Identification of subsets of proliferating low Thy 1 cells in thymus cortex and medulla

Subsets of proliferating thymocytes were identified in the normal mouse thymus by in vivo labeling with [³H]TdR and by cell separation according to relative amounts of Thy 1 antigen. In order to resolve apparent discrepancies in the literature, parenteral and topical application of [³H]TdR were compared as labeling methods for dividing thymocytes, and limited complement lysis and fluorescence-activated cell sorting were compared as separation principles for high Thy 1 and low Thy 1 thymocyte subsets. The separated cells were further characterized by immunofluorescence for terminal deoxynucleotidyltransferase (TdT), which normally is restricted to cortical thymocytes, and for H2 alloantigens, which are preponderant on medullary thymocytes. Four subsets of proliferating cortical thymocytes were identified after application of [³H]TdR to the thymus capsule. The major subset, which comprised about 92% of dividing cortical thymocytes, had a high Thy 1, low H2 phenotype. Most were also TdT + ve. The three minor subsets of proliferating cortical thymocytes each had a low Thy 1 phenotype, but differed according to H2 and TdT markers. Systemic injection of [³H]TdR also labeled the above subsets of dividing cortical thymocytes, but in addition it detected a subset of proliferating low Thy 1, low H2, TdT — ve cells in the thymus medulla. The latter subset comprised about one-third of the pool of proliferating low Thy 1 cells. In their aggregate the four subsets of low Thy 1 cells constituted approximately 13% of total proliferating thymocytes and 1.1% of total thymocytes. The identification of discrete subsets of proliferating low Thy 1 cells in the thymus cortex as well as in the thymus medulla is compatible with the hypotheses that all thymocytes are descended from low Thy 1 precursors and that separate precursor cell subsets exist for cortical and medullary thymocytes.     

Ontogeny of murine T lymphocytes: I. Maturation of thymocytes induced in vitro by tumor necrosis factor-positive serum (TNF+)

One question which is unresolved in developmental immunology is whether cortical thymocytes are the precursor cells which give rise to medullary thymocytes and peripheral T cells. Cortical thymocytes display a characteristic surface antigen phenotype (high TL and Thy-1, low H-2, no Qa-2, no Qa-3), are agglutinated by peanut agglutinin (PNA), and are unresponsive to concanavalin A (Con-A). The functionally more mature medullary thymocytes express a surface phenotype more closely resembling peripheral T cells (no TL, low Thy-1, high H-2, and some Qa-2), are not agglutinated by PNA, and are responsive to Con-A. An in vitro induction system has been devised in which mouse thymocytes undergo quantitative changes in surface antigens in less than 24 hr and increase their mitogen response to Con-A. The phenotypic changes are characterized by a decrease of TL and Thy-1 and an increase in H-2, Qa-2, and Qa-3. Studies in which thymocytes were fractionated on BSA gradients and by PNA agglutination demonstrate that the inducible cells have the properties of cortical thymocytes. Our data show that a subpopulation of cortical thymocytes can acquire phenotypic characteristics similar to medullary thymocytes and peripheral T cells.     

Antigenic phenotype of TdT-positive cells in human peripheral blood

Double immunofluorescence studies for terminal deoxynucleotidyl transferase (TdT) and leucocyte surface membrane antigens have been used to characterize the small subpopulation of TdT-positive cells in human peripheral blood. The predominant antigens demonstrated were those coded for by the major histocompatibility complex, namely HLA-A,B and Ia-like antigens. A small proportion of TdT+ cells expressed antigens restricted to B lymphocytes and their precursors (BA-1+ CALLA+). In contrast, antigens associated with T-lymphocyte differentiation were not detected using a panel of T-cell-specific monoclonal antibodies. These results preclude the possibility that circulating TdT+ cells are immature cortical thymocytes that have "leaked" into the bloodstream. Although bone marrow-derived prothymocytes, which have not yet acquired T-cell lineage markers, may be included amongst this subset, the expression of B-cell related antigens by some TdT+ cells indicates the likely existence of lineage heterogeneity amongst this population of lymphoid cells. The relevance of these findings to the monitoring of human acute lymphoblastic leukaemia is discussed.     

