Alloreactive T cells: Expression of HLA-D antigens, stimulation of autologous MLR, and possible immunoregulatory function

Primed in MLC with allogeneic stimulators T cells acquire the capacity of expressing HLA-D and DR antigens and of stimulating the MLC response of autologous lymphocytes When primed T cells from HTCs are used as stimulators, a bimodal distribution of response with clear-cut “typing responses” and no significant “back stimulation” is observed This pattern may be due to the expansion during priming of a population of HLA-D restricted suppressors since irradiated primed T cells inhibit the MLC responsiveness of HLA-D “compatible” lymphocytes. The development and size of such a population is not dependent, however, on the strength of the antigenic stimulus used for priming since no differences were seen between the pattern of reactions induced by T cells primed against HLA-D identical or HLA-D different cells Primed OKT4⁺ and OKT8⁺ T cells share the capacity of expressing Ia antigens and of inducing “HLA-D restricted suppression.” We suspected that a similar phenomena accounted for the behavior of two HLA-D heterozygous cells as if they were HTCs Although no suppression was found, the fact that these cells typed for their “silent” antigen when tested as responders, yet failed to express it when tested as stimulation, supports the theory that different genes control the MLC-responding and stimulating capacities.     

Transfer of latent atopy by bone marrow transplantation? A case report

A previously healthy 8-year old girl was diagnosed with acute myelogenous leukemia, and, while she was in first remission, she received a bone marrow transplant from her atopic brother. Studies 1 to 2 years after transplantation revealed that the marrow recipient had a specific-IgE production of donor-type pattern, indicated by the similar skin prick test results and RAST scores in the donor and recipient demonstrating allergy to animal dander and house dust. The recipient's own immunity had been destroyed by the preparative regimen for marrow transplantation, and no lymphoid cells of host origin could be found after transplantation in the chromosome analysis. A sensitization of the recipient to animal dander after transplantation was very unlikely because no animal contacts were present, and the chronic liver graft-versus-host disease of the patient additionally suggested a delayed immunologic recovery. The case history suggests that atopy can be transferred by bone marrow transplantation from donor to recipient. A possible mechanism appears to be a passive transfer not only of lymphoid precursors but also of mature memory cells within the bone marrow inoculum. The donor memory B cells are presumably capable of starting specific-IgE production when the cells are stimulated in the host environment by factors still unknown.     

Detection of HLA-D region products by primed lymphocyte typing (PLT)

In vitro priming experiments were pormed with lymphocytes from members of two different families carrying Dw-,DR2 or Dw2,DR2 haplotypes. It was demonstrated that lymphocytes could be primed to allogeneic HLA-D determinants without detectable priming to the associated HLA-DR determinant, even when the priming cell was also HLA-DR incompatible to the responding cell. It was further shown that the unknown HLA-D determinants of the two families (e.g., Dw-) were different, one of them showing cross-reactivity to Dw2. Priming to MT1 determinants or to Lewis antigens could not be detected.     

Production of human T cell growth factor

We provide here a protocol for production of T cell growth factor (TCGF) using cells isolated from defibrinated blood. Whether combined in allogeneic pools or tested as single donors, these cells consistently yield high activity TCGF, following PHA stimulation. Protocols using cells isolated from heparinized blood have often included addition of indomethacin or removal of adherent cells, or both, to overcome the problem of frequent nonproducing cultures. Our failure to find nonproducing cultures using this source of cells suggests that inhibitory cells or factors are removed by the blood clot formed during defibrination.

A distincteconomic advantage is gained by using these cells, not only because of the reliability of obtaining active supernatants, but also because the same blood can be used for preparation of a serum pool for cell cultures and the erythrocytes are useful for absorption of contaminating PHA present in the TCGF-containing media. Not only can defibrinated blood leukocytes be stimulated by PHA to release TCGF, but when cultured in allogeneic mixtures containing no PHA, they also release active TCGF.     

