Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.     

Functional and Vbeta repertoire characterization of human CD8+ T-cell subsets with natural killer cell markers,CD56+ CD57- T cells,CD56+ CD57+ T cells and CD56- CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK-type T cell); CD56 single positive (CD56)-T cells, CD56/CD57 double positive (DP)-T cells and CD57 single positive (CD57)-T cells in the peripheral blood. All NK-type T-cell populations expressed CD122 and intermediate levels of T-cell receptor (TCR; regular CD8+ T cells are CD122- and express high levels of TCR). The number of both DP-T cells and CD57-T cells, but not CD56-T cells, gradually increased with age. All NK-type T-cell populations produced larger amounts of interferon-gamma than did regular CD8+ T cells after stimulation with interleukin (IL)-2, IL-12 and IL-15. However, CD56-T cells and CD57-T cells but not DP-T cells showed a potent antitumour cytotoxity to NK-sensitive K562 cells, whereas only CD56-T cells showed a potent cytotoxity to NK-resistant Raji cells. Furthermore, although NK-type T cells produced large amounts of soluble Fas-ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VbetaT cells were found in each NK-type T-cell population. The non-variant CDR3 region(s) for the TCRbeta chain(s) showed CD57-T cells and CD56-T cells to be derived from distinct origins, while the DP-T cell population consisted of a mixture of the clones seen in both CD56-T cells and CD57-T cells. Our results suggest that CD57-T cells and CD56-T cells are functionally and ontogenically different populations while DP-T cells appear to originate from both CD56-T cells and CD57-T cells.     

Inhibition of anti-GD3-ganglioside antibody-induced proliferation of human CD8+ T cells by CD16+ natural killer cells

The ganglioside GD3 has been described as a membrane component of human T cells which is involved in T cell growth. In the present study the activating function of GD3 for human CD4⁺ and CD8⁺ T cells was analyzed by five different monoclonal antibodies (mAb) directed against the GD3 molecule. Three mAb U5, Z21 and R24 induced strong proliferation of peripheral blood mononuclear cells and purified CD8⁺ and CD4⁺ T cells of normal donors containing less than 5% CD16⁺ natural killer (NK) cells. In contrast to CD4⁺ T cells, CD8⁺ T cells proliferated only weakly in the presence of 15% CD16⁺ NK cells. The proliferative response of purified CD4⁺ and CD8⁺ T cells (<5% NK cells) correlated with the antibody-dependent induction of integral and soluble interleukin-2 (IL-2) receptors and was reduced to 20% by an anti-IL-2 receptor antibody. Our results show, that the GD3 molecule represents an activation molecule for both CD4⁺ and CD8⁺ T cells and that CD16⁺ NK cells selectively inhibit anti-GD3 antibody-induced proliferation of CD8⁺ T cells.     

Evidence that CD4+, but not CD8+ T cells are responsible for murine interleukin-2-deficient colitis

Mice deficient in interleukin-2 production (IL-2^null mice) develop colonic inflammation closely resembling ulcerative colitis in humans. Although this disease is marked by substantial infiltration of the colon by CD8⁺ and CD4⁺ T lymphocytes, no function has yet been assigned to these T cell subsets in the development of colitis in the IL-2^null mouse. For the present study, we investigated the involvement of T lymphocytes in the onset of colitis in IL-2^null mice, and examined the possible role played by cytotoxic T cells. Both lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) of the colon of IL-2^null mice were potently cytotoxic ex vivo in short-term redirected cytotoxic lymphocyte (CTL) assays. In contrast, colonic T cells of wild-type animals showed little or no constitutive cytotoxic T cell activity. Colonic CTL were detectable prior to the appearance of disease in IL-2^null animals and CTL activity was confined to the TcRαβ, rather than to the TcRγδ IEL subset. IL-2^null animals crossed with major histocompatibility complex class I-deficient mice [IL-2^null × β₂ microglobulin (β₂m^null] mice also developed colitis, which appeared even earlier than in most IL-2^null mice. These findings suggest that neither CD8⁺ IEL nor LPL were causal in the onset of colitis in IL-2^null animals. In IL-2^null × β₂m^null mice, an ulcerative colitis-like disease was evident from histological studies and immunohistological staining which showed very large numbers of CD4⁺ lymphocytes within the intestinal mucosa. Significant ex vivo killing by CD4⁺ T cells was observed in IL-2^null × β₂m^null animals, although this required an extended incubation time compared to colonic CD8⁺ T cells. Peripheral as well as colonic CD4⁺ T cells in IL-2^null and IL-2^null × β₂m^null animals, were activated as judged by their cell surface phenotype (CD45RB^lo, L-selectin^lo and CD69⁺). In light of these findings, we propose that infiltrating CD4⁺, but not CD8⁺ T cells are central to the inflammation observed in the intestinal mucosa in IL-2^null colitis.     