Hematopoietic thymocyte precursors. III. A population of thymocytes with the capacity to return ("home") to the thymus.

A subpopulation of thymocytes with the capacity to return (“home”) to and repopulate the thymus of an irradiated mouse has been identified. These cells differ from the majority of cortical thymocytes in that they are of a lower buoyant density and have higher cortisone and X-ray resistance. They also bear an antigen common to mouse brain but not found on the majority of cortical thymocytes. This subpopulation is derived from a hematopoietic thymocyte progenitor and provides an intrathymic reserve of thymocyte precursors. It appears to be a non self-sustaining “transit” population in the pathway of T-cell development.     

Differentiation of human T lymphocytes. I. Acquisition of a novel human cell surface protein (p80) during normal intrathymic T cell maturation

The thymus is thought to be the primary central lymphoid organ in which T cells mature. Although thymic cortical and medullary compartments are distinct histologically, few antigens have been described that are absolutely acquired during the presumed intrathymic maturation pathway from cortical to medullary thymocytes. In this paper, we describe the acquisition during human intrathymic T cell maturation of a novel protein (p80) defined by a monoclonal antibody (A1G3). Although the p80-A1G3 antigen is distributed throughout the body and is not T cell specific, our study demonstrates that expression of p80-A1G3 antigen in normal human thymus is associated with thymocyte functional maturity and location in the thymus medulla. Moreover, in contrast to other markers of mature human T cells, the p80-A1G3 cell surface protein is not expressed on T6+ cortical thymocytes, and, therefore, is absolutely acquired by medullary thymocytes during T cell maturation. Thus, the p80-A1G3 antigen and the A1G3 antibody provide a heretofore unavailable system for the study of molecular events that transpire during the maturation of thymocytes.     

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.     

Evidence for the cellular origin of Gross virus-induced leukemia in the rat: description of a unique LDH isozyme sub-band in leukemic lymphoid cells and lymphohemopoietic precursor cells

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross-leukemia virus (RAGV) were analyzed for a variety of differentiation markers to define their differentiation state and possible cellular origin. A majority of thymocytes from leukemic rats had the phenotypic characteristics of subcapsular cortical thymocytes that are the most ancestral of the thymocytes. These cells exhibited readily detectable levels of Thy-1 and histocompatibility antigens on their surfaces, they contained terminal deoxynucleotidyl transferase (TdT) and they contained low adenosine deaminase (ADA) and high purine nucleoside phosphorylase (PNP) specific activity. The leukemic thymocytes also contained a sub-band of the LDH-5 isozyme (LDH-5') that was not detected in normal thymocytes but that was present in lymphocyte-rich fractions of postnatal bone marrow, fetal and prepubertal spleen, and fetal and neonatal liver. The tissue distribution and ontogeny of LDH-5'-containing cells is similar to prethymic TdT+ cells in the rat and both TdT and LDH-5' are enriched in a subset of bone marrow "null" cells. These results suggest that TdT+ thymocyte progenitors or their precursors are the targets of leukemic transformation of RAGV.     

A homing receptor-bearing cortical thymocyte subset: implications for thymus cell migration and the nature of cortisone-resistant thymocytes

The thymus exports a selected subset of virgin T lymphocytes to the peripheral lymphoid organs. The mature phenotype of these thymus emigrants is similar to that of medullary thymocytes and has been cited as supporting a medullary rather than cortical exit site. Using the monoclonal antibody MEL-14, we identify a 1%-3% subpopulation of thymocytes that expresses high levels of a receptor molecule involved in lymphocyte homing to peripheral lymph nodes. We present evidence that these rare MEL-14hi thymocytes are predominantly of mature phenotype and represent the major source of thymus emigrants. Surprisingly, MEL-14hi thymocytes are exclusively cortical in location, although their mature phenotype may allow them to masquerade as medullary cells in conventional studies. We also demonstrate that unlike medullary thymocytes, many cortisone-resistant thymocytes (CRT) are MEL-14hi. Thus, in contrast to current dogma, CRT do not represent a sample of medullary thymocytes as they are found in situ and their level of immunocompetence does not necessarily reflect that of the medullary population. Our findings refute the hypothesis that phenotypically and functionally mature cells are restricted to the medulla, and support our proposition that most thymus emigrants are derived from the MEL-14hi cortical subset.     