Heterogeneity of HLA-D(DR)4 detected by primary and secondary mixed-lymphocyte-culture reactions and by individual HLA-DR4 antisera

One of our panel families (Sb), in which the paternal haplotypes Dw4, DR4 (a) and Dwblank, DR4 (b) segregate, was tested in primary mixed lymphocyte culture (1.MLC) and in the primed lymphocyte test (PLT). In the 1.MLC, cells which carry the a haplotype strongly stimulate b-haplotype cells, and vice versa. For the PLT, lymphocytes of two family-members were primed against the a haplotype and two against the b haplotype. A strong positive restimulation (RR greater than or equal to 60%) occurred only with cells bearing the original stimulating haplotype. The PLs were tested later against families St and Sm, which possess DR4 haplotypes, and against a panel of 73 unrelated persons. The results show heterogeneity of D(DR)4, suggesting at least three different subgroups: D(DR)4a, present on DR4 cells which strongly restimulate the anti-a PLs; D(DR)4b, on DR4 cells which strongly restimulate the anti-b PLs, and D(DR)4c, on the DR4 cells, which do not restimulate any of the PLs tested here. It seems also possible to differentiate between these subgroups with conventional DR-serology, as the 8W sera 903 and 981 react only with a-haplotype cells of family Sb, and ths 8W sera 872 and 1045 react only with b-haplotype cells.     

Subtypes of HLA-B27 detected by cytotoxic T lymphocytes and their role in self-recognition

In the present study cytotoxic T lymphocytes were generated in MLC of lymphocytes from two unrelated HLA-A, B, C-identical, B27-positive, but D/DR-different, individuals. These CTL were shown to detect subtypes of HLA-B27. CTL specific for influenza virus lysed infected target cells matched for HLA-B27 only when they shared the same subtype. This indicates that the two subtypes of HLA-B27 detected by CTL function also as distinct elements in a self-restricted CTL response. Both subtypes were found among patients with ankylosing spondylitis.     

茴香霉素对小鼠T细胞反应性及其杀伤作用的影响

目的 茴香霉素对小鼠T细胞活化、反应性、杀伤作用及其同种异基因皮肤移植的影响.方法 利用荧光标记的单克隆抗体双染技术结合流式细胞仪检测茴香霉素对小鼠CD3 T细胞早期及中期活化标志分子CD69和CD25表达的影响; 用MTT法检测茴香霉素刺激下T细胞在单向或双向混合淋巴细胞反应中T细胞的反应性及其对大鼠肝癌(7919)细胞的杀伤效应;建立小鼠同种异基因皮肤移植动物模型,观察茴香霉素对移植皮肤存活时间的影响.结果 在最佳剂量10.0 ng/mL时,茴香霉素能够明显抑制T细胞表面分子CD69和CD25的表达,且能抑制单向或双向混合淋巴细胞反应中T细胞的反应性(P＜0.01);10.0 ng/mL茴香霉素亦能明显抑制T细胞对大鼠肝癌细胞的杀伤效应(P＜0.01);5.0和15.0 mg/kg茴香霉素能够明显延长小鼠移植皮肤的存活时间 (P＜0.01).结论 茴香霉素对T 细胞的活化、反应性、杀伤效应及同种异基因皮肤移植排斥反应均有明显抑制作用,可能为治疗免疫反应性疾病提供新的策略 .     

Change in functional phenotype of cloned human alloreactive cytolytic T cells

Alloreactive T cell clones primed in vivo were tested for the expression of T cell differentiation antigens CD2, CD3, CD4, and CD8. Each of 29 different clones were found to express CD2 and CD3, but were variable in their expression of CD4 (7 positive clones) and CD8 (15 positive clones). Six clones were positive for both CD4 and CD8. One of the 29 clones expressed neither CD4 or CD8. Over a period of 12–18 weeks of culture, these clones began to lose their alloreactivity but acquired NK-like activity. By changing the concentration of TCGF, the “allo” and “NK-like” lytic activities could be modulated. After 18 weeks of culture, these clones lost their alloreactive specificity, but not their NK activity. The expression of surface markers was unchanged. CD2 and CD3 molecules were determined to play a role in both the alloreactive and NK activity of these clones.     

