Thymocyte maturation following interaction with thymus-derived macrophages.

Murine C57BL/6 thymocytes were cultivated together with syngeneic thymus-derived macrophages (TDM phi) for up to 96 hr to determine whether TDM phi participate in thymocyte maturation. The expression level of H-2b and Thy-1.2 antigens served as thymocyte differentiation surface markers as analyzed by flow cytometry. Indirect immunofluorescent staining profiles of the thymocytes demonstrate a dramatic increase in H-2b expression and a profound decrease in Thy-1.2 expression during cultivation with TDM phi. A similar phenomenon was observed when enriched populations of immature thymocytes were cocultivated with TDM phi. These changes were not observed when thymocytes were cultivated alone or with trypsin-treated TDM phi; neither were they observed when cortisone-resistant thymocytes manifesting mature characteristics were cultivated together with TDM phi. These findings suggest that interaction of thymocytes with TDM phi, involving binding and engulfment, results in the appearance of mature thymocyte subsets.     

Inhibition or acceleration of tumor growth by subpopulations of thymus cells separable by a peanut lectin.

The effect of different lymphocytes subpopulations on tumor growth in mice was investigated using an in vivo adoptive neutralization test (Winn test). Thymocytes from non-tumor-bearing mice accelerated the growth of the tumors tested [Lewis lung carcinoma (3LL), thymoma 40-127-299 and fibrosarcoma P-14] when injected into syngeneic or F₁ mice in a mixture with the tumor cells. The thymocytes were separated with the aid of peanut agglutinin into immunologically mature and immature subpopulations (Reisner Y., Linker-Israeli and Sharon N., Cell. Immunol. 25, 129, 1976). The immature thymocytes accelerated tumor growth to an extent similar to that of the unfractionated cells, whereas the mature subpopulation exhibited pronounced inhibitory activity. Our findings demonstrate that the murine thymus contains two thymocyte subpopulations with opposite activities on tumor growth and that the mature thymocytes have an inhibitory effect on tumor growth similar to that of spleen cells.     

Interaction between thymocytes and thymus-derived macrophages. II. Engulfment of thymocytes by macrophages

A high percentage (80-90%) of immature thymocytes were engulfed by syngeneic thymus-derived macrophages (TDM phi) following cocultivation for 3 days. Elimination occurred via internalization of thymocytes by the macrophages. We unequivocally demonstrated the presence of many live thymocytes inside the TDM phi by means of specific staining. Mature PNA- thymocytes were phagocytized to a lower degree than immature thymocytes, and T splenocytes were not eliminated at all. Bone marrow-derived macrophages internalized immature thymocytes to a degree similar to TDM phi. Since thymocyte survival was not at all affected by M phi culture supernatants alone, we conclude that cell to cell contact is necessary for thymocyte elimination. To identify the surface molecules which participate in internalization of thymocytes by the macrophages, both cell types were pretreated with a variety of agents. Treatment of thymocytes with tunicamycin (N-glycosylation inhibitor) and anti-Lyt-2 mAb decreased their elimination by M phi. Similarly, treatment of M phi with neuraminidase, trypsin, and anti-Ia mAb markedly suppressed their capacity to engulf thymocytes. On the other hand, thymocyte elimination was unaffected by (1) cell cultivation in syngeneic serum rather than heterologous serum; (2) use of allogeneic rather than syngeneic thymocytes; and (3) use of X-irradiated M phi and LPS-activated M phi rather than nontreated M phi.     

Requirement for macrophages in lipopolysaccharide-stimulated mixed thymocyte cultures.

Cortisone-resistant thymocytes are stimulated by allogeneic thymocytes in the presence of lipopolysaccharide or small numbers of syngeneic, allogeneic or third party macrophages, as measured by ³H-thymidine uptake and by the generation of specifically cytotoxic effector cells. Lipopolysaccharide (LPS) has no stimulating effects on macrophage-depleted mixed thymocyte cultures. It also has no stimulating effect on syngeneic thymocytes in the absence of allogeneic thymocytes.Macrophages and lipopolysaccharide are only required in the induction phase of mixed thymocyte reactions. The possible role of both lipopolysaccharide and macrophages in lymphoproliferative reactions involving pure thymocyte populations is discussed.     

The majority of CD4+8- thymocytes are functionally immature.

The thymus is the major site of T cell development and repertoire selection. During these processes, T cells segregate into two subsets that express either CD4 or CD8 accessory molecules, the phenotype of peripheral T cells. Analysis of CD4+8- thymocytes revealed that the majority of these cells express the heat-stable Ag (HSA) but not the nonclassical class I Ag, Qa-2. This HSA+, Qa-2- phenotype is similar to that of the less mature, CD4+8+ thymocytes. The remaining CD4+8- thymocytes possess the HSA-, Qa-2+ phenotype of peripheral T cells. To determine whether the Qa-2-, CD4+8- thymic subset is fully mature, we have analyzed the functional status of these CD4+8- subpopulations. The results indicate that only those thymocytes which express Qa-2 are fully responsive to anti-TCR stimulation in a manner analogous to peripheral T cells. The Qa-2- subset is nonresponsive to stimulation by anti-TCR antibodies that have been immobilized to plastic, even in the presence of lymphokines or syngeneic APC. This subset is, however, capable of proliferating to allogeneic cells or to anti-TCR on the surface of syngeneic APC, although not to the levels achieved by Qa-2+ thymocytes. Thus, the Qa-2- subset appears to require additional interactions which are not necessary for peripheral T cells or Qa-2+ thymocytes. Relevant to this issue, the Qa-2+ thymocyte subset does not appear until relatively late in development, and does not reach adult frequencies until several weeks after birth. These results would suggest that there is a progression from HSA+, Qa-2- to HSA-, Qa-2+ which parallels the maturation of functional responsiveness. These findings are important to understanding T cell selection since thymocytes with such a decreased responsiveness may have a differential capacity for tolerance induction. The results presented suggest that the bulk of CD4+8- thymocytes are not fully mature and that Qa-2 may serve as a marker for T cells with a more complete functional competence.     

Decreased natural killer-type activity of spleen cells against immature thymocytes in autoimmune strains of mice

Recently we have reported that normal natural killer (NK)-enriched murine spleen cells have the capacity to lyse immature thymocytes of syngeneic or allogeneic origin. The studies presented in this paper show a different pattern of NK-type cytolysis and thymocyte sensitivity in mice which later in life develop autoimmune disease. Such mice appeared to have reduced effector NK capacity and thymocytes from these mice seemed to have reduced sensitivity as targets. This may allow presence and persistence of autoreactive T cell subpopulations which would normally be eliminated.     

Preferential expression of fibronectin receptors on immature thymocytes

Fibronectin-adherent (FNR+) thymocytes are enriched for immature (CD4-8-) and large (CD4+8+) cells, and depleted of mature (CD4-8+ and CD4+8-) and nonmature small (CD4+8+) cells. Among purified CD4-8- thymocytes, cells with the surface marker J11d and the IL-2 receptor, which can give rise to all other thymocyte subsets, showed selective attachment to fibronectin. Analysis of FNR+ thymocytes showed that such cells are greatly enriched for cells in cycle. Additionally, FNR+ cells expressed low levels of T cell receptor. These results suggest a role for the fibronectin receptor during the early, proliferative phase of thymocyte differentiation. The data suggest that loss of the fibronectin receptor is a hallmark of cells that have become committed either to functional maturation or to programmed cell death.     

Expression of asialo GM1 on a subset of adult murine thymocytes: histological localization and demonstration that the asialo GM1-positive subset contains both the functionally mature and the proliferating thymocyte subpopulations

A small population (10 to 14%) of adult murine thymocytes expresses the glycolipid asialo GM1 (aGM1). Flow cytometric analysis of the aGM1+ cells present in thymus demonstrates the expression of a mature or medullary phenotype by 50% of the aGM1+ cells. Analysis of cytotoxic T lymphocyte precursor activity, proliferative capacity, and IL 2 production displayed by aGM1+ and aGM1- thymocyte fractions isolated by cell sorting indicates that these functional compartments of the thymus are contained within the aGM1+ subset. The aGM1+ population also contains virtually all mitotically active thymocytes, as measured by incorporation of bromodeoxyuridine. The immature IL 2 receptor-bearing thymoblasts are also included in the aGM1+ population. Immunohistochemical labeling of thymic tissue sections reveals that the majority of aGM1+ cells are located in the medulla. Clusters of aGM1+ cells are found scattered throughout the cortical and subcapsular areas. The aGM1+ population therefore contains the functionally mature thymocytes as well as some immature thymocytes, particularly those that are mitotically active. It is suggested that the aGM1+ subset of thymocytes represents those cells that are mature or actively maturing. This hypothesis is discussed in the context of current concepts of intrathymic T cell differentiation pathways.     