Distribution of terminal deoxynucleotidyl transferase and purine degradative and synthetic enzymes in subpopulations of human thymocytes

Terminal deoxynucleotidyl transferase (TdT) and purine metabolic enzymes were examined in subsets of human infant thymocytes (defined by surface cell antigens) and normal peripheral T lymphocytes. Putative prothymocytes (RFB-1+, HTA-1+/- large blast-like cells), medium and high density cortical thymocytes (RFB-1+, HTA-1+), and medullary thymocytes (RFB-1-, HTA-1-, OKT3+) were isolated by density gradient centrifugation, monoclonal antibody and complement-mediated cytolysis, and cell-antibody affinity chromatography. Peripheral T lymphocytes were isolated from normal adult mononuclear cells using nylon fiber technique. Adenosine deaminase (ADA) and TdT were highest in prothymocytes 48.8 +/- 14.7 mumol/hr/10(8) cells (mean +/- SE) and 22.9 +/- 1.4 U/10(8) cells, respectively. Both enzymes decreased progressively down the maturation pathway. In peripheral T lymphocytes, ADA was 3.9 +/- 1.5 mumol/hr/10(8) cells, and TdT was undetectable. Purine nucleoside phosphorylase (PNP) and ecto-5'nucleotidase (5'NT) were lowest in cortical thymocytes (27.5 +/- 11.0 nmol/hr/10(6) cells and 2.8 +/- 1.3 nmol/hr/10(6) cells, respectively) and increased with T cell maturation. The PNP level was 124.9 +/- 17.2 nmol/hr/10(6) cells and 5'NT was 30.1 +/- 3.9 nmol/hr/10(6) cells in peripheral T lymphocytes. The deoxynucleoside kinases (deoxyguanosine, deoxyadenosine, and deoxycytidine kinases) paralleled the changes in ADA and TdT activity among the different T subsets. The proliferative activity (labeling index) was highest in the prothymocyte fraction and lowest in peripheral T cells. Variation in the distribution of these enzymes in T cell subsets may explain their different sensitivities to deoxyadenosine and deoxyguanosine toxicity and the different effects on T cell development of ADA or PNP deficiency.     

Tissue localization and biochemical characteristics of a new thymic antigen recognized by a monoclonal thymocytotoxic autoantibody from New Zealand black mice

Naturally occurring thymocytotoxic autoantibodies (NTA) have been described in both humans and mice with SLE. To define further the role of anti-thymic autoantibodies in murine lupus, we studied the cellular and molecular specificity of a spontaneous monoclonal NTA, designated TC-17, derived from a 4-mo-old New Zealand Black mouse. TC-17, an IgM autoantibody, has been shown previously to be unreactive with Lyt-1, Lyt-2, and L3T4 (T helper) antigens. We have shown further that it is also unreactive with Thy-1. TC-17 recognizes a new thymic antigen that appears to mark a distinct subpopulation of cortisol-sensitive cortical thymocytes. The antigen consists of a single glycoprotein chain with an apparent m.w. of 88,000. TC-17 shows reduced binding to thymocytes treated with tunicamycin, indicating either that glycosylation of TC-17 antigen is necessary for TC-17 to bind to it or that glycosylation is required for expression of the antigen on the cell surface. TC-17 uniquely reacts with two of 17 murine lymphoid tumor cell lines of intermediate cellular maturity. The thymocytotoxic activity of TC-17 is absorbed by single cell suspensions of murine stomach, small intestine, large intestine, kidney, and thymus. Moreover, the specific binding of TC-17 to gut tissue of normal and germfree mice can be demonstrated by indirect immunofluorescence, suggesting antigenic cross-reactions between thymic and gut tissue. TC-17 reacts with rat thymocytes as well as it does with murine cells, indicating moderate evolutionary conservation of the TC-17 antigen. The expression of this glycoprotein by a discrete thymocyte subset may prove to be a valuable probe for the study of murine T cell differentiation.     

