Vav-1 regulates NK T cell development and NK cell cytotoxicity

The hematopoietic-specific Rho-family GTP exchange factor Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells.Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, we show that Vav-1 is also expressed in murine NK cells. Vav-1(-/-) mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as wild-type mice. Unexpectedly, IL-2-activated Vav-1(-/-) NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1(-/-) NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, the results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK natural cytotoxicity. Unlike the findings for NK cells, NK T cells were dramatically diminished in Vav-1(-/-) mice and splenocytes from Vav-1 mutant mice failed to produce IL-4 in response to in vivo CD3 stimulation. These data highlight the important role of Vav-1 in NK T cell development and NK cell function.     

Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow

Viable Lin(-) CD27(+) c-kit(Hi) Sca-1(Hi) GFP(+) cells recovered from heterozygous RAG1/GFP knockin mice progressed through previously defined stages of B, T, and NK cell lineage differentiation. In contrast to the GFP(-) cohort, there was minimal myeloid or erythroid potential in cells with an active RAG1 locus. Partial overlap with TdT(+) cells suggested that distinctive early lymphocyte characteristics are not synchronously acquired. Rearrangement of Ig genes initiates before typical lymphoid lineage patterns of gene expression are established, and activation of the RAG1 locus transiently occurs in a large fraction of cells destined to become NK cells. These early lymphocyte progenitors (ELP) are distinct from stem cells, previously described prolymphocytes, or progenitors corresponding to other blood cell lineages.     

CTLA4Ig Primed Donor Lymphocyte Infusion: A Novel Approach to Immunotherapy after Haploidentical Transplantation for Advanced Leukemia

CTLA4Ig attenuates T cell activation by co-stimulation blockade, but natural killer (NK) cells are not only resistant to CTLA4Ig, they also may demonstrate better antileukemia effect in the presence of CTLA4Ig. To explore this phenomenon we used sequential CTLA4Ig primed donor lymphocyte infusion (DLI) after post-transplant cyclophosphamide–based haploidentical transplantation. Thirty patients (CTLA4Ig-DLI group) with advanced leukemia received CTLA4Ig on day –1 and subsequently on days +7, +21, and +35, followed 12hours later by DLI of 1 to 10?×?10⁶ CD3⁺ T cells/kg containing .1 to 3.27?×?10⁶/kg CD56⁺ NK cells, with low dose cyclosporine for 60days. The incidences of acute graft-versus-host disease (GVHD), chronic GVHD and nonrelapse mortality (NRM) were 6.7%, 21%, and 4.5 %, respectively, with disease progression of 23.3% and overall survival of 79% at 18 months. Patients without disease progression had a significant early surge in CD56^dimCD16⁺NK cells with lower NKG2A expression. CTLA4Ig primed DLI was associated with an upregulation of CD86 in mature NK cells that was not witnessed with CTLA4Ig administration alone. Thus, CTLA4Ig primed DLI resulted in early proliferation of mature NK cells with cytotoxic potential enabling early institution of adoptive immunotherapy to mitigate the risk of relapse in advanced leukemia with reduced GVHD and NRM.     

Maturation of lymphocyte immunophenotypes and memory T helper cell differentiation during development in mice

The goal of this study was to systematically investigate the ontogeny of lymphoid populations throughout postnatal development. In CD-1 mice, peak lymphocyte numbers occurred in blood on postnatal day 10 (d10) including those for natural killers (NK1.1), B cells (CD19), T helper (CD3CD4), na?ve T helper (CD4CD62LposCD44low), memory T helper (CD4CD62LnegCD44high), and T cytotoxic (CD3CD8) cells. As percent of total lymphocytes, peaks were achieved by d10 for all T helper subtypes but not B cells which declined to a nadir. In spleen, lymphocyte numbers increased exponentially after d10. Proportionately, NK and T cells peaked on d10, declined by d20, and increased 2-3-fold by d45. Naive T cells constituted the majority of lymphocytes during development while memory cells gained to 2.2% (blood) and 12% (spleen) by d20. C57BL/6 mice had similar profiles except that the B cell nadir and T cell subset peaks were at d5. Peripheralization of critical numbers of lymphocytes by d10, and importantly, development of a repertoire of memory cells by d20, may define immune response capabilities that close the period of immaturity for the neonate.     

Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

AIMS: To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. METHODS: Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). RESULTS: CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. CONCLUSIONS: Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts.     

Severe combined immunodeficiency in man with an absence of immunoglobulin gene rearrangements but normal T cell receptor assembly

An autosomal recessive type of severe combined immunodeficiency disease (SCID) was characterized by an absence of immunoglobulins (Ig) in the serum and of Ig+ lymphocytes in bone barrow (BM) and peripheral blood. In the BM CD10+/terminal deoxynucleotidyl transferase-positive lymphocytes were identified. Epstein-Barr virus-transformed B lymphoblastoid cell lines (BLCL) obtained from BM and peripheral blood did not synthesize Ig. The Ig heavy and light chain gene complexes in the BLCL had retained the germ-line configuration. Mature T cells were present but their numbers in peripheral blood were decreased. T lymphoblastoid cells derived from peripheral blood expressed normal T cell receptor (TcR) CD3 complexes and manifested various genomic TcR rearrangements. It was concluded that this type of SCID entailed a complete arrest of B lymphocyte differentiation in an early stage prior to Ig rearrangements and a quantitative defect of T lymphocytes which nevertheless allowed development of mature T cells. Repeated failures of BM transplantation and the striking absence of Ig assembly suggested that this SCID defect resides in the BM microenvironment.     

Human clones with natural killer function can activate B cells and secrete B cell differentiation factors

Large granular lymphocyte clones were prepared from normal human mononuclear cells. Seven clones were studied in detail. All had high cytotoxic activity against the natural killer (NK) cell target K562 and all were T11- and DR-positive but T4- and T8-negative: none released migration inhibitory factor, a characteristic of helper T cell clones. When these NK cell clones were co-cultured with autologous B cells in a 1:1 ratio, 4 of the 7 induced both IgM and IgG synthesis. Ig production could not be further enhanced by the addition of B cell differentiation factors. The cells stimulated included antigen-specific memory B cells, as the Ig induced contained specific antibody to an antigen, tetanus toxoid, to which the B cell donors had recently been immunized. Supernatants from the helper/NK clones contained B cell differentiation factors, and were able to induce IgG synthesis from the lymphoblastoid cell line CESS and from co-cultured T and B cells. However, NK clone supernatants, unlike the clones themselves, were not effective at inducing Ig synthesis from purified B cells. Instead it appears that NK clones first activate B cells and thus render them responsive to the factors that the clones secrete.     

胃癌患者红细胞C3b受体与淋巴细胞免疫功能的相关性研究

目的：研究胃癌患者红细胞C3b受体（RBC-C3bR）与淋巴细胞免疫功之间的关系。方法：利用酵母菌花环试验、MTT法及克隆抗体致敏花环法对51例胃癌患者及30例正常人的RBC-C3bR、自然杀伤细胞（NK）活性及T淋巴细胞（TC）亚群进行测定。结果：胃癌患者RBC-C3bR阳性率、NK细胞杀伤活性、CD3^＋、CD4^＋、CD4^＋／CD8^＋比值均比正常对照组显著低下（P〈0．05、P〈0．01）。经统计学相关性分析显示，RBC-C3bR阳性率与CD4^＋／CD8^＋比值、NK细胞杀伤活性间呈正相关（P〈0．05，P〈0．01）。结论：胃癌患者红细胞免疫功能与淋巴细胞免疫功能一样受到抑制，二者免疫功能密切联系、相互影响。     

Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen

To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.     

Immunological reconstitution after bone marrow transplant with Campath-1 treated bone marrow.

