Martine Papiernik

EVIDENCE FOR A SERUM-FACTOR SECRETED BY THE HUMAN THYMUS

Abstract A factor in human serum modifies the rosette-forming cells present in the spleen of adult thymectomised mice. The level of this factor is higher in children than in adults, and this factor is not found in subjects over 50 years old. The level of this factor is normal in patients with myasthenia gravis who are less than 30 years old, but in these patients it does not decrease with age. In eight patients with myasthenia gravis the factor diasppeared from the serum within 24 hours of thymectomy. These results agree with those in mice and suggest that there is a thymic hormone in human serum.

Thymocyte subpopulations in young and adult mice. III. Qualitative and quantitative electron microscopic study of steroid resistant and steroid sensitive populations.

Abstract In an attempt to correlate morphological aspects of thymic lymphocytes with functional criteria, thymocytes from normal and steroid-treated mice were studied. Electron microscopy was performed on the whole cell suspension or after separation on a density gradient. Steroid sensitivity and phytomitogen responses of the different density subpopulations were compared to the morphological aspect. Steroid-resistant (Sr) and steroid-sensitive (Ss) populations were shown to be heterogeneous populations on the basis of their morphological structure and density. Sr cells are highly phytomitogen sensitive in all the density fractions. The three different cell types, which may represent different steps in the maturational procedure, are present in the Sr and in the Ss populations, but in different proportions. These results represent an additional argument for the hypothesis that the two functional pools of thymic lymphocytes may be two different types of cells, following different developmental pathways.

Thymic medullary lymphocytes: I. Lymphokines produced by thymic medullary lymphocytes as a second signal for intrathymic stem cell homing

Abstract Lymphokines produced by thymic medullary cells (TMC) or by normal spleen cells after allogeneic stimulation have been tested for their chemotactic properties in an in vitro migration test. Lymphokines produced by TMC specifically attract cells from nude spleen or from T-cell-deprived spleen depleted of macrophagic cells, but not from normal adult spleen. Supernatants produced by normal adult spleen cells did not attract any of the cells tested (nude spleen cells, T-cell-deprived spleen cells, or normal spleen cells). These results suggest a role for mature TMC in intrathymic stem cell homing. These cells could deliver a second signal to the stem cell, complementary to those provided by thymic epithelium.

Control of helper-T-cell proliferation by recognition of la and Mac-1 antigens on phagocytic cells of the thymic reticulum

Phagocytic cells of the thymic reticulum (P-TR) have been previously described as being Ia-positive, Mac-1-positive accessory cells which pursue a close relationship with thymocytes. They form rosettes with thymocytes, and these rosettes are inhibited by antibody directed against the complement receptor type 3 CR3 (anti-Mac-1). P-TR induce the proliferation of syngeneic thymocytes. In the present paper, we show that thymocytes enriched in mature medullary type are induced to proliferate in coculture with syngeneic P-TR, while the cortical type does not. After 5 days of culture, 85% of the thymocytes are of helper L3T4+Lyt-2- phenotype. As previously shown by others for syngeneic reactions, antibodies directed against related class II antigens (anti-I-A and anti-I-E) block this helper-T-cell syngeneic proliferation. A new finding is the blockage of helper-T-cell proliferation by anti-Mac-1 as well as with anti-LFA-1 antibodies, showing that accessory molecules may be as important as specific recognition of class II antigen molecules in the control of thymocyte proliferation and hence in thymocyte selection. Mac-1, like LFA-1, belongs to a novel family of differentiation antigens involved in cell interactions. The blockage of cell recognition and interaction between P-TR and thymocytes by either anti-Ia or anti-Mac-1 during the early induction phase of the syngeneic response leads to its inhibition. We demonstrate that P-TR/thymocyte interaction stimulates the enhanced expression of IL-2 receptors on thymocytes, a step which is necessary for helper-T-cell proliferation. The mechanism of syngeneic proliferation inhibition by anti-Ia, anti-Mac-1, and LFA-1 antibodies may be the prevention of IL-2 receptor expression on thymocytes, and/or the inhibition of IL-2 secretion. Although this is an in vitro model, which may not totally reflect in situ situation, our results indicate that thymic accessory cells may participate in a positive selection process which leads to helper-T-cell proliferation.

Role of the spleen in ontogenic development of phytomitogen response in thymus of CBA mice.

Abstract Postnatal splenectomy increases the thymic proliferative response to concanavalin A. This effect is maximum when splenectomy is performed during the first 10 days of life at the time of the maximum response of thymocytes to the mitogen. These data suggest the existence of a negative control of the spleen on thymus development.

Thymic lymphocytes: II. Phenotypic modifications of thymocytes after concanavalin A stimulation in the presence of interleukin 2: Early modifications of Lyt 1+2+ subset and later proliferation of cells with more mature phenotypes

Cortical thymocytes are devoid of any immune function, as tested by presently available techniques. The ability of this subpopulation to respond to mitogens or antigens in the presence of interleukin 2 (IL-2) produced by activated mature T lymphocytes has been claimed but is still questioned. In an attempt to study the participation of the different thymocyte subsets and especially that of the cortical type, phenotypic modifications were examined during concanavalin A activation in the presence of IL-2. An immunofluorescent double labeling technique with anti-Lyt 1 and anti-Lyt 2 antibodies was used which led to the determination of four different phenotypes: Lyt 1+2+, Lyt 1+2-, Lyt 1-2+, and Lyt 1-2-. Careful analysis of cell viability in culture and expression of the results in absolute numbers of living cells per culture allowed us to follow modifications of small cellular subsets. Cultures of total thymocytes and PNA-agglutinated (enriched in Lyt 1+2+ cells) and non-PNA-agglutinated cells (enriched in Lyt 1+2-, Lyt 1-2+, and Lyt 1-2- cells) were studied. It was shown that thymocyte activation began by early phenotypic modifications which took place within the first 2 hr of culture but only when Con A plus IL-2 were used. These modifications imply the reduction of the Lyt 1+2+ pool and a compensatory enhancement of Lyt 1-2+ and Lyt 1-2- cells, without modification of the total cell number or [3H]thymidine incorporation. These early phenotypic changes are interpreted as the modulation of antigens on the surface of Lyt 1+2+ cells. The second phase of thymocyte activation implies cell death (essentially Lyt 1+2+ cells) and cell proliferation. The cells which specifically proliferate in the presence of Con A and IL-2 are Lyt 1+2- and Lyt 1-2+, the latter always being present in greater number. Cell survival and absolute number of Lyt 1+2- and Lyt 1-2+ cells in the activated PNA- -enriched population are always higher than in total thymocyte and PNA+ cells cultures. Thus, if Lyt 1+2+ cortical thymocytes do not proliferate by themselves, they seem to intervene by providing Lyt 1-2+ cells which proliferate secondarily.

Effect of cytokines on thymic hematopoietic precursors

SummaryWe have previously shown that the interaction of thymocytes with thymic accessory cells (macrophages and/or interdigitating cells) is one of the factors required for thymocyte activation. Precursors of both thymic accessory cell and thymocytes are included in the CD4- CD8- Mac-1- Ia- subpopulation, and their respective maturation and/or activation may be modulated by granulocyte-macrophage colony-stimulating factor, interleukin 1 and interleukin 2. When CD4- CD8- thymic cells are activated with granulocyte-macrophage colony-stimulating factor plus interleukin 2, both macrophages and interdigitating-like cells are present, as shown by electron microscopy. When activated with interleukin 1 plus interleukin 2, the interdigitating-like cells is the only accessory cell present. In both culture conditions, large clusters are formed between interdigitating cells and lymphoid cells. These results have led us to propose two-step signals for thymocyte proliferation: first, the maturation of macrophages under granulocyte-macrophage colony-stimulating factor control and the production of interleukin 1, and secondly, the maturation of interdigitating cells under interleukin 1 control, their clustering with thymocytes which are then activated.

Definition of a differentiation antigen on the surface of phagocytic cells of thymic reticulum which is down-regulated by interferon γ☆

Monoclonal antibodies (MoAb) were raised against phagocytic cells of thymic reticulum (P-TR) grown in vitro. Each of the two MoAb (TR-1N, TR-3N) defined two polypeptides of 46-57 kDa on P-TR membrane. TR-1N and TR-3N recognize respectively 48 and 81% of P-TR, but do not recognize any cells in spleen, lymph node, thymic lymphocytes, or bone marrow. They bind to part of peritoneal macrophages and to macrophage cell lines J 774 and P 388 D1. Cell binding of TR-1N and TR-3N was compared by immunofluorescence to that of anti-CR3 antibody (Mac-1) which recognizes P-TR, a small number of cells in bone marrow and spleen, and a much higher percentage of peritoneal macrophages. The polypeptides recognized by TR-1N/TR-3N may be defined as differentiation antigens on accessory cells as they appear on bone marrow cells during maturation in vitro in the presence of L-cell supernatant which contains colony stimulating factor (CSF-1). Interferon gamma is able to down-regulate the expression of TR-1N/TR-3N antigen on P-TR membrane while that of Mac-1 is unchanged and that of Ia is up-regulated.

Thymic lymphocytes: III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation☆

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.

Abnormal T-lymphocyte proliferation and cytotoxic responses induced by a neonatal injection of hydrocortisone: Normalization by interleukin-2 addition

Spleen cells from mice neonatally treated with hydrocortisone hemisuccinate (HHC) had an abnormal pattern of cell-mediated immunity when tested two weeks later. Mitogen-induced proliferation was reduced. The proliferative responses to allo-antigens were normal but the cytotoxic response thereby induced was impaired. The mechanisms of these anomalies are complex: reduction of cytotoxic precursors as measured by limiting dilution assays; enhancement of suppressor cells which are able to reduce the cytotoxic response of normal spleen cells; reduction of interleukin-2 (Il-2) production. The addition of exogenous Il-2 restores normal proliferative and cytotoxic responses.