Immunoregulation of lymphoproliferation in vitro by monocytes and their subpopulations. I. The induction of suppressor T cells in long term cultures and the role of MHC class II determinants and preliminary characteristics

Peripheral blood monocyte (MO) subpopulations isolated on a basis of functional expression or a lack of Fc receptor (FcR+ and FcR- MO) were used to study the regulation of the antigen (PPD)-driven lymphoproliferation. Long-term cultures of T lymphocytes with FcR+ MO, but not FcR- MO, led to the induction of T suppressor (Ts) cells that inhibited antigen-driven lymphoproliferation in the test cultures. These Ts cells were resistant to mitomycin C, belonged to afferent-acting category of Ts cells, expressed CD8 and HLA-DR determinants, and showed no antigenic specificity nor genetic restriction in action. The expression of MHC class II molecules (HLA-DR and HLA-DP but not HLA-DQ determinants) on MO which were used for antigen presentation was critical for Ts cell induction. It was concluded that specialized MO subpopulations may regulate the lymphoproliferation by inducing Ts cells (or Ts cell circuit) that in turn inhibit antigen-driven immune response.     

Behaviour of guinea pig T cells stimulated by antigen, allo-antigen and mitogen

The expression of major histocompatibility (MHC) antigens on guinea pig T cells was used as a functional marker for lymphocyte activation. Antigen-stimulated lymphocytes were recovered from guinea pigs responding to the contact sensitizer DNFB, and isolated T cells were then phenotyped using a new antiguinea pig monoclonal antibody, MSgp7. The level of expression of MHC class II, as defined by the monclonal antibody, MSgp8, was increased on T cells recovered 4 days after sensitization, as compared with unsensitized controls. The value of this experiment was extended by measuring MHC class II expression on T cells stimulated in vitro by the mitogen concanavalin A, where a clear increase in MSgp8 binding was also observed. Confirmation of the specificity of MSgp8 for guinea pig MHC class II antigens was achieved by studying the inhibitory capacity of this antibody on an MHC class II restricted mixed leucocyte reaction. The combination of antibodies MSgp7 and MSgp8 with flow cytometry could be applied to other guinea pig experimental models to quantitate the expression of MHC class II antigens on T cells to determine their putative value in disease manifestation.     

Professional presentation of antigen by activated human T cells

Activated human T cells express class II molecules, but their capacity to present soluble antigens and stimulate T cells has been repeatedly questioned. Two lines of evidence indicate that T cells may indeed function as professional antigen-presenting cells. First, T cells that have been recently activated can efficiently capture, process and present tetanus toxoid to class II-restricted T cell clones. This capacity correlates with the rate of class II synthesis. Second, activated T cell clones express high levels of B7, are powerful stimulators in mixed lymphocyte reactions, and their stumulatory capacity is inhibited by soluble CTLA4 or anti-B7 antibody. Furthermore, expression of B7 can be detected in vivo on T cells from biopsies of patients with liver disease. Presentation of soluble antigen by activated T cells may play a role in the amplification of the specific response, and possibly in immunopathological states.     

卵巢癌细胞株冻融抗原负载的树突状细胞诱导CTL抗卵巢癌免疫应答的体外研究

目的研究卵巢癌冻融抗原负载的树突状细胞(dendriticcells,DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤卵巢癌细胞的细胞毒性效应。方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34 细胞并在体外诱导分化为DC,用反复冻融法从卵巢癌细胞系SKOV3中提取的可溶性相关抗原负载DC。流式细胞学检测负载抗原后DC表面各种分化相关抗原的表达,ELISA法检测DC上清中IL12的表达,混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力,MTT法检测抗原负载DC激活的抗原特异性CTL对卵巢癌细胞的杀伤作用。结果与未经抗原负载的DC相比,经卵巢癌抗原负载的DC不仅能更高地表达各种DC分化相关抗原CD1α(73.35%±2.94%vs34.1%±2.35%)、CD83(73.9%±8.46%vs54.68%±3.26%)、CD80(91.95%±2.48%vs52.53%±3.18%)、HLADR(70.05%±2.35%vs48.7%±2.07%)以及CD54(88.9%±5.52%vs71.45%±2.29%),同时具有更强的刺激同种异体T淋巴细胞增殖和IL12分泌的能力(P均<0.05)。此外,卵巢癌细胞SKOV3冻融抗原负载DC激活的CTL在体外对SKOV3的杀伤率为77.35%,显著高于未经抗原负载的DC(P=0.0001)。结论经卵巢癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤卵巢癌细胞的作用。     

Stimulation of activated rat T cells in vitro by Listeria monocytogenes antigens.