HIV感染后CD4~+ T细胞表面NK相关受体表达的变化

目的研究人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染后CD4+T细胞表面NK相关受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV感染者、11例AIDS患者、15例进行高效抗逆转录病毒治疗(highly activeantiretroviral therapy,HAART)者和13例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血CD4+T细胞表面NKG2D、NKG2A和KIR3DL1的表达。结果 AIDS患者CD4+T细胞表面NKG2D表达的百分比显著高于其他各组,且NKG2D表达的百分比与CD4+T细胞的绝对数量呈负相关(R=-0.352,P<0.05),与HIV病毒载量呈正相关(R=0.426,P<0.05)。AIDS患者CD4+T表面NKG2A表达的百分比显著高于其他各组,NKG2A+NKG2D-表达的百分比显著高于其他各组,且NKG2A表达的百分比与CD4+T细胞的绝对数量呈负相关(R=-0.432,P<0.01)。结论 HIV感染机体后,CD4+T细胞NK相关受体表达变化与疾病进展相关。     

Evidence that human CD8+CD45RA+CD27- cells are induced by antigen and evolve through extensive rounds of division.

We recently showed that circulating human CD8(+) effector cells have a CD45RA+CD27(-) membrane phenotype. In itself this phenotype appeared to pose a paradox: CD45RA, a marker expressed by unprimed cells, combined with absence of CD27, characteristic for chronically stimulated T cells. To investigate whether differentiation towards the CD45RA+CD27(-) phenotype is dependent on antigenic stimulation and involves cellular division, TCR Vbeta usage and telomeric restriction fragment (TRF) length were analyzed within distinct peripheral blood CD8(+) subsets. FACS analysis showed that the TCR Vbeta repertoire of CD8(+)CD45RA+CD27(-) cells differed significantly from that of unprimed CD8(+)CD45RA+CD27(+) cells. Moreover, in two out of six individuals large expansions of particular Vbeta families were observed in the CD8(+)CD45RA+CD27(-) subset. CDR3 spectrotyping and single-strand confirmation analysis revealed that within the CD8(+)CD45RA+CD27(-) population most of the 22 tested Vbeta families were dominated by oligoclonal expansions. The mean TRF length was found to be 2.3+/-1.0 kb shorter in the CD8(+)CD45RA+CD27(-) subset compared with the unprimed CD8(+)CD45RA+CD27(+) population, but did not differ substantially from that of memory type, CD8(+)CD45RA-CD27(+) T cells. These findings indicate that the CD8(+)CD45RA+CD27(-) cytotoxic effector population consists of antigen-induced, clonally expanded cells and confirm that the expression of CD45RA is not a strict marker of antigen non-experienced T cells.     

HIV感染后CD8~+T细胞表面NK相关受体表达的变化

目的深入了解人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染后CD8+T细胞表面NK相关受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV感染者、11例AIDS患者、15例进行高效抗逆转录病毒治疗(highly active antiretroviral therapy,HAART)者和13例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血CD8+T细胞表面NKG2D、NKG2A和KIR3DL1的表达。结果 HIV感染后CD8+T细胞表面NKG2D表达的百分比显著低于健康对照,NKG2D+NKG2A-表达的百分比随疾病进展逐渐下降,且NKG2D+NKG2A-表达的百分比与CD4+T细胞的绝对数量呈正相关。AIDS患者CD8+T细胞表达NKG2A显著高于其它各组,随着疾病的进展CD8+T细胞表达NKG2A+NKG2D-百分比逐渐上升,AIDS患者显著高于其它各组,经抗逆转录病毒治疗后下降至健康对照的水平,且NKG2A+NKG2D-表达的百分比与CD4+T细胞的绝对数量呈负相关。HIV感染后CD8+T细胞KIR3DL1+表达的百分比较健康对照并无显著差异。结论 HIV感染机体后,CD8+T细胞NK相关受体表达变化与疾病进展相关,抗病毒治疗后可恢复其变化。     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

CXCR5+ CCR7- CD8 T cells are early effector memory cells that infiltrate tonsil B cell follicles

Naive and central memory CD8 T cells use CCR7 to recirculate through T cell zones of secondary lymphoid organs where they can encounter antigen. Here we describe a subset of human CD8 T cells expressing CXCR5 which enables homing in response to CXCL13 produced within B cell follicles. CXCR5+ CD8 T cells were found in tonsil B cell follicles, and isolated cells migrated towards CXCL13 in vitro. They expressed CD27, CD28, CD45RO, CD69, and were CD7low, and produced IFN-gamma and granzyme A but lacked perforin, a functional profile suggesting that these cells are early effector memory cells in the context of contemporary T cell differentiation models. Receptors important in the interaction with B cells, including CD70, OX40 and ICOS, were induced upon activation, and CXCR5+ CD8 T cells could to some extent support survival and IgG production in tonsil B cells. Furthermore, CXCR5+ CD8 T cells expressed CCR5 but no CCR7, suggesting a migration pattern distinct from that of follicular CD4 T cells. The finding that a subset of early effector memory CD8 T cells use CXCR5 to locate to B cell follicles indicates that MHC class I-restricted CD8 T cells are part of the follicular T cell population.     

