Limiting dilution analysis of the suppressive effect mediated by alloantigen-primed cells

T cells primed in mixed lymphocyte culture exert both positive and negative allogeneic effects on B cells expressing the appropriate alloantigens. The positive and negative effects can be separated by limiting dilution analysis: positive effects, measured by production of anti-sheep erythrocyte antibody, are revealed when low numbers of primed T cells are added to cultures of B cells and sheep erythrocytes, while suppression of the response occurs at higher T-cell inputs. In the present report, these negative allogeneic effects have been analysed in detail. Suppression was qualitatively and quantitatively similar when helper T cell activity was provided from any of several sources. Helper T cells in the alloantigen-primed population gave rise to active T-cell replacing factors even under conditions in which all microcultures were suppressed and suppressor cells were present at a high multiplicity in every well. The degree of suppression was influenced by the multiplicity of B cells in culture; as the number of B cells increased, more suppressor cells were required to inactivate a microculture. Taken together, these data indicate that the targets of the suppressor cells are B cells and not helper T cells or T-cell replacing factors. Although suppressor cells can prevent the activation of B cells by the more frequent helper cells in the primed T-cell population, detailed analysis of the stoichiometry of the suppression demonstrated that a single suppressor cell is capable of inactivating only a limited number of B cells, suggesting that a 'ratio-dominance' model of suppression is operative in this system.     

Specificity of antigen recognition by normal thymus cells in nude mice

If injected with normal thymus cells from BALB/c or BALB/c-Ig^b, nude mice from a stock which is partially backcrossed to BALB/c make antibodies to many antigens to which untreated nude mice do not respond. Anti-Ig^b can be made if the thymus donor is BALB/c, but not if the thymus donor is BALB/c-Ig^b. If cells from both donor strains are injected, a response is obtained which shows that the unresponsiveness of recipients of BALB/c-Igb cells is not due to tolerance of donor antigens.     

Idiotype-specific neonatal suppression of phosphorylcholine-responsive B cells.

The effect on neonatal anti-idiotypic suppression on the expression of B cells of the T15 clonotype has been investigated at the level of individual clonal precursor cells. The results indicate that B cells of the T15 clonotype are almost completely eliminated from the repertoire for four months after neonatal injection of allogeneic anti-idiotypic serum. The degree of this suppression is dependent on the amount of anti-idiotypic antibody administered and is less profound if anti-idiotypic antibody is given after the first week of life. No suppression was observed when anti-idiotypic antisera were administered to mice 30 days of age or older, which may indicate that immature B cells are the population most susceptible to suppression. However, since suppression could be reversed by administration of T15 myeloma protein several days after injection of anti-idiotype, the inability to suppress adult BALB/c mice may have been due to the high level of T15 idiotype normally present in their serum. Finally, phosphorylcholine-responsive B cells of identifiable clonotypes other than T15, even a clonotype sharing antigen-combining site determinants with T15, appear unaffected by anti-T15 suppression.     

Differential effect of cyclosporin application on epithelial cells of the rat thymus. Immunohistochemical study.

Young adult male Wistar rats were given 30 mg per kg of cyclosporin (CS) for 21 consecutive days. A panel of monoclonal antibodies was used to study the phenotype of thymic epithelial cells. After treatment with CS, subcapsular epithelial cells, although phenotypically similar to medullary epithelial cells, were changed in a similar manner to phenotypically distinct epithelial cells of the deep cortex. These cells became enlarged, stockier and their cytoplasmic prolongations were thicker and coarser compared with control cells and their number was not decreased. In contrast, the number of medullary epithelial cells was markedly reduced, whereby the cells with the most mature phenotype (CK8+10-19- and CK8+10+19-) were the most prominently depleted. No proliferation of thymic epithelial cells was detected as monitored by incorporation of 5-bromodeoxyuridine.     

The effect of antigen stimulation on the migration of mature T cells from the peripheral lymphoid tissues to the thymus

Although the maturation and export of T cells from the thymus has been extensively studied, the movement of cells in the opposite direction has been less well documented. In particular, the question of whether T cells which have been activated by antigen in the periphery are more likely to return to the thymus had been raised but not clearly answered. We examined this issue by activating T cells present in the periphery with their cognate antigen, and assessing migration to the thymus. TCR-transgenic cells from OT-I mice (Thy1.2+), which recognise the ovalbumin peptide OVA257-264 in the context of H-2Kb, were transferred into otherwise unmanipulated Thy1.1+ C57BL/6 mice. Recipient mice were injected i.v. with 5 microg peptide (SIINFEKL) approximately 24 hours later. The numbers of donor-derived (Thy1.2+) cells in the thymus and peripheral lymphoid tissue were determined. The results clearly show increased numbers of transgenic cells in the thymus 3 days after antigenic stimulation. However, since numbers of transgenic cells increased in the spleen and LN in about the same proportion, the data do not support the notion that there is highly increased selective migration of activated T cells to the thymus. Rather, they suggest that a sample of peripheral cells enters the thymus each day, and that the mature immigrants detected in the thymus merely reflect the contents of the peripheral T cell pool.     

Contribution of thymus lymphocytes to the peripheral lymphoid tissues and the effect of antigen on the rate of cell exit from the thymus

Some of the important questions concerning the development of T cells in the thymus can be answered by a study of the different thymocyte subpopulations and a comparison of their properties with those of the cells exported to the peripheral lymphoid tissues. What is the relationship between cortical and medullary thymocytes? Why do most cortical cells die? Which subpopulation gives rise to thymus migrants? How many cells are exported from the thymus? Are the exported cells fully mature? Are any of these functions affected by antigen stimulation or other peripheral events? In this paper we review the background to some of these questions and focus on the effect of peripherally administered antigen on the export of cells from the thymus. Experimental data are presented which suggest that the overall rate of emigration is not grossly affected by large doses of intravenous protein antigens. Nor is there any obvious qualitative change, at least in terms of the size of the cells released. The possibility of changes in the specificity of the exported cells is discussed, but as yet there are no data which throw light on this point.     

Anergic T cells effect linked suppression

Although the phenomenon of T cell-mediated suppression is well established, particularly in experimental models of transplantation, the mechanisms involved in this form of immunoregulation remain controversial. We have recently demonstrated, using an in vitro system, that anergic T cells can act as suppressor cells by competing for the membrane of the antigen-presenting cell (APC) and for locally produced interleukin-2. In the experiments described here we have explored the ability of anergic T cells to effect linked suppression in antigen-specific and allospecific responses. We observed that anergic antigen-specific CD4⁺ T cells can inhibit T cells restricted by a different major histocompatibility complex (MHC) class II molecule provided that both restriction elements are expressed by the same APC. In addition, anergic allospecific clones could also effect linked suppression since they could regulate not only T cells specific for the same alloantigen but also responder T cells with direct allospecificity for a second allogeneic MHC molecule or with indirect, self MHC-restricted allospecificity for a processed MHC class I alloantigen. Furthermore, the regulatory effect of the anergic T cells was dependent on cell contact, was not dependent upon irradiation, and was maintained during in vitro culture. These data demonstrate that linked suppression can be effected by anergic T cells in vitro. In the clinical context this raises the possibility that induction of tolerance to a single alloantigen could serve to regulate the immune response to an allograft carrying several MHC and minor antigen differences.     

Immune suppression induced by acetoacetylated antigen.

Injection of acetoacetylated antigen into rabbits with an ongoing reagin response abrogated this response. The possibility that this phenomenon might be due to activation of suppressor cells was studied in mice. Thymus or spleen cells from animals which had been primed with acetoacetylated antigen were able to suppress the IgG and IgM antibody response upon transfer to syngeneic mice. Maximal suppressive effect was observed 12-14 days after priming with native as well as acetoacetylated antigen. The IgG and IgM responses were equally affected by the transferred suppressor cells.     

Stimulation and suppression of contact dermatitis in mice by low-molecular-weight thymus humoral factor

The effect of a low-molecular-weight lymphocytosis-stimulating substance (LSS) from the thymus on the development of contact sensitivity to picryl chloride was investigated in mice. Small doses of LSS were found to potentiate, whereas large doses suppressed this type of delayed hypersensitivity. Contact sensitivity can be transferred passively by means of lymph node and spleen cells isolated on the 6th day after immunization. The experiments showed that mice receiving large doses of LSS contain cells which suppress the passive transfer of contact sensitivity by immune cells. This suppression was absent after treatment of the cells with -antiserum and complement. It is concluded that the suppressor cells influence the effector phase of contact sensitivity.Laboratory of Immunochemistry of Hormones, Institute of Endocrinology and Metabolism, Ministry of Health of the Ukrainian SSR, Kiev. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Gorev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 572–575, May, 1978.     

Implication of the Thy-1 antigen on mouse thymus cells in the recognition of 'self' structures

Thymus cells of mice form rosettes with autologous and allogeneic erythrocytes. The nature of the thymus cell receptors which mediate the binding of erythrocytes is not known. The aim of the present study was to determine the effect of various antisera to T-cell specific antigens on the formation of rosettes by mouse thymus cells. In all strains of mice tested, the exposure of thymus cells to rabbit anti-mouse brain serum (RABR) was found to inhibit autorosette formation. Similarly, monoclonal Thy-1 antibodies inhibited the formation of autorosettes by thymus cells in all strains tested. Monoclonal antibodies against Lyt-1, Lyt-2, and TL determinants had no such effect. Monoclonal Thy-1 antibodies inhibited the formation of rosettes with allogeneic erythrocytes only when the thymus contained the Lyt-2.2 allele (BALB/c, C57BL), but not when it contained the Lyt-2.1 allele (AKR/J, C3H, DBA/2). These results indicate that Thy-1 determinants on thymus cells are involved in the recognition of 'self' structures, shared by erythrocytes of all strains of mice. Lyt-2 determinants may play a role in the recognition of 'non-self', allogeneic determinants, but the thymus cell surface structures encoded by the two Lyt-2 alleles may differ in their affinity to allogeneic erythrocytes.     

Inside the thymus,Mls antigen is exclusively presented by B lymphocytes

The ability to stimulate an Mls-1 mixed lymphocyte reaction (MLR) is predominantly expressed by low density B lymphocytes in the spleen and peritoneal cavity of normal adult mice, and is absent in splenic B cells 1 month after lethal irradiation and reconstitution from autologous bone marrow. Coreconstitution of these mice with normal syngeneic peritoneal cells restores the stimulatory potential of splenic B cells, but sorted CD5⁺ or CD5⁻ IgM⁺ lymphocytes from peritoneum are equally good stimulators, suggesting that functional Mls-1 expression may require long life spans and selection. Bone-marrow-reconstituted DBA/2 mice that fail to express Mls-1 antigens in the periphery nevertheless maintain T-cell receptor Vβ 6 and 8.1 deletions among the newly formed T cells. These findings led us to directly investigate the Mls stimulatory ability of purified antigen-presenting cell populations inside the thymus. We report here that thymic B lymphocytes seem to represent the only intrathymic cell population able to stimulate Mls-1 MLR.     

人工抗原提呈细胞的制备和应用的研究

目的 制备人工抗原提呈细胞（artificial antigen presenting cell,aAPC）并用它从HLA-A2阳性健康个体外周血单个核细胞（PBMC）中诱导和扩增特异性细胞毒性T淋巴细胞（CTL）.方法 将HLA-A2-EBV四聚体分子和抗CD28抗体分子吸附固定在细胞大小的聚苯乙烯乳胶微球（5μm）表面制成aAPC;采用流式细胞仪表型分析;aAPC和HLA-A2阳性个体人外周血单核细胞进行混合淋巴细胞反应;用HLA-A＊0201-EBV四聚体染色法检测特异性CTL的频率;应用细胞内细胞因子染色法检测特异性CTL功能性细胞因子IFN-γ的分泌;采用LDH释放法检测特异性CTL的特异杀伤活性.结果 流式细胞仪分析显示微球表面吸附有HLA-A2-EBV四聚体分子和抗CD28抗体分子;四聚体检测及细胞内细胞因子染色法检测与经典细胞毒试验结果一致,结果表明aAPC在体外可诱导抗原特异性CTL的生成.结论 aAPC制备成功,并在体外有效地诱导抗原特异性CTL的生成.     

Progressive restriction of antigen binding by human foetal thymus lymphocytes.

The proportion of foetal thymus lymphocytes (FTL) that binds the bacterial antigens beta-galactosidase and flagellin is high in early foetal life. Binding of beta-galactosidase, and the response by FTL in mixed lymphocyte culture falls during gestation. Some FTL bound both antigens, suggesting that immature lymphocytes are not fully restricted in their capacity to recognize antigens. Such findings have been reported in foetal lymphocytes from other species. We suggest that cellular diversity may partly be generated by progressive restriction of antigen recognition by individual lymphocytes, which may result from progressive stabilization of genetic repression during lymphocyte multiplication.     

Comparison of antigen presentation by lymph node cells from protein and peptide-primed mice.

Lymph node cells from mice primed with peptides from the allergens Der p I and Der p II (the group I and II allergens of Dermatophagoides pteronyssinus) were unable to recall responses to the protein antigen when cultured in vitro despite being able to mount large responses to the peptides. The T cells could however recall responses to the protein when spleen-adherent cells were added into culture. Treating the spleen accessory cells with the monoclonal antibody (mAb) 33D1 and complement largely abrogated the protein response of peptide-primed T cells which indicates that dendritic cells were mainly responsible for the antigen-presenting function. If mice were primed with two injections of peptide the lymph node cells obtained could respond to both protein and peptides in vitro without the need for exogenous accessory cells. Using either negative depletion with the J11D mAb or positive purification, it was found that the presentation of protein antigen to lymph node T cells primed with either protein or peptide was limited to antigen-specific B cells. Peptide antigens could however be presented by both B and non-B populations. In one case the peptide 105-129 from Der p II which contains a T-cell epitope could not be shown to induce T-cell responses in the lymph node unless presentation was mediated by spleen-adherent or B-specific cells. These results are important for peptide-based immunomodulation and in interpreting results obtained from lymph node cultures.     

Immunoglobulin synthesis by thymus B cells.

Rabbit lymphoid cells transferred to newborn recipients synthesized donor-type immunoglobulin. Elimination of donor T cells did not affect this synthesis of donor immunoglobulin, while elimination of B cells abolished or significantly decreased the synthesis. The synthetic capacity of B cells from thymus was thirty-four times greater than the synthetic capacities of B cells from spleen, fifty-five times greater than that of mesenteric lymph nodes and 180 times greater than that of appendix cells. Synthetic activity of spleen cells ceased before the tenth day after transfer, while thymus cells might continue to synthesize immunoglobulin for a longer time. This was shown by comparing half-lives of donor immunoglobulin in the recipients' sera. Increasing the number of injected spleen cells (from 2 x 10(6) to 120 x 10(6)), resulted in a corresponding increase in donor Ig synthesis. With thymus cells, donor immunoglobulin increased with cell numbers up to 2 x 10(7) cells, above this dose there was no further donor immunoglobulin increase in the recipients' serum.     

Rat thymus macrophages: an immunohistochemical study on fetal, neonatal and adult thymus

The pre- and postnatal development of the macrophage population of rat thymus is investigated applying enzyme-histochemical and immunohistochemical techniques, both on tissue sections and cell suspensions. A set of three monoclonal antibodies (ED1, ED2 and ED3), each of which recognizes cells of the monocyte-macrophage lineage in the rat, enabled us to distinguish between macrophages in the various compartments of the thymus. The medulla is characterized by ED1-positive dendritic cells, the corticomedullary region comprises numerous monocyte-like ED1-positive macrophages and the cortex contains a particular subpopulation of branched ED2-positive macrophages. Both the medullary dendritic cells and the cortical branched cells show Ia-membrane staining. ED3-positive cells are only occasionally present. During fetal life ED1-positive monocyte-like macrophages and dendritic cells are present. Just after birth ED2-positive cortical macrophages start to develop. Their number increases strongly during the first week after birth. The role of the various subpopulations of thymic macrophages is discussed.