A soluble extract of Listeria monocytogenes bound firmly and in similar amounts to a variety of rat cells. Cells that bound this material differed in their capacity to stimulate the in vitro proliferation of lymphocytes obtained from the thoracic duct of Listeria-immune donors. The capacity of cells to serve as antigen-presenting cells in this system coincided or closely overlapped the expression on these cells of an Ia antigen-like structure. Three lines of evidence indicate that T cells respond to L. monocytogenes antigen: the responder cells are members of a nylon-wool nonadherent population that lacks readily detectable surface immunoglobulin; they express determinants recognized by the W3/25 monoclonal antibody (a surface marker of rat peripheral T cells); and they are stimulated optimally by L. monocytogenes antigen when the latter is displayed on cells that share a haplotype with the responder lymphocytes.     

T lymphocyte subpopulations and Ia-positive T cells in patients with immunodeficiency.

T lymphocyte subpopulations (T gamma and Tmu) were studied in a group of 36 adult patients with immunodeficiency. Proportions and numbers of Ia(+) T cells were also studied in comparison to 46 normal adult controls. Values for per cent and total numbers of T gamma and Tmu cells indicated no uniform abnormality. Mean normal percentage of Ia(+) T cells was 2.4% whereas 16 to 29 immunodeficient patients showed elevated proportions and absolute numbers of Ia(+) T cells. Striking fluctuation in proportions of Ia(+) T cells was noted in serial studies of five immunodeficient subjects in contrast to similar analyses of normal controls. A correlation (P less than 0.01) was recorded between absolute numbers of Ia(+) T cells in immune deficiency patients and numbers of T mu cells. Depletion of T gamma cells by EA rosetting in patients with late-onset primary acquired hypogammaglobulinaemia did not result in significant change in IgG or IgM synthesis with T gamma-depleted T cells were co-cultured with normal B cells. Depletion of Ia(+) T cells likewise did not significantly influence Ig synthesis in co-culture with normal or immune-deficient B cells. These studies emphasize the complexity of defects present among any large group of patients with immune deficiency.     

Suppression of TNF by V antigen of Yersinia spp. involves activated T cells

The 39-kDa V antigen (Vag) of pathogenic Yersinia species has been described to be a potent suppressor of TNF production. The underlying cellular and molecular mechanisms, however, are completely undefined. Here we show that Vag does not act directly on macrophages, the primary source of TNF, but rather requires help of activated T cells for TNF suppression. Suppression of TNF strictly required the presence of T cell stimuli, i.e. concanavalin A or immobilized anti-CD3 antibody. In controls, suppression of TNF was completely blocked by anti-recombinant polyhistidine V antigen (rVagHis) IgG. As determined by transwell chamber experiments, suppression of TNF by rVagHis did not depend on cell-to-cell contact, indicating that it is mediated by an as yet unknown soluble factor. This is the first report to show a suppressive effect of rVagHis on TNF production in tissue culture. The results demonstrate the importance of activated T cells for suppression of TNF expression by rVagHis in vitro.     

In vitro stimulation with mitogen and antigen in patients with monoclonal gammopathy

Summary Peripheral blood lymphocytes from 32 patients with defined paraproteinaemia (16 IgG, 9 IgA and 7 IgM) and from 15 healthy donors were studied for their in vitro response to various stimuli, including unspecific mitogens such asPhytohaemagglutinin (PHA),Pokeweedmitogen (PWM) and Concanavalin A (ConA) as well as specific antigens such as purified Tuberculin, Candida, Varidase, Tetanus Toxoid, Vaccinia antigen and Vaccinia-control antigen.Mitogens and antigens were lyophilized in Microtiter plates. The lymphocytes of all tested patient-groups responded (measured by H3-Thymidin-up-take) significantly lower towards the unspecific mitogens than those of the control group. If the patients' lymphocytes were stimulated by the specific antigens, their in vitro response was significantly diminished to candida and vaccinia. Macroglobulinaemia showed significantly lower response to ConA if compared to myelomas of IgG- and IgA-type. No correlation was found between mitogen and antigen response and the serum concentration of the paraproteins or immunoglobulins. The results show that monoclonal gammopathy and especially macroglobulinaemia are associated with abnormalities of the cellular immunity which correlates with the clinical observation of increased fungal and viral infections.Supported in part by SFB 37 München and Euratom/GSF BIAD I 031-64     

High antigen dose and activated dendritic cells enable Th cells to escape regulatory T cell-mediated suppression in vitro