Immunological reconstitution after allogeneic bone marrow transplant (BMT) was studied in 20 patients who received Campath-1 treated bone marrow. The peripheral blood lymphocyte phenotype was analysed with a panel of monoclonal antibodies at 3, 6 and 12 months. T cell proliferative capacity was evaluated by stimulation with PHA and Con A and in the mixed lymphocyte reaction (MLR). Natural killer (NK) cell activity was analysed against the K562 cell line at specified times after BMT in nine patients. Absolute numbers of T lymphocytes were reduced in all patients at 3 and 6 months. A marked decrease in the number of CD4+ cells persisted beyond 12 months. CD8+ cells regenerated more rapidly and reached normal at 6 months. No correlation was found between changes in lymphocyte subpopulations and the presence of graft-versus-host disease or cytomegalovirus infection. B cells recovered rapidly and maintained normal numbers throughout the study. A moderate increase in HNK1+ (Leu7) cells was observed at 3 and 6 months simultaneously with a low expression of NK15 (Leu11) and OKM1 antigens at 3 and 6 months, suggesting the presence of immature NK cells early after the transplant. A profound decrease of T cell proliferative capacity was observed both after mitogen stimulation and in the mixed lymphocyte reaction. NK cell activity was raised during the first month after transplant in all but one patient but no correlation was found with the presence of GVHD or cell marker analysis.     

The NK receptor KLRG1 is dispensable for virus‐induced NK and CD8+ T‐cell differentiation and function in vivo

The killer cell lectin‐like receptor G1 (KLRG1) is expressed by NK and T‐cell subsets and recognizes members of the classical cadherin family. KLRG1 is widely used as a lymphocyte differentiation marker in both humans and mice but the physiological role of KLRG1 in vivo is still unclear. Here, we generated KLRG1‐deficient mice by homologous recombination and used several infection models for their characterization. The results revealed that KLRG1 deficiency did not affect development and function of NK cells examined under various conditions. KLRG1 was also dispensable for normal CD8⁺ T‐cell differentiation and function after viral infections. Thus, KLRG1 is a marker for distinct NK and T‐cell differentiation stages but it does not play a deterministic role in the generation and functional characteristics of these lymphocyte subsets. In addition, we demonstrate that E‐cadherin expressed by K562 target cells inhibited NK‐cell reactivity in transgenic mice over‐expressing KLRG1 but not in KLRG1‐deficient or WT mice. Hence, the inhibitory potential of KLRG1 in mice is rather weak and strong activation signals during viral infections may override the inhibitory signal in vivo.     

Maturation of Lymphocyte Immunophenotypes and Memory T Helper Cell Differentiation During Development in Mice

The goal of this study was to systematically investigate the ontogeny of lymphoid populations throughout postnatal development. In CD-1 mice, peak lymphocyte numbers occurred in blood on postnatal day 10 (dl0) including those for natural killers (NK1.1), B cells (CD19), T helper (CD3CD4), naïve T helper (CD4CD62L^posCD44^low), memory T helper (CD4CD62L^negCD44^high), and T cytotoxic (CD3CD8) cells. As percent of total lymphocytes, peaks were achieved by d10 for all T helper subtypes but not B cells which declined to a nadir. In spleen, lymphocyte numbers increased exponentially after d10. Proportionately, NK and T cells peaked on d10, declined by d20, and increased 2–3-fold by d45. Naive T cells constituted the majority of lymphocytes during development while memory cells gained to 2.2% (blood) and 12 % (spleen) by d20. C57BL/6 mice had similar profiles except that the B cell nadir and T cell subset peaks were at d5. Peripheralization of critical numbers of lymphocytes by d10, and importantly, development of a repertoire of memory cells by d20, may define immune response capabilities that close the period of immaturity for the neonate.     

