍ Ｃ Ｓ Ｆ 可使外周血单核细胞分化为 Ｃ Ｄ１４ ＋ 、 Ｃ Ｄ１ａ 或低表达、 Ｃ Ｄ８０ 、 Ｃ Ｄ８６ 低表达、 Ｈ Ｌ Ａ Ｄ Ｒ＋ 、 Ｃ Ｄ１１ａ ＋ 、 Ｃ Ｄ４５ Ｒ Ａ＋ , 和非特异性酯酶呈强阳性反应的细胞, 符合巨噬细胞的形态及表型特征。在 Ｍ Ｌ Ｒ 体系中加入上述细胞, 发现其 Ｍ Ｌ Ｒ 的结果因巨噬细胞含量的不同而异, 巨噬细胞与反应细胞比例为１∶２ 时抑制 Ｍ Ｌ Ｒ（ Ｐ＜ ００５ ） , １∶４或１∶８ 时刺激 Ｍ Ｌ Ｒ（ Ｐ＜ ００５ ） ; Ｐ Ｈ Ａ 和 Ｉ Ｌ ２ 能够逆转大剂量巨噬细胞对 Ｍ Ｌ Ｒ 的抑制作用, 抗 Ｃ Ｄ３ 单抗可增强其对 Ｍ Ｌ Ｒ 的抑制作用, 消炎痛不影响巨噬细胞对 Ｍ Ｌ Ｒ 的作用。     

Relationship of Arachidonic Acid Metabolism to Indomethacin-Sensitive Immunoregujatory Function and Lymphocyte PGE Sensitivity in Peripheral Blood Mononuclear Cells of Disseminated Solid Tumor Cancer Patients

Abstract

The relationship between the conversion of arachidonic acid (AA) to E series prostaglandins (PGE), indomethacin-sensitive immunoregulation and lymphocyte PGE sensitivity was investigated in the peripheral blood mononuclear cells (PBMC) of normal subjects and disseminated solid tumor patients. Production of PGE was assessed by thin layer chromatography of ether-extracted glass adherent cells following a 24-hour pulse with ³H-AA. Immunoregulatory cell function was assessed in PHA-stimulated PRMC cultured in the presence of the prostaglandin synthetase inhibitor, indomethacin. Lymphocyte PGE sensitivity was assessed in PHA stimulated glass nonadherent cells cultured in the presence of 10^?8 M PGE. The cells from cancer patients demonstrated greater AA conversion to PGE and greater indomethacin-sensitive immunoregulatory cell function than the cells of normal subjects. However, lymphocyte PGE sensitivity was comparable for both groups. When levels of arachidonic acid conversion to PGE were correlated to levels of indomethacin-sensitive immunoregulatory cell function by linear regression analysis, a significant correlation was found. These data suggest that the increased indomethacin-sensitive immunoregulatory cell function seen in PBMC from cancer patients can be directly correlated with increased production of E series prostaglandins by cancer patient peripheral blood monocytes.     

Relationship of Arachidonic Acid Metabolism to Indomethacin-Sensitive Immunoregujatory Function and Lymphocyte PGE Sensitivity in Peripheral Blood Mononuclear Cells of Disseminated Solid Tumor Cancer Patients

The relationship between the conversion of arachidonic acid (AA) to E series prostaglandins (PGE), indomethacin-sensitive immunoregulation and lymphocyte PGE sensitivity was investigated in the peripheral blood mononuclear cells (PBMC) of normal subjects and disseminated solid tumor patients. Production of PGE was assessed by thin layer chromatography of ether-extracted glass adherent cells following a 24-hour pulse with ³H-AA. Immunoregulatory cell function was assessed in PHA-stimulated PRMC cultured in the presence of the prostaglandin synthetase inhibitor, indomethacin. Lymphocyte PGE sensitivity was assessed in PHA stimulated glass nonadherent cells cultured in the presence of 10^-8 M PGE. The cells from cancer patients demonstrated greater AA conversion to PGE and greater indomethacin-sensitive immunoregulatory cell function than the cells of normal subjects. However, lymphocyte PGE sensitivity was comparable for both groups. When levels of arachidonic acid conversion to PGE were correlated to levels of indomethacin-sensitive immunoregulatory cell function by linear regression analysis, a significant correlation was found. These data suggest that the increased indomethacin-sensitive immunoregulatory cell function seen in PBMC from cancer patients can be directly correlated with increased production of E series prostaglandins by cancer patient peripheral blood monocytes.     