Development and ontogeny of hamster T cell subpopulations

The Syrian hamster is unique among laboratory animals because products of class I MHC genes are monomorphic. Thus, this species may be a model in which to test the relationship between MHC polymorphism and the T cell antigen receptor repertoire. Recently, cytotoxic and helper T cell subpopulations have been distinguished on the basis of cell surface phenotype detected with monoclonal antibodies (mAb). We used these reagents (mAb 110 detects all peripheral T cells and mAb 38 detects cytotoxic T cells) to dissect and categorize thymic populations according to relative maturational status. The two mAb divide thymocytes into four subpopulations in the young adult. Two (110+ 38+, 110+ 38-) were peripheral-like and were housed in the medulla, exclusively; another subset (110- 38+) consisted almost entirely of TdT+ cortical thymocytes. The fourth subset (110- 38-), bearing neither marker, was heterogeneous and consisted mostly of medium-large-size thymocytes, including cells with an early phenotype (nuclear TdT+). Cells with the cortical phenotype proved to be the most sensitive to cortisone treatment, whereas those which expressed the medullary marker, 110, were most resistant. To ascertain the relationship between 110- and 110+ T lineage cells, we followed the appearance of the four thymic subpopulations during ontogeny of the hamster thymus. Adult-like thymic architecture (delineation of cortex and medulla) as well as the two 110- subsets were established before expression of 110 antigen was apparent in the thymus. However, lymphocytes bearing the 110 antigen were found in lymph nodes prior to thymus during ontogeny, concomitant with developing T cell function in peripheral tissue. This finding implies that cells lacking 110 antigen were exported from the thymus and subsequently acquired expression of the molecule in the periphery, and we suggest that acquisition of 110 antigen may be a stage of postthymic maturation. Although 110+ cells appeared to be the most mature subset by several criteria, all functional thymocytes of adults or neonates were not 110+. Thus, we conclude that the 110 marker is acquired after T cells reach functional maturity. Moreover, the response profile of isolated 38+ thymocytes was analogous to peripheral 38+ T cells, suggesting that the dichotomy of function detected with our mAb also occurs before acquisition of 110 antigen. We have modeled what is known about hamster T cell development into a hypothetical scheme.     

Proliferation of phenotypically immature human thymocytes with and without interleukin 2 receptors

Previous studies have indicated that the human thymus is composed of several discrete compartments. Cortical thymocytes are reactive with the monoclonal antibody anti-T6, whereas most medullary cells, unreactive with anti-T6, stain brightly with anti-T3, which defines mature T cell populations. Only a minor thymocyte population lacks both T3 and T6 but expresses T11 antigens. Within the thymus, several proliferating lymphoblasts are present. In addition a distinct subset shows the capacity to proliferate in response to mitogens. By continuous Percoll density gradient centrifugation, we have obtained a cell fraction comprising the vast majority of cells able to proliferate spontaneously or after PHA stimulation. By a panning procedure performed with anti-T3 and anti-T6 antibodies, three phenotypically distinct thymocyte subsets were separated from this fraction, and their functional capabilities were tested. The spontaneous proliferating activity was found to be mainly attributable to thymocytes unable to respond to mitogen, expressing the cortical T6 marker and lacking receptors for IL 2. T3-positive cells are able to respond to mitogen. However, these thymocytes are incapable of producing the adequate amount of IL 2 required to fully saturate their intrinsic proliferative capability. Surprisingly, the phenotypically least mature intrathymic T lymphocytes (T3 and T6 negative) respond to phytomitogen, at least in part, in an interleukin-dependent manner. It is noteworthy that a large proportion of these T3- and T6-negative thymocytes express IL 2 receptors and class II MHC antigens without in vitro activation. These novel findings have potential implications in the context of current models of differentiation pathways within the human thymus.     

Antigenic relationship between bone marrow lymphocytes cortical thymocytes and a subpopulation of peripheral T cells in the rat: description of a bone marrow lymphocyte antigen.

Populations of rat bone marrow lymphocytes (BML) consisting of approximately 90 percent, “tnull” cells were prepared by density gradient centrifugation, passage through a column of fine glass beads, and treatment with anti-T cell and anti-B cell serum plus complement. Antisera to these bone marrow lymphocytes were raised in rabbits. After absorption with RBC and peritoneal exudate cells, the anti-BML sera were found by immunofluorescence to react selectively with “null” cells in bone marrow, with cortical thymocytes, and with a cortisone-sensitive subset of T cells in blood and in spleen, possibly in red pulp. The antigen that is common to these cell types is designated the rat bone marrow lymphocyte antigen (RBMLA). Lymphocytes that are positive fur KBMLA are negative for another lymphocyte-specific heteroantigen, rat musked thymocyte antigen (RMTA). As shown previously, RMTA is present on medullary thymocytes and ou cortisone-resistant T cells in white pulp of spleen, paracortex of lymph node and thoracic duct lymph. It is postulated that two developmentally and functionally distinct lines of T cells exist in peripheral lymphoid tissues of the rat, one derived from cortical thymocytes and one derived from medullary thymocytes. It is further postulated that the “null” population of bone marrow lymphocytes contains the lymphopoietic stem cells from which these two lines of T cells originate.     

Prothymocytes in postirradiation regenerating rat thymuses: a model for studying early stages in T cell differentiation

Prothymocytes were obtained from regenerating thymuses of intrathymic-irradiated, bone marrow-shielded rats. In contrast to cortical thymocytes, which are small nondividing cells containing nuclear TdT, prothymocytes are characterized by their large size, high mitotic activity, lack of natural attachment, absence of PNA-binding capacity, nonexpression of membranal thymic specific antigens, and absence of nuclear TdT. In addition, these cells are capable of responding to the mitogens Con-A and PHA, and are sensitive to in vitro lysis by physiologic concentrations of corticosterone and cortisol. Prothymocytes incubated for 3 days on thymic monolayers differentiated into small lymphocytes expressing cortical thymocyte characteristics. Light and electron microscopy studies demonstrated the infiltration of prothymocytes from the circulation via the thymic blood vessel wall into the perivascular sinuses. Prothymocytes isolated from the thymuses, however, did not exhibit specific "homing" to the thymus when transfused back into the animals. In view of the observed accelerated thymic repopulation in adrenalectomized rats, and the high in vitro glucocorticoid sensitivity of the prothymocytes, it is suggested that thymic homeostasis is regulated by specific effect of adrenocortical hormones on the prothymocyte subset.     

The influence of dexamethasone treatment on the lymphoid and stromal composition of the mouse thymus: a flowcytometric and immunohistological analysis

The effect of injection of a range of doses of dexamethasone on the distribution of T-cell subpopulations and stromal cells in the thymus of BALB/c mice was investigated with flowcytometry and immunohistology. To this purpose we used monoclonal antibodies directed to the T-cell differentiation antigens Thy-1, T200, Lyt-1, Lyt-2, T4, MEL-14, and monoclonal antibodies directed to various classes of stromal cells. Injection of dexamethasone in increasing doses of 5-130 mg/kg body weight gradually leads to a depletion of the cortical thymocyte population, i.e., bright Thy-1 + ve, dull T-200 + ve, bright Lyt-2 + ve, and bright T4 + ve cells. These cortical cells are very dull MEL-14 + and express variable numbers of Lyt-1 molecules. Also the medulla is affected by dexamethasone although to a lesser extent. Dexamethasone injection at 130 mg/kg selects for a dull Thy-1 + ve, bright T-200 + ve, and bright Lyt-1 + ve medullary population. These cells are either T4 + ve Lyt-2-ve or T4-ve Lyt-2 + ve. Under these conditions, MEL-14 + ve cells were no longer present in the cortex but accumulated in medullary perivascular spaces. Staining of sequential sections showed that this particular subpopulation has a typical "helper" phenotype. This observation provides strong evidence that perivascular compartments are an exit pathway for emigrating T cells. The medullary population contains a phenotypically distinct, dexamethasone-sensitive subpopulation. This conclusion is based on two findings: 130 mg/kg dexamethasone depletes the thymus of all but 4% of the thymocytes, which form a much smaller subpopulation than the population of dull Thy-1 + ve cells (amounting to 15% of the total thymocytes). The medulla contains a subpopulation of dull Lyt-2 + ve cells, which are resistant to 20 mg/kg dexamethasone, but depleted by 130 mg/kg. Dexamethasone also has a severe effect on thymic nonlymphoid cells. Even at low doses, dexamethasone induces TR4 + ve cortical epithelial-reticular cells to become spherical ("nurse cell-like") structures, depleted of lymphoid cells. These stromal cells no longer express MHC antigens in a membrane-bound fashion. In contrast, the medullary epithelial cells appear morphologically unaffected even at a dexamethasone dose of 130 mg/kg.     

Immunohistochemical characterization of nurse cells in normal human thymus.

Lymphoepithelial complexes known as thymic "nurse" cells (TNC) have been isolated and described in the thymus of several animal species including man. Most of the investigations on TNC have been carried out in enzymatically digested thymuses in which TNC were isolated by differential sedimentation. In the present study we demonstrate TNC in immunohistochemically stained sections of human thymus as ring-shaped cells completely enclosing thymocytes and localized not only in the cortex, but also at the corticomedullary junction where they have not been previously described. TNC expressed epithelial markers [low and high molecular weight keratins identified by 35 beta H11 and 34 beta E12 monoclonal antibodies, a cortical antigen shared with neuroectodermal neoplasms recognized by the GE2 monoclonal antibody, and tissue polypeptide antigen (TPA:B1)], class II histocompatibility antigens (HLA-DR), and thymosin alpha 1. Double staining experiments with the nuclear proliferation-associated antigen Ki-67 and the cortical epithelium marker GE2 showed that most thymocytes enclosed in these cortical TNC were not proliferating. The antigens expressed by TNC indicate that not only cortical, but also medullary epithelial cells are part of the TNC system. The possible role of TNC in the education and maturation of thymocytes is discussed.     

Control of helper-T-cell proliferation by recognition of Ia 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.     

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.     

Opposing reactivities of subpopulations of T lymphocytes toward syngeneic tumor cells: separation of early thymocytes.

The present communication is a continuation of earlier studies which indicated that interaction between syngeneic tumors and those lymphocytes in the early stages of thymic processing can result in enhanced tumor growth in vivo. The thymocytes involved in this tumor enhancement were found previously in the rapidly dividing subpopulation of subcapsular cortical thymocytes, both in the untreated thymus and in the thymus undergoing repopulation after cortisone depletion. In the present experiments we have isolated this small subpopulation of early thymocytes. After cortisone injection such cells could be separated from the medullary cortisone-resistant thymocytes since the latter cells exhibit a high level of surface H-2 antigens and were thus lysed preferentially by anti-H-2 serum and complement. The repopulating subcapsular early thymocytes, which were resistant to this treatment, were incapable of responding to PHA while their basal proliferation rate was undiminished, and the majority of the cells were found to be dividing. When such low H-2 early thymocytes were injected together with three different tumors into syngeneic mice their tumor-enhancing activity was evident. It is clear that such early thymocytes are not devoid of biologic reactivity and their release from the thymus could have decisive results.