宫颈癌患者外周血T淋巴细胞及NK细胞的检测及其临床意义

为探讨宫颈癌患者外周血T淋巴细胞亚群和NK细胞活性检测的临床意义,采用流式细胞仪测定宫颈癌患者外周血T淋巴细胞亚群和NK细胞活性,以正常人作对照分析。结果表明,宫颈癌患者外周血CD3＋、CD3＋CD4＋、NK细胞数量及CD3＋CD4＋/CD3＋CD8＋比值较正常对照组均明显下降（P〈0.05）,而CD3＋CD8＋细胞水平显著升高。外周血T淋巴细胞亚群和NK细胞数量的改变与宫颈癌临床病理分期有关,分期越晚,CD3＋细胞、CD3＋CD4＋细胞、CD3＋CD4＋/CD3＋CD8＋细胞比值及NK细胞数量越低,CD3＋CD8＋细胞水平越高;Ⅰ、Ⅱ期宫颈癌患者与Ⅲ、Ⅳ期患者之间有显著差异（P〈0.05）。实验结果提示,宫颈癌患者细胞免疫功能低下,且临床病理分期越晚,其免疫功能越低,检测T淋巴细胞亚群、NK细胞可用于宫颈癌患者的免疫监测。     

自体外周血造血干细胞移植后淋巴细胞亚群的恢复

目的探讨自体外周血造血干细胞移植(auto-PBSCT)后免疫重建的规律。方法测定血液肿瘤患者auto-PBSCT前后淋巴细胞各亚群的绝对值。结果部分亚群在移植前低于正常。移植后各细胞亚群恢复时间不同,其中NK细胞在6个月恢复;B细胞在9个月恢复正常。T细胞总数在9个月恢复正常,其中主要为CD3 CD8 细胞。CD3 CD4 细胞在18个月恢复,而CD4 CD28 细胞在2年内仍未恢复。CD3 CD8 细胞在移植后早期有所下降,在3个月时恢复并高于正常,至9个月时逐渐下降至正常。CD3 CD8 细胞的主要部分是免疫表型呈活化的T细胞,即CD8 HLA-DR 细胞和CD8 CD38 细胞。两者分别在1个月和3个月时高于正常,在9个月时恢复正常。结论血液肿瘤患者移植前即存在淋巴细胞亚群异常的前提下,移植后NK细胞最先恢复,其次为B细胞,T细胞各亚群中CD3 CD8 细胞恢复较早,其中主要为活化T细胞。     

Phenotypic and functional characterization of a panel of cytotoxic murine NK cell clones that are heterogeneous in their enhancement of Ig secretion in vitro

NK cells not only function as cytotoxic effector cells, but also have immunoregulatory roles including the enhancement of Ig secretion. To have a stable and uniform population of NK cells to study their role in Ig secretion, we generated murine NK clones. Thus, culture of splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene (p53-KO) with IL-2 and poly(IC) resulted in a long-term NK line, from which four stable clones were derived. This approach also yielded a long-term NK line from splenocytes of normal C57BL/6 mice. Identification of the clones as members of the NK lineage was based on large granular morphology, expression of NK-TR and absence of TCR gene rearrangement. Flow cytometry revealed that all clones expressed IL-2R alpha and beta, chains and B220, but no CD3, NK1.1, DX5 or Ly-49. RT-PCR analysis showed heterogeneity in NK1.1 gene expression, and demonstrated expression of perforin and several granzymes in all clones. Three out of four clones lysed YAC-1, but not P815 target cells, corresponding to a pattern of NK specificity. All NK clones enhanced Ig secretion in an in vitro model for T cell- independent type 2 antigens, albeit to varying degrees. We found no correlation between the degree of helper activity of the NK clones and the level of their cytotoxic activity on YAC-1 targets. Thus, we established murine NK clones, and show that they mediate both cytotoxicity and enhancement of Ig secretion.      