Combined CD4+ Donor Lymphocyte Infusion and Low-Dose Recombinant IL-2 Expand FOXP3+ Regulatory T Cells following Allogeneic Hematopoietic Stem Cell Transplantation

CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4⁺ lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4⁺DLI alone (P = .001). FOXP3 expression correlated with absolute CD4⁺CD25⁺ cell counts. Moreover, expanded CD4⁺CD25⁺ T cells displayed normal suppressive function and treatment with CD4⁺DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4⁺ cellular therapy may provide a mechanism to expand Treg in vivo.     

Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study

The Multicenter AIDS Cohort Study (MACS), an ongoing prospective study of the natural history of human immunodeficiency virus (HIV), has stored biologic specimens, including peripheral blood mononuclear cells (PBMC), from 5,622 participants for up to 12 years. The purpose of the present analysis was to evaluate the quality of the PBMC in the MACS repository in order to test the validity and feasibility of nested retrospective studies and to guide the planning of future repositories. PBMC were collected from MACS participants at four centers at 6-month intervals from 1984 to 1995, cryopreserved, and transported to a central repository for storage. A total of 596 of these specimens were subsequently tested for viability and used to evaluate cell function, to conduct immunophenotype analysis, or to isolate HIV. Simple linear regression models were applied to evaluate trends in recovery and viability over time and by center. Results indicated that from a nominal 10⁷ cells cryopreserved per vial at all four centers, the median number of viable cells recovered was at least 5 × 10⁶ (50% of the number stored) and the median viability was at least 90%. Results suggested that cryopreserved cells can be stored for at least 12 years with no general tendency toward cell loss over time. Furthermore, there were no statistically significant changes in the percent cell viability according to the length of time frozen, regardless of HIV serostatus or the level of CD4⁺ lymphocytes. Storing 10⁷ PBMC per vial yields sufficient viable cells for phenotypic and/or functional analysis. Results from the MACS provide the basis for the planning of future repositories for use by investigators with similar research goals.     

Analysis of CD28 and bcl-2 Expression on Peripheral Blood and Liver-Infiltrating Mononuclear Cells in Patients with Autoimmune Hepatitis

Because the underlying mechanism of hepatocellular damages in autoimmune hepatitis (AIH) still remains unclear, analysis of CD28 and bcl-2 molecules, which are critical for T cell activation and survival, was performed in patients with AIH. The number of CD28(+)CD4(+) peripheral blood mononuclear cells (PBMC) in corticosteroid (CS)-treated patients was comparable to normal control individuals but decreased in untreated AIH patients. In contrast, the number of CD28(+)CD8(+) PBMC was decreased in both CS-treated and untreated AIH patients. Analysis of liver-infiltrating mononuclear cells (LIMC) showed that the number of CD28(+)CD4(+) and CD28(−)CD8(+) LIMC were positively correlated with the histology activity index score. Bcl-2(+)CD4(+) LIMC were observed in the portal area of the liver and the numbers fluctuated with disease activity during the time course after CS administration. By contrast, CD8(+) LIMC were shown not to express bcl-2. Taken collectively, these results suggest that bcl-2(+)CD28(+)CD4(+) and bcl-2(−)CD28(−)CD8(+) cells may play critical and distinct roles in hepatocellular damage in AIH.