Interaction between thymocytes and thymus-derived macrophages. I. Surface components participating in mutual recognition

Incubation of C57BL/6 thymus-derived macrophages (TDM phi) with syngeneic thymocytes resulted in binding of thymocytes to macrophages and rosette formation. Up to 60% of the TDM phi formed rosettes with thymocytes after 6 hr of interaction at 4 degrees C. Rosette formation of the immature PNA+ thymocyte fraction was up to fivefold higher than that of PNA- and cortisone-resistant thymocytes. Pretreatment of PNA- thymocytes with neuraminidase enhanced thymocyte binding to macrophages up to sevenfold, whereas a marked reduction of rosette formation was seen following (1) incubation of thymocytes with tunicamycin; (2) incubation of macrophages with 20 mM D-galactose, GLCNaC, or GalNaC; (3) treatment of macrophages or thymocytes with trypsin; (4) treatment of macrophages with anti-1-Ab mAb and its F(ab')2 fragment; (5) treatment of thymocytes with anti-Lyt-2.2 mAb; and (6) addition of EDTA and EGTA to the interacted two cell populations.     

Thymocyte differentiation activity from the cloned monocyte/macrophage cell line RAW 264.7. Alterations in the expression of immature thymocyte surface antigens

The cloned monocyte/macrophage cell line RAW 264.7 was previously shown to produce thymocyte mitogenic and co-mitogenic activity that eluted from a Sephadex G-75 column not only at approximately 16,000 daltons, the m.w. described for interleukin 1 (IL 1), but also at 30,000 to 40,000 daltons. The studies reported here indicate that the 30,000 to 40,000 dalton molecule has thymic differentiating activity. Thymocytes from A/J mice were fractionated on discontinuous BSA gradients, which yielded populations of cells enriched for immature and mature cells. The cells found at the interface between 35 and 29% BSA (band 1 cells), which are the most immature, were cultured for 48 hr with highly purified IL 1, with the 30,000 to 40,000 dalton form of thymocyte co-mitogenic activity obtained after Sephadex G-75 chromatography and chromatofocusing chromatography, or with media alone. The surface antigens TL-3, H-2Kk, Thy-1.2, Lyt-1, and Lyt-2 were examined by immunofluorescence. It was found that the highly purified 30,000 to 40,000 dalton species of co-mitogenic activity induced a significant increase in the content of surface H-2Kk, a decrease in TL-3, and a very small decrease in Thy-1.2 on the cell surface, whereas IL 1 was not capable of inducing a change in these surface antigens. There was no change in Lyt-1 on the surface of band 1 thymocytes after incubation with either IL 1 or the 30,000 to 40,000 dalton species. The 30,000 to 40,000 dalton species caused a significant decrease in the percentage of cells staining positive for Lyt-2, whereas IL 1 caused a smaller but significant decrease in Lyt-2. These changes in the surface markers TL-3, H-2Kk, and Thy-1.2 are consistent with changes that occur during thymocyte differentiation. It was also observed that the proliferative response to the 30,000 to 40,000 dalton form and IL 1 increased with increasing functional maturity of each band of thymocytes when used in the thymocyte mitogenic assay. However, only the 30,000 to 40,000 dalton form was capable of inducing a proliferative response in the immature band 1 thymocytes in the thymocyte co-mitogenic assay. These results indicate that the RAW 264.7 cells produce a factor that has, in addition to thymocyte co-mitogenic activity, thymocyte differentiation activity, and this factor is distinct from IL 1.     

Suppression of mixed lymphocyte reactions by alloantigen-activated spleen-localizing thymocytes.

Heavily irradiated BALB/c and C57BL/6 mice were injected intravenously with BALB/c thymocytes. At varying times thereafter spleen and lymph node cell suspensions from these animals were treated with mitomycin C and added as regulator populations to mixed lymphocyte reactions (MLR) with syngeneic responder cells. Alloantigen-activated spleen-localizing thymocytes suppressed MLR responses 40 to 95%. Suppressor activitity, manifested as a quantitative reduction in peak proliferative responses, was maximal 4 days after cell transfer. Antigenic specificity for the stimulator cell strain in MLR was not demonstrated. The effect of lymph nodelocalizing thymocytes as regulators in MLR was variable, but in most experiments these cells slightly enhanced responses. We conclude that splenic localization is an intrinsic property of the thymocyte subpopulation capable of suppressing MLR responses, and that the suppressor activity of this subpopulation is substantially enhanced by activation in allogeneic hosts.     

The immunocompetence of murine stromