MTS-32 monoclonal antibody defines CD4+8- thymocyte subsets that differ in their maturation level, lymphokine secretion, and selection patterns.

We have previously described MTS-32 as identifying an Ag on both thymic stromal cells and thymocytes. In contrast with CD4+8+ and CD4-8+ thymocytes, of which the vast majority are MTS-32+, a notable subset of CD4+8- thymocytes is MTS-32-. Here we show that with regard to heat-stable Ags, Qa-2, and CD69 expression CD4+8- MTS-32- thymocytes are phenotypically enriched in mature cells when compared with their MTS-32+ counterparts. Moreover, sorted CD4+8- MTS-32+ thymocytes are unable to respond to anti-CD3 cross-linking, whereas MTS-32- CD4+8- thymocytes respond to the same stimulus by producing IL-4, IL-5, IL-10, IFN-gamma, and trace amounts of IL-2. In addition, MTS-32- CD4+8- and CD4-8- TCR-alpha beta+ thymocytes differ in their TCR V beta repertoire on a Mls-1a selecting background. We therefore suggest that the MTS-32 ligand is involved in signals consecutive with TCR recognition in the thymus, i.e., selection, activation, and lymphokine production.

A kinetic study on the deletion of thymic, peripheral, and gut-associated V beta 6+ T cells in an Mls-1b BALB/c colony infected with an exogenous mouse mammary tumor virus.

Mls-1a expression results in the deletion of T cells bearing V beta 6 chains of the TCR. However, V beta 6+ T cells are also deleted in Mls-1b BALB/c mice that have been infected with an exogenous mouse mammary tumor virus (swiss mice) via maternal milk intake, and whose open reading frame region is markedly similar to that of the provirus Mtv-7. In this report we describe the kinetics of V beta 6+ T cell deletion in the thymus, spleen, lymph nodes, and gut-associated lymphoid populations of these BALB/c mice from the early weeks of life to 6 mo of age. Deletion of V beta 6+ T cells within the CD4+ T cell population was more obvious in the thymus than in the spleen at 8 wk of age. However, the earliest incidence of deletion was observed in the gut intraepithelial lymphocyte population at 5 wk of age. Furthermore Mtv-7 (SW) transcripts were only found in the gut in the first wk of life, whereafter they could be detected in the thymus, spleen, and lymph nodes. This report suggests that after entering the intestinal tract of host mice, mouse mammary tumor virus (swiss mice) is subsequently transferred to the thymus and peripheral lymphoid organs resulting in the deletion of CD4+V beta 6+ T cells in that order.

Tissue distribution of Mtv-7-like exogenous retroviral transcripts and clonal deletion of V beta 6+ T cells in Mls-1b BALB/c mice.

We have previously described an Mls-1a-like clonal deletion of mature CD4+ T cells which express V beta 6 and V beta 8.1 chains of the TCR in half of the mice of a BALB/c, Mls-1b colony (BALB/c IC). This occurs in the absence of the Mtv-7 provirus which is responsible for the clonal deletion in Mls-1a mice. We developed a polymerase chain reaction assay in order to study the presence of retroviral transcripts homologous to the viral superantigen gene (vSAG) of Mtv-7 and Mtv-6 in various tissues. Mtv-7 homologous transcripts were present in the mammary glands of lactating BALB/c IC mice and in the thymuses and/or spleens of BALB/c IC virgin mice with deletion of V beta 6+ lymph node T cells, and not in BALB/c IC with normal V beta 6 expression. These results indicate that this BALB/c colony is infected with an exogenous mouse mammary tumour virus (MMTV) whose vSAG is similar to Mtv-7, as recently reported. Thymectomies performed at 4-5 weeks of age (at least 4 weeks before detection of clonal deletion), did not affect the occurrence of clonal deletion in peripheral lymph nodes when tested 20 weeks later. This suggests that clonal deletion can be achieved without further intrathymic contact with the antigen. Since MMTV is transmitted through milk and is likely to be present in the gut, we evaluated the percentage of V beta 6+CD4+ T cells within the gut intraepithelial lymphocyte (IEL) population. Mice with normal V beta 6 expression in lymph nodes may show partial deletion of V beta 6+CD4+ IEL.(ABSTRACT TRUNCATED AT 250 WORDS)

Five novel antigens illustrate shared phenotype between mouse thymic stromal cells, thymocytes, and peripheral lymphocytes.

