J. Rozing

Intrathymic Differentiation in the Rat

In view of our interest in the role of various subpopulations of T lymphocytes in the process of immunological tolerance induction or rejection after allogeneic renal transplantation in an experimental rat model, we have started to produce monoclonal antibodies against cell surface determinants of cell types at various stages along the T cell lineage. Such cell types may represent an early phase in T cell differentiation, such as thymocytes or even prothymocytes, or more mature and functionally differentiated stages, such as T helper or T suppressor/cytotoxic cells. This paper describes five new mouse anti rat monoclonal antibodies. Three of these recognise distinct subpopulations in peripheral lymphoid organs. From comparative studies with existing markers in the rat [1], it can be concluded that ER2 reacts with the helper subset, whereas ER3 and ER10 react with the suppressor/cytotoxic subset. ER4 and ER14, on the other hand, do not react with peripheral cells in adult animals. These antibodies recognise different determinants on the 25 KD THY-1 glycoprotein, which are eaqpressed dissimilarly during T cell differentiation. Both are present on almost all thymocytes, but in contrast to ER14 the expression of the ER4 marker on cortical cells exceeded that of medullary cells by manyfold.

LYMPHOCYTE MIGRATION ACROSS MAJOR HISTOCOMPATIBILITY BARRIERS IN SPLENECTOMIZED RATS

Localisation and migration patterns of iv injected radio-labelled thoracic duct (TD) lymphocytes were studied in particular with regard to passage through lymph nodes and re-entry into thoracic duct lymph. To avoid unwanted splenic sequestration of migrating lymphocytes presenting alloantigens to the recipient, only splenectomized recipients were used. Donor cells and recipients differed at the MHC (RT-1) locus, either in fully allogeneic (AO -- greater than BN and v.v.) or semi-allogeneic (AO -- greater than AO X BN and v.v.) combinations. In two of these combinations (BN -- greater than AO and AO X BN -- greater than AO) deficient output in TD lymph correlated with deficient localisation in lymph nodes and high amounts of radioactivity in the liver. In the other allogeneic combination (AO -- greater than BN), however, high TD output (i.e. when compared with the syngeneic combination BN -- greater than BN) correlated with good localisation in lymph nodes and low (control) levels of radioactivity in the liver. It was postulated that lymphocyte migration from blood to lymph under these circumstances can only be studied as an artifact secondary to whether or not migrating cells are removed from the circulation before they can reach and cross HEVs. These Allogeneic (or Altered) Lymphocytes Removing Tissues (by definition: Extranodular) may (conceptually) be comprised within one system: ALERT. It is our working hypothesis that the study of lymphocyte migration across (major) histocompatibility barriers is seriously impaired by the functioning of ALERT. It might be worthwhile to try and create conditions in which interference by this system is prevented, e.g. by using tolerant animals or bone-marrow chimeras.

“T-Cells” in Nude Rats

There are several reports that congenitally athymic nude mice and rats have cells with T-cell markers in spleen and lymph nodes. For the nude mouse, the numbers of these cells increase with age.1 In athymic rats we found cells with T-cell markers in T-dependent areas of mesenteric lymph node and Peyer’s patches, especially at ages older than 35–40 weeks.2 This observation prompted us to study in detail the presence of cells with T-cell phenotypes in lymphoid organs of nude rats as a function of age. Lymphoid organs of 8, 12, 20, 36 and 68-week-old nude rats were analysed by both flow cytometry (FACS) on cell suspensions and by immunohistochemistry on tissue sections. A panel of monoclonal antibodies to T-cell subpopulations was applied. The proliferative response after mitogenic stimulation was simultaneously investigated. In these assays athymic nude rats were compared with age- and sex-matched immunocompetent littermates.

Pre B Cell Leukaemia in the Rat

Recently a case of spontaneous B cell leukaemia has been reported in mice (1). In rats, a transplantable lymphoid leukaemia, which arose originally in a rat undergoing chronic internal beta-irradiation of the spleen, has been described by Dibley et al. (2,3). Based on the absence of surface immunoglobulin molecules and Fcreceptors, and the presence of the Thy-1 determination the surface of the leukaeraic cells, as well as on the appearance of pyroninophilic lymphoblasts in T dependent lymphoid areas after inoculation of syngeneic recipients with tumour cells, this malignancy has been designated as a T cell leukaemia (2). We report here some of the characteristics of a pre B cell leukaemia derived from the successfully transferred in (AOxBN)F1 hybrid rats.

The Recovery of the B Cell Compartment in Lethally Irradiated and Reconstituted Mice

Reappearance of immunological responsiveness after lethal irradiation and reconstitution with hemopoietic stem cells is dependent on the recovery of two types of lymphoid cells: B and T cells. The B cell compartment seems to recover faster than the T cell compartment (1, 2). Nossal and Pike compared the rate of recovery of anti-immunoglobulin binding cells in different lymphoid organs. They found a somewhat faster repopulation in the spleen than in lymph nodes and bone marrow (3, 4). Everett and Tyler (5) provided evidence that in normal mice the marrow is the major site of lymphocyte production. These lymphocytes are probably immature stages of the B cell line (6).

PRE-B-CELLS IN RAT BONE-MARROW - IDENTIFICATION, SURFACE-MARKERS AND ISOLATION

In adult rats, as in other species, bone marrow has the highest potential for B-lymphocyte genesis as determined by a long-term repopulation assay for B-lymphocyte stem cells (1). These cells carry the Thy-1 and W3/13 antigens (2, 3). Pre-B cells characterized by the presence of µ heavy chains in the cytoplasm (cµ) but not on the cell surface (sµ) have been described in mice (4), men (5) and rabbits (6) but not in rats, and appear to be the immediate precursors of B-lymphocytes (7–10). The relation between pre-B cells and B-lymphocyte stem cells is not clear, however. Therefore, the aims of the present study were to identify cµ+sµ- pre-B cells in rat bone marrow and to find surface markers that would permit isolation of viable pre-B cells to subsequently assay their functional potential in vivo.

Monoclonal Antibodies Against Rat T Cells

In view of our interest in the role of various subpopulations of T lymphocytes in the process of immunological tolerance induction or rejection after allogeneic renal transplantation in an experimental rat model, we have started to produce monoclonal antibodies against cell surface determinants of cell types at various stages along the T cell lineage. Such cell types may represent an early phase in T cell differentiation, for example thymocytes, or more mature and functionally differentiated stages, such as T helper (1) or T suppressor cells (2). The present paper describes a primary analysis of three monoclonal antibodies, derived after fusion of the spleens of BALB/c mice immunized with rat thymocytes, with the mouse fusion line FO.