Human suppressor cells: evidence of functionally distinct subsets

Human suppressor cells (SC) differing in both kinetic characteristics and degree of specificity were induced in vitro either by Concanavalin A (CON A) (10 μg/ml) or by repeated stimulation with allogeneic cells. CON A SC caused a characteristic early peak in blastogenesis (Day 2) when cocultured with fresh autologous cells in the presence of either CON A or allogeneic cells, although CON A SC alone did not respond to these stimuli. This early augmented response fell rapidly to values 50–90% below positive controls by the usual optimal day for the particular stimulus. Treatment with 5-bromodeoxyuridine and light during rapid proliferation ablated the early responses but increased the later responses (i.e., reduced the suppression). The cells mediating both the early rise and late suppression were found to belong to a subset of T cells that lost their ability to form SRBC rosettes (theophylline sensitive) in the presence of the phosphodiesterase inhibitor, theophylline, and were inactivated by mitomycin C. In contrast, SC induced by repeated allogeneic stimulation were suppressive in MLC but not CON A cultures, did not initiate early blastogenesis in the presence of naive cells and were partially mitomycin resistant. The mitomycin resistant SC induced by allogeneic stimulation are found in the theophylline resistant T-cell subset. These data are consistent with a model of human SC differentiation in which at least two subsets may be present: (1) a mitomycin sensitive, nonspecific cell that participates in an obligatory early autologous recognition event (induction?) and (2) a later occurring, more mitomycin resistant effector cell that appears to acquired specfficity for the response that it affects.     

人CK8&18阳性胸腺上皮细胞对脐血CD34~+细胞向T细胞分化的影响

目的:通过CK8/18阳性胸腺上皮细胞与人脐血单个核细胞在Transwell板的共培养,探讨CK8/18阳性TEC对T细胞增殖分化的影响。方法:用胶原酶消化法分离纯化胸腺上皮细胞、免疫组化鉴定分离纯化的TEC,采用密度梯度离心法分离获得脐血单个核细胞,并采用免疫磁珠分选CD34+细胞,将TEC种植培养于Transwell双层培养板上层,使其均匀平铺于上层板底部,再将分选后的细胞加入上层小室,经过48小时的共培养,流式细胞术检测进入下室细胞的表型变化。结果:分离纯化的TEC经免疫组化鉴定CK8/18阳性。TEC与脐血单个核细胞共培养后,CD3+CD4-CD8-双阴性、CD3+CD4+CD8-单阳性细胞显著增加,CD3+CD4+CD8+双阳性细胞亚群细胞减少,CD8单阳性细胞增加不明显;CD45RA阳性细胞比率无显著变化,CD45RO阳性细胞比率显著增加。结论:CK8/18阳性TEC能选择促进脐血单个核细胞CD4+T细胞和CD45RO+细胞增殖。     

Five HLA-D clusters associated with HLA-DR4

In order to investigate the HLA-D clusters associated with DR4, 54 DR4-positive, Dw4- and Dw10-negative responders, together with selected Dw4- or Dw10-positive responders, were tested with 22 HTCs that define DR4-associated D specificities. The results are consistent with previous data defining four distinct D clusters--Dw4, Dw10, DB3, and DYT--and have identified a new cluster provisionally termed LD40. In addition, the DB3 cluster is complex and appears to give typing response patterns overlapping those of the KT2 cluster originally defined as being associated with DR4 in Japanese populations. Of 116 DR4-positive haplotypes tested, 44% typed as Dw4, 18% were LD40, 16% were Dw10, 9% were DB3, 3% were DYT, and 10% gave no typing response to the HTCs defining any of these clusters. These studies are informative not only in defining the DR4-associated D clusters and in supporting the concept that D and DR cannot be considered identical but also in emphasizing the complexity of the D region.     