Previously we characterized by immunohistology a group of rat anti-mouse thymic stromal mAbs (MTS 12, 32, 33, 35, and 37), which recognized novel plasma membrane determinants on both thymic stromal cells (TSCs) and thymocytes. The present study investigates in more detail this incidence of shared phenotype by an extensive flow cytometric analysis of MTS mAb reactivity on TSCs, thymocyte subsets, peripheral lymphocytes, and bone marrow cells. Examination of freshly isolated or cultured heterogeneous TSCs and TSC clones confirmed that the mAb identified plasma membrane molecules on distinct subsets of these cells. All but MTS 12 reacted with epithelial cells. Triple-labelling illustrated that MTS 32, 33, and 37 were also reactive with more than 90% of total thymocytes, but varied in their distribution on the four major CD4 and CD8 defined subsets. MTS 12, staining with thymic vascular endothelium by immunohistology, labelled more than 95% of each subset. MTS 35 reactivity in each subset correlated strongly with only the immature populations. Examination of peripheral lymphocytes by triple- and double-labelling unexpectedly showed that MTS 33, 35, and 37 did not recognize peripheral T cells but labelled all B cells. MTS 32 was negative for B cells, but positive for all CD8+ T cells, yet only a subset of CD4+ T cells. Further, MTS 33, 35, and 37 were present on a significant percentage of bone marrow cells. MTS 12 reacted with virtually all peripheral T and B cells, and about 50% bone marrow leukocytes. Collectively these results reveal the same novel epitopes on different thymic cell types and subsets thereof, highlighting specific similarities between cells of apparently diverse lineages. These findings may be of importance in the delineation of intercellular communications within the thymus and emphasize the integrated nature of the microenvironment in this organ.

Thymic shared antigen-1. A novel thymocyte marker discriminating immature from mature thymocyte subsets.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.

The phenotypic heterogeneity of mouse thymic stromal cells.

Sixteen monoclonal antibodies (mAb) were produced against mouse thymic stromal elements. These mAb fell into two groups of reactivity: (i) thymic epithelial markers (screened and presented according to the guidelines proposed in the 1989 Rolduc Thymic Epithelial Workshop); and (ii) non-epithelial thymic markers. Specificities of these mAb included extensive subpopulations of both epithelial and non-epithelial thymic stromal cells, as well as isolated stromal cells, demonstrating some of the complex microspecificities in existence within the thymic microenvironment. Furthermore, six of these mAb demonstrated shared antigenicity between thymocytes and thymic stromal cells, revealing greater similarities than previously recognized between these two components. Three mAb detected antigens illustrating three consecutive layers of the blood-thymus barrier: the vascular endothelium; connective tissue of the capsule and perivascular spaces; and the connective tissue associated with the basal laminae lining these regions. This study illustrates unequivocably that there are indeed complex and varied microenvironments existing within the thymus, and emphasizes the need for reclassification of these cells.

Surface expression of CD4 and Thy-1 on mouse thymic stromal cells.

Subpopulations of thymic stromal cells (TSC) and T lymphocytes have been antigenically defined as a basis for understanding intrathymic cellular communications which result in the mature T-cell repertoire. In the current study we present data that the markers CD4 and Thy-1, standard T lymphocyte markers in mice, are also expressed by subpopulations of freshly isolated and cultured TSC. This was shown by flow cytometric analyses and fluorescence microscopy respectively. Use of F(ab')2 fragments of anti-CD4 mAb demonstrated specific antibody binding and not that due to FcR binding. Double-labelling experiments illustrated that the subpopulation of CD4+ stromal cells includes cells bearing the MAC-1 antigen. The majority of Thy-1+ stromal cells were epithelial cells (determined by double-labelling cultured TSC with anti-cytokeratin); however, there was also a novel subpopulation of non-epithelial stromal cells present. Whilst actual synthesis of these glycoproteins has not yet been demonstrated, their existence as surface antigens on TSC to any degree could have major relevance to the understanding of intrathymic intercellular events. It also cautions that experiments on T cell lineage relationships based on in vivo injection of anti-CD4 antibodies be interpreted with care.

Antigens shared by thymic stromal cells and T lymphocytes are abnormally expressed in AKR thymuses.

Accumulating evidence has shown that thymic stromal-lymphoid interactions play a major role in the development of AKR thymic leukemia. A normal thymic stromal cell (TSC) line B6TE-A, which has been shown to support the in vitro growth of AKR leukemic T cells by forming multicellular complexes with them, was used to raise monoclonal antibodies. Three of these mAb, MTS 31, 32 and 38, in addition to 2 other MTS mAb, are abnormally expressed in the preleukemic and/or leukemic stages in AKR mice. These 5 MTS mAb, which detect antigens on both subpopulations of TSC and T cells, show some reduced cortical reactivity from the pre-leukemic period (MTS 32 and 35) to markedly depressed reactivity in the leukemic period (MTS 31, 32, 33, 35 and 38). While it appears that the major reduction is due to the loss of antigens from the cortical thymocytes, there is some indication that the stromal elements may be affected also. In addition, MTS 29, which was also produced in this study, while only reacting with rare thymic medullary cells in situ was densely distributed on cultured stromal cells from both normal and leukemic thymuses. In this report, the value of these MTS mAb for documenting various stages of AKR leukemogenesis has been clearly demonstrated: their possible modulatory effects on in vitro T cell leukemia growth is currently being investigated.