CD28 is not directly involved in the response of human CD3- CD56+ natural killer cells to lipopolysaccharide: a role for T cells

We have previously shown that human CD3- CD56+ and CD3+ CD56+ cells from some individuals mount vigorous proliferative responses to lipopolysaccharide. Such responses have been blocked by the presence of cytotoxic T-lymphocyte antigen-4 immunoglobulin fusion protein in the cultures, implicating a role for B7-mediated costimulation. Here we confirm this inhibition of natural killer (NK) expansion using antibodies against B7-1 and B7-2. We were unable to specifically detect CD28 on the surface of resting or stimulated human peripheral blood NK cells, however, in either lipopolysaccharide-responsive or non-responsive individuals, using a panel of four different anti-CD28 monoclonal antibodies. T-cell depletion from peripheral blood mononuclear cell cultures resulted in a reduction in the induction of CD25 on activated CD3- CD56hi cells and in the expansion and proliferation of CD3- CD56+ NK cells. Furthermore, reconstitution experiments using peripheral blood dendritic cells and purified NK cells demonstrated that NK expansion could only be achieved in the presence of purified T cells.     

Impaired in vivo CD4+ T cell expansion and differentiation in aged mice is not solely due to T cell defects: decreased stimulation by aged dendritic cells

CD4+ T cells regulate humoral and cell-mediated immune responses, which are progressively impaired in aging, resulting in susceptibility to infections and cancer. Dendritic cells (DCs) are major activators of T cells, providing signals that drive their expansion and differentiation. In this study, we asked if decreased CD4+ T cell responses were influenced by the age of DCs rather than being exclusively due to T cell defects. Old T cells transferred to young recipients expanded and differentiated similarly to young T cells. However, aged recipients were poor stimulators of both old and young T cells, which failed to acquire CD44 expression and produce interferon gamma (IFN-γ). DCs in aged hosts expressed fewer MHC-peptide complexes. The CD86 expression in the DCs of both hosts was similar; however, CD40 levels were reduced in old DCs. Finally, old DCs failed to produce inflammatory cytokines in response to LPS. Our results indicate that the impairment of aged CD4+ T cell function is intimately related to multiple alterations in aged DCs, rather than being caused solely by intrinsic T cell defects, suggesting that the function of aged T cells may be partially rescued in vivo when appropriate stimulation is applied. These findings are relevant to vaccination design for elderly populations.     

卵巢癌细胞培养上清诱导CD4+CD25-T细胞分化为CD4+CD25+调节性T细胞

目的研究卵巢癌细胞培养上清液是否能诱导外周血CD4^＋CD25^- T细胞转变为CD4^＋CD25^＋调节性T细胞。方法将外周血CD4^＋CD25^- T细胞分离后，对照组用CD3和CD28单抗活化，实验组在对照基础上加用卵巢癌细胞株SKOV3培养上清，72h后分离各组的CD25^＋和CD25^-T细胞，溴化脱氧尿嘧啶掺入标记法测定增殖能力及对静息的自体同源CD4^＋CD25^- T细胞的增殖抑制能力，流式细胞仪测定细胞糖皮质激素诱发型TNF受体（glucocorticoid-induced TNFR,GITR）与CTLA-4分子的表达，RT-PCR检测细胞卿mRNA的表达。结果与对照组相反，实验组的CD4^＋CD25^＋T细胞具有免疫抑制功能，自身增殖能力下降，GITR和CTLA-4分子的表达和CD4^＋CD25^＋调节性T细胞相似，并被诱导表达转录因子Foxp3 mRNA。结论卵巢癌细胞分泌的可溶性物质能诱导外周血CD4^＋CD25^-T细胞转化为CD4^＋CD25^＋调节性T细胞。     

The expression of killer cell inhibitory receptors on natural killer cells and activation status of CD4+ and CD8+ T cells in the decidua of normal and abnormal early pregnancies.

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer cells within the maternal decidua. These NK cells have an unusual phenotype, CD3- CD16- CD56(bright), distinguishing them from peripheral blood NK cells. They may control trophoblast migration and placentation. Using a panel of monoclonal antibodies to several members of the KIR family and flow cytometry, we found that KIRs are expressed on decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. In anembryonic pregnancy, the proportions of decidual NK cells with a particular KIRs (GL183 and EB6) decreased significantly when compared with normal pregnancy (p = 0.01 and 0.01, respectively), raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site. In the decidua, more CD4+ and CD8+ T cells expressed CD69 and HLA-DR than in blood, indicating that T cells are regionally activated during early pregnancy. When compared with normal pregnancy, decidual HLA-DR+CD4+CD3+, CD69+CD8+CD3+ and HLA-DR+CD8+CD3+ T lymphocytes are significantly increased in anembryonic pregnancy. The over-activation of decidual T cells during anembryonic pregnancy may thus contribute to the increased NK cytotoxicity activity.     

Parainfluenza type 1 virus-infected cells are killed by both CD8+ and CD4+ cytotoxic T cell precursors.

The memory cytotoxic T lymphocyte (CTL) response to human parainfluenza type 1 virus (hPIV-1), a prominent cause of respiratory infection in young children, has been analysed for a panel of healthy adults. The CTL response to the parainfluenza viruses has not been investigated previously. Precursor CTL (CTLp) with activity against hPIV-1-infected Epstein-Barr virus (EBV)-transformed B lymphoblastoid target cells were found at a relatively high precursor frequency (approximately 1/2500-1/4700 CD8+ and CD4+ subsets respectively) in peripheral blood. Both CD4+ and CD8+ CTLp were detected by the analysis of individual microcultures set up under limiting dilution conditions from freshly isolated blood, the phenotype of the responder cell from individual wells being determined by flow cytometry. Further characterization of the CTL response demonstrated MHC restriction by the HLA-A2 glycoprotein in 3/4 HLA-A2+ donors. The presence of effective, hPIV-1-directed T cell memory may explain, in part, the protection observed in the adult population.     

Low-dose cyclophosphamide combined with IL-2 inhibits tumor growth by decreasing regulatory T cells and increasing CD8+ T cells and natural killer cells in mice

Interleukin-2 (IL-2) benefits some cancer patients by promoting the proliferation of cytotoxic effector T cells, but this process is limited by the expansion of regulatory T cells (Tregs). Low-dose cyclophosphamide (CTX) can inhibit the number and function of Tregs. We treated carcinoma-bearing mice with Vehicle, CTX, IL-2 and CTX + IL-2 to investigate the effects of low-dose CTX combined with IL-2 in antitumor treatment. In comparison to monotherapy, CTX + IL-2 significantly limited tumor growth, via tumor cell proliferation inhibition and increased apoptosis. The infiltration of CD8⁺ T cells in tumor tissues was significantly increased in the CTX + IL-2 group. CTX + IL-2 safely increased CD8⁺ T and natural killer cells in the spleen, lymph nodes and peripheral blood, and CTX attenuated the increase in Tregs induced by IL-2 in the spleen.     

Both T cell receptor (TcR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56lck. CD2 can mediate TcR-CD3-independent and CD45-dependent activation of p56lck.

T cell activation by triggering the T cell receptor (TcR)-CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti-CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR-CD3 in CD2-induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR-CD3. We found that cell surface expression of TcR-CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced-electrophoretic-mobility form of p56lck observed after CD2 triggering. By isolating CD45- mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4-induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.     

Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4+CD25highFOXP3+ regulatory T cells

Adult bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells that are the subject of intense investigation in regenerative medicine. In addition, MSCs possess immunomodulatory properties with therapeutic potential to prevent graft-versus-host disease (GvHD) in allogeneic hematopoietic cell transplantation. Indeed, MSCs can inhibit natural killer (NK) function, modulate dendritic cell maturation, and suppress allogeneic T-cell response. Here, we report that the nonclassic human leukocyte antigen (HLA) class I molecule HLA-G is responsible for the immunomodulatory properties of MSCs. Our data show that MSCs secrete the soluble isoform HLA-G5 and that such secretion is interleukin-10-dependent. Moreover, cell contact between MSCs and allostimulated T cells is required to obtain a full HLA-G5 secretion and, as consequence, a full immunomodulation from MSCs. Blocking experiments using neutralizing anti-HLA-G antibody demonstrate that HLA-G5 contributes first to the suppression of allogeneic T-cell proliferation and then to the expansion of CD4(+)CD25(high)FOXP3(+) regulatory T cells. Furthermore, we demonstrate that in addition to their action on the adaptive immune system, MSCs, through HLA-G5, affect innate immunity by inhibiting both NK cell-mediated cytolysis and interferon-gamma secretion. Our results provide evidence that HLA-G5 secreted by MSCs is critical to the suppressive functions of MSCs and should contribute to improving clinical therapeutic trials that use MSCs to prevent GvHD.