Impaired expression of CD28 on T cells in hairy cell leukemia

T cells from patients with active hairy cell leukemia (HCL), a chronic B cell malignancy, show poor proliferation in response to allogeneic peripheral blood mononuclear cells (PBMC). In order to study the T cell dysfunction, the expression of several adhesion and costimulatory molecules was analyzed by flow cytometry. Circulating T cells from HCL patients showed increased percentages of CD28(-) in all T cell subsets. In some patients the percentage of CD28(-) T cells within the CD4(+) subset was increased up to 80%. These CD4(+)CD28(-) T cells did not proliferate in a mixed lymphocyte culture (MLC) against allogeneic PBMC. After enrichment for CD4(+)CD28(+) T cells, the proliferative response in the MLC was recovered, but this response was still lower than the proliferative response from control T cells. In conclusion, lack of CD28 on T cells and a restricted T cell repertoire may contribute to immune deficiency in patients with HCL.     

Cytotoxic T lymphocytes directed against HLA-Bw35-linked target determinants show differences in sensitivity toward antibiotics during sensitization period

Recently, we demonstrated that the outcome of human CML^* was influenced by the presence of antibiotics in the culture medium. In earlier studies we had found that cytotoxic T lymphocytes could recognize HLA-Bw35-linked target determinants. These so-called Bw35 a and b determinants showed Mendelian segregation.

We describe in this article a difference between the Bw35 a and b cytotoxic T cells in their sensitivity toward antibiotics. The lysis against the Bw35 b cytotoxic determinant was not influenced by either the presence or the absence of antibiotics during the sensitization period. whereas the lysis against the Bw35 a cytotoxic determinant was drastically diminished when the effector cells were cultured in the absence of antibiotics during the sensitization period.     

自身LAK和肿瘤细胞在TIL大量扩增中的作用

将肿瘤浸润淋巴细胞（ＴＩＬ）分别与自身ＬＡＫ细胞、肿瘤细胞以及后二者共同混合培养，结果均使ＴＩＬ的体外扩增能力和细胞毒活性大为提高，体外扩增有效期和高细胞毒活性持续时间均较单纯ＩＬ－２培养的ＴＩＬ延长。以与自身ＬＡＫ和肿瘤细胞混合培养的ＴＩＬ对自身肿瘤的杀伤活性提高最明显。     

Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones

A serum-free lymphocyte culture medium is described in which serum is replaced by bovine serum albumin, transferrin, insulin, ethanolamine and a mixture of saturated and unsaturated fatty acids (linoleic acid, oleic acid and palmitic acid). In this serum-free medium proliferative and cytotoxic responses induced in mixed lymphocyte culture were comparable with those obtained in medium containing serum. Antigen-specific cytotoxic and helper T cells were isolated and could be propagated in serum-free medium without loss of function.     

An anti-I-A murine monoclonal antibody which cross-reacts with HLA-DR2

A murine monoclonal antibody, 12.7G3, directed against an Ia antigen encoded by genes in the I-Ab subregion of the H-2 genetic complex, was found to be cytotoxic against human B lymphocytes. When tested against a random panel of normal human donors, the reactivity of 12.7G3 exhibited a correlation coefficient of 0.58-0.68 with cells expressing HLA-DR2. Antibody reactivity segregated with HLA-DR2 in two families studied. Binding of 12.7G3, as detected by immunofluorescence using flow microfluorometry, was positive for two human cell lines, GM 3161 and HFB-1, both expressing HLA-DR2, and negative for two other cell lines, GM 3104 (DR1,1) and GM 3164 (DR4,4).     

Removal of phytohemagglutinin from conditioned medium by affinity chromatography

Supernatants of lymphocytes cultured with phytohemagglutinin (PHA-induced conditioned medium) are known to contain residual lectin. Studies with T cells specifically sensitized against a given antigen and maintained in culture by the T cell growth factor in conditioned medium may be hampered by the presence of PHA since the lectin could induce polyclonal activation of T cells.We developed a procedure for removing lectin from conditioned medium by affinity adsorption on porcine thyroglobulin-Sepharose. The affinity method was capable of removing detectable amounts of lectin since the mitogenic capacity for peripheral blood lymphocytes was lost after adsorption. In contrast, thyroglobulin-Sepharose adsorbed CM retained good mitogenic activity and growth-supporting capacity for human cultured T cells.     