淋巴结外恶性淋巴瘤外周血T淋巴细胞亚群与免疫指标测定分析

目的：检测原发性淋巴结外恶性淋巴瘤（Primary extranodal lymphoma，PENL）患者外周血T淋巴细胞亚群比例和免疫球蛋白（Ig）水平的变化。方法：分别应用流式细胞术（FCM）和免疫速率比浊法测定T淋巴细胞亚群比例和免疫球蛋白（Ig）以及补体C3、C4含量。结果：PENL组CD3^＋、CD4^＋和CD8^＋明显降低（P〈0．01），原发性结性恶性淋巴瘤（结性组）CD4^＋／CD8^＋比值和NK细胞比较其他两组有显著意义（P〈0．01）；提示PENL组、结性组中CD3^＋、CD4^＋和NK细胞与IgA、IgG、C3比较有显著的相关性（P〈0．01）。结论：结外恶性淋巴瘤患者免疫功能的改变，在其发病机制中的作用不容忽视，可以作为病情进展的免疫学指标。     

Malignant histiocytosis. A phenotypic and genotypic investigation.

Ten cases of malignant histiocytosis (MH) were evaluated for clinical and histopathologic features, phenotype, and rearrangement of T cell receptor (TCR) beta, gamma, and alpha and immunoglobulin (Ig) genes (7/10). All cases were HLA-DR+ and CD30-positive. Four cases had molecular evidence of T cell lineage such as TCR beta, gamma, and alpha rearrangements, and one additional case synthesized the cytoplasmic TCR beta chain. The remaining five cases did not show unequivocal T, B, natural killer (NK) cell, or macrophagic origin, and three of them had germline TCR and Ig genes. Ultrastructural analysis was not helpful for the definition of the cell lineage. Most myelomonocytic markers (MAC387, CD13, CD14, CD64, CD68) were either negative on the MH cells or were expressed on cells with rearranged TCR gene. Precursor (CD34, CD7) and NK (CD16, CD56, and CD57) cell markers were not found. The lineage of a number of cases of MH remains unresolved.     

鳖血提取物对小鼠免疫功能正向调节作用的研究

目的 :研究鳖血提取物对小鼠的免疫调节功能。方法 :采用环磷酰胺为免疫抑制剂 ,获得免疫功能低下的小鼠动物模型。比较鳖血提取物作用前后 ,小鼠T细胞亚群的数量、NK细胞的杀伤功能 ,淋巴细胞的增殖功能以及细胞因子水平的变化。结果 :鳖血提取物对免疫功能低下小鼠的CD4 T细胞在外周血中的比例、NK细胞的杀伤活性、淋巴细胞的增殖功能 ,均具有正向调节作用 (P <0 .0 5 ) ,并呈剂量依赖性 ;能提高淋巴细胞分泌IFN γ的能力。结论 :鳖血提取物具有正向调节小鼠免疫功能的能力     

T lymphocyte subsets and NK cell cytotoxicity in chronic hemodialysis patients. The effect of recombinant human erythropoietin (rHu-EPO) treatment.

We investigated subpopulations of T lymphocytes, NK cell number and cytotoxic activity in 14 chronic uremic patients on regular hemodialysis treatment. We observed a significantly decreased absolute lymphocyte number and percentage of CD3 cells. Relative numbers of CD16 cells were significantly elevated, but NK cell cytotoxic activity was within a normal range. Nine patients with chronic renal anemia on maintenance hemodialysis were enrolled in rHu-EPO treatment trial. The treatment was continued till the hematocrit level reached 30%. Each of the patients had corrected anemia and well-being. After 12 weeks of the treatment we observed in these patients decreases in CD3, CD4, CD8 and CD16 cell numbers and elevation of CD4/CD8 ratio. Cytotoxic activity of NK cells did not change significantly. Presented results indicate that chronic hemodialysis patients have significantly diminished lymphocyte number. rHu EPO treatment affects the T lymphocyte subsets inducing a deep decrease of CD8 and CD16 cell percentage leading to normalisation of the CD4/CD8 ratio.