CD4+CD25+ regulatory T cells (Tregs) are critical for peripheral tolerance and prevention of autoimmunity. In vitro coculture studies have revealed that increased costimulation breaks Treg-mediated suppression in response to anti-CD3 or antigen. However, it was unclear whether loss of suppression arose from inactivation of Tregs or whether increased stimulation caused Th cells to escape suppression. We have investigated conditions that allow or override Treg-mediated suppression using DO11.10 TCR-transgenic T cells and chicken ovalbumin peptide 323-339-pulsed antigen-presenting cells. Treg suppression of Th proliferation is broken with potent stimulation, using activated spleen cells and high antigen dose, but is intact at low antigen dose. Costimulation with CD80 and CD86 expressed on activated dendritic cells was essential for Th cell escape from suppression at a high antigen dose. Potently stimulated Tregs were functional since they reduced levels of IL-2, IFN-gamma, IL-4 and Th CD25 expression in cocultures. Furthermore, Tregs responding to high antigen dose and activated splenocytes retained the ability to suppress proliferation, but only of Th cells responding to a sub-optimal dose of independent antigen. Together, our results demonstrate that under conditions of strong antigen-specific stimulation, Tregs remain functional, but Th cells escape Treg-mediated suppression.     

Generation of antigen specific cytotoxic T lymphocytes (CTL) directed against class II molecules expressed by activated T cells

Mitogen-activated T cells were used to provide a source of Class II antigens to CTL originally stimulated against mononuclear cells expressing both foreign Class I and Class II determinants. Our results indicated that 7-10-day-old activated T antigen-presenting cells, which shared only Class II antigens with the original priming cell, were able to stimulate the differentiation of CTL-recognizing Class II determinants. The use of 14-day-old activated T cells as target cells in the CML assay, compared with 72 h PHA blasts or 7-day-old activated T cells, enabled a more sensitive detection of the anti-Class II CTL.     

In vitro activation of human T and B lymphocytes by pokeweed mitogen.

In previous in vitro studies the DNA synthesis in human blood lymphocytes induced by low concentration of PWM correlated with the percentage of bone marrow-derived (B) lymphocytes in cell suspensions. No correlation with lymphocyte subpopulations was noted when lymphocytes were activated by high concentrations of PWM. In this paper the hypothesis that low concentration of PWM mainly activates B lymphocytes was tested by measuring the [14C]thymidine incorporation into purified T or B lymphocytes from healthy donors. B lymphocytes were purified to 90--95% by buoyant density centrifugation of T lymphocytes rosetted with sheep red blood cells. T lymphocytes were enriched by passage of lymphocytes through an IgG-anti-IgG-coated column. Low concentration of PWM-stimulated B lymphocytes but not T lymphocytes, while high concentrations of the stimulant activated both cell types. It was also noted that the B- but not the T-lymphocyte fraction contained cells which synthesized DNA in the absence of PWM.     

Lymphocyte subpopulations in man: induction of cytotoxic T lymphocytes in vitro by allogeneic B and T cells

Testing B and T cells as allogeneic stimulators of cytotoxic T lymphocytes in primary as well as secondary in vitro cultures, reveals that fresh, nonactivated B cells isolated from peripheral blood have an enhanced cytotoxic T cell stimulating capacity compared to T cells, although target determinants are present both on B and T cell blasts. Similarly, the capacity of T and B cells to stimulate proliferation in MLC is also quantitatively different. These results are in accordance with the hypothesis concerning the in vitro generation of alloreactive cytotoxic T lymphocytes, which postulates concurrent stimulation by strong lymphocyte activating determinants and target determinants for the generation of cytotoxic effector T lymphocytes, as both determinants are simultaneously found on B lymphocytes. Three cell experiments performed by coculturing allogeneic stimulating B and T cells with responding T cells show that strong lymphocyte activating determinants found on B cells enhance the cytotoxicity against target determinants on cocultured B cells but not on cocultured T cells, indicating qualitative differences between target determinants on B and T cells with respect to specific CTL stimulating capacity. Furthermore, primed resting CTLs in secondary cultures could unspecifically be restimulated by third party B cells or pokeweed mitogen. These results are the basis for a hypothesis concerning activation of CTLs, postulating nonspecific triggering of cytotoxic precursor cells by lymphocyte activating properties intrinsic to target determinants (TD) on B cells, preferentially activating clones of cytotoxic cells. The clonal proliferation is further unspecifically amplified by products of the T cell recognition of strong lymphocyte activating determinants (LAD).     

An antigen expressed by murine CD8+ T cells and activated B cells

A monoclonal antibody, H1F5, is described that reacts with a subset of Lyt-2 (CD8) mouse T cells and LPS-activated B cells. In both lymph nodes and spleen of BALB/c mice, the H1F5 antigen is coexpressed approximately on 20%-30% of the CD8+ T cells and approximately on 91% of LPS-activated B cells. In the thymus, few cells (less than 1%) are positive for the marker, but no correlation could be demonstrated with markers for mature T cells such as MEL-14 and PNA expression. Elimination of H1F5+ cells by complement lysis led to a 30%-50% reduction of specific lysis as measured in a primary allo CTL, indicating that the cytotoxic effector cells are injured. The relationship of this marker and other antigenic determinants on lymphocytes is discussed.     

Signal transduction by HLA class II antigens expressed on activated T cells.

Human T cells express HLA class II antigens upon activation. Although activated, class II+ T cells can present alloantigens under certain circumstances, the functional role of class II antigens on activated T cells remains largely unknown. Here, we report that cross-linking of HLA-DR molecules expressed on allospecific, CD4+ T clones and cell lines can function as transduction elements that trigger rapid cellular responses including tyrosine phosphorylation of cellular proteins and mobilization of Ca2+ from internal stores. The proteins phosphorylated on tyrosine were distinct from those observed after cross-linking CD4. Ligation of CD4 and class II molecules generated a synergistic effect of the intracellular free Ca2+ concentration response that required an interaction between the molecules on the cell surface. Since class II is the natural ligand for CD4, the present data suggest that class II is induced on activated T cells to regulate CD4 function, possibly by specific interaction with the CD4-associated p56lck protein tyrosine kinase.     

In vitro suppression of lymphocyte blastogenic response to mitogen and antigen by Leishmania tropica.

Cell-mediated immune (CMI) responses are important in the immunity against Leishmania spp. infection in man. However, an infection continues to persist for a limited or indefinite period of time in spite of demonstrable CMI. The factors which allow the infection to persist in the presence of the CMI are hitherto unknown. Evidence is presented here that Leishmania tropica or their products suppress the in vitro proliferative response of normal human lymphocytes to mitogen and specific antigens. The suppressive effect of L. tropica is neither due to a direct toxic action on lymphocytes nor to competition for nutrients or antigens. In vivo such an immunosuppressive effect could both facilitate macrophage parasitization and the intracellular survival of L. tropica, even after the CMI develops to processed L. tropica antigen. Persistence of infection is seen in many other bacterial, viral and fungal infections. The in vitro suppressive effect of L. tropica on the immune response observed in our study therefore becomes relevant to the understanding of the host-parasite interaction, which may determine the eventual outcome of infection in many other intracellular infections.     

Phenotypic and functional modulation of T cells in vivo by extrathymic T cells when T cells with MHC class II disparity were injected into athymic nude mice

TCR^high cells are generated by the mainstream of T cell differentiation in the thymus, whereas TCR^int cells (or NK1.1⁺ T cells) are generated extrathymically in the liver and by an alternative intrathymic pathway. It is still unknown how these T cell populations interact in vivo with each other. To investigate the interaction of TCR^int cells with TCR^high cells, we used congenitally athymic nude (B6-nu/nu) mice which carry only TCR^int cells in all immune organs. When TCR^high cells from B6-C-H-2^bm12 (bm12) mice (i.e. I-A^bm12) were injected into B6-nu/nu mice (i.e. 1-A^b), the expanding T cell population was a mixture of TCR^high cells of donor origin and TCR^int cells of recipient origin. However, 9 Gy-irradiated nude mice permitted a full expansion of TCR^high cells which expressed the IL-2Rα⁺β⁺ phenotype, namely, they were at the most activated state. These mice died of acute graft-versus-host disease (GVHD) within 5 days. On the other hand, non-irradiated nude mice suppressed the expansion of TCR^high cells of donor origin and such TCR^high cells continued to have the IL-2Rα^±β⁺ phenotype. These mice could survive but showed signs of chronic GVHD thereafter. In both situations, CD4⁺αβ T cells expanded irrespective of donor or recipient origin. These results suggest that TCR^int cells in the recipient mice possess a regulatory function in relation to donor TCR^high cells; as a result, fully activated TCR^high cells acquired the IL-2Rα⁺β⁺ phenotype and injured the host, but TCR^high cells suppressed in vivo remained as the IL-2Rα^±β⁺ phenotype and only partially injured the host.     

Immunoregulation by targeting T cells in the treatment of allergy and asthma

T-cell responses to allergens are crucial in determining the choice between health and disease. Th2 responses drive synthesis of IgE and the recruitment, maturation, survival and effector function of accessory cells such as eosinophils, basophils and mast cells. Allergen-specific strategies for targeting T-cell responses in established allergic diseases have been employed with success for almost a century. Recently, new insight into the mechanism of action of such approaches has revealed modulation of the delicate and complex regulatory mechanisms including suppression of Th2 responses through antagonistic Th1 responses and regulation through IL-10 and TGFbeta production.