腰椎间盘退变性疾病与外周血淋巴细胞亚群的关系

文题释义：淋巴细胞亚群：淋巴细胞是白细胞的一种,是机体免疫应答功能的重要细胞成分,是淋巴系统几乎全部免疫功能的主要执行者,占外周血白细胞总数的20%-40%,按其发生迁移、表面分子和功能的不同,主要分成T淋巴细胞、B淋巴细胞和自然杀伤细胞三大类。 自然杀伤细胞(natural killer cell,NK)：是机体重要的免疫细胞,不仅与抗肿瘤、抗病毒感染和免疫调节有关,而且在某些情况下参与超敏反应和自身免疫性疾病的发生。 背景：既往研究认为椎间盘退变的主要原因为遗传、衰老、营养不良和负荷史,免疫系统在椎间盘退变过程中的作用尚不清楚。 目的：观察腰椎间盘退变患者外周血淋巴细胞亚群的变化,并研究腰椎间盘退变程度与外周血各淋巴细胞亚群的关系。 方法：收集76例腰椎间盘退变性疾病患者和56例健康志愿者(对照组)的血样,用流式细胞仪检测外周血各淋巴细胞亚群,包括CD3⁺T细胞、CD4⁺T细胞、CD8⁺T细胞、CD19⁺B细胞、CD3^-CD16⁺CD56⁺自然杀伤细胞等淋巴细胞亚群的百分率,计算CD4⁺/CD8⁺比值。采用Pfirrmann分级标准评估2组腰椎间盘退变程度和分级,进一步评估外周血各淋巴细胞亚群与腰椎间盘退变程度的相关性。研究经郑州大学第一附属医院伦理审查委员会批准(伦理批号：2019-KY-285),所有受试者都签署了知情同意书。 结果与结论：①腰椎间盘退变性疾病组的腰椎间盘退变程度明显高于对照组(P < 0.05);②腰椎间盘退变性疾病组CD4⁺T细胞百分率、自然杀伤细胞百分率和CD4⁺/CD8⁺比值明显高于对照组(P < 0.05);腰椎间盘退变性疾病组CD8⁺T细胞百分率较对照组显著降低(P < 0.05);③对照组腰椎间盘退变程度与外周血各淋巴细胞亚群无相关性;腰椎间盘退变性疾病组腰椎间盘退变程度与CD4⁺ T细胞百分率、CD4⁺/CD8⁺比值、自然杀伤细胞百分率成线性正相关(r =0.412,P=0.000;r=0.715,P=0.000;r=0.494,P=0.000),与CD8⁺ T细胞百分率成线性负相关(r=-0.737,P=0.000);④结果表明,腰椎间盘发生退行性改变可能与外周血各淋巴细胞亚群改变有关,且CD4⁺T细胞增多、自然杀伤细胞增多以及CD4⁺/CD8⁺比值增高可能加速腰椎间盘退变。提示,免疫系统改变预示腰椎间盘退变发生的可能,其有望成为腰椎间盘退变性疾病的防治靶点。 ORCID: 0000-0002-8087-2356(冯阳) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Age-dependent separation of class-specific suppressor cells in thymus of SJL/J mice

The thymus of SJL/J mice of age 3-6 weeks has been previously shown to contain suppressor cells that inhibit the antibody response to lymph node cells to SRBC. The effect of these suppressor cells disappear as the animals age (24 weeks or more). We find that these aged animals acquire thymic suppressor cells which suppress the generation of cytotoxic T-cells both in vitro and in vivo. Although such suppressors are not present in the thymuses of young SJL/J mice, suppression can be induced by treatment with estrogen and progesterone. The differentiation of functionally different suppressor cell populations in thymus may be affected by both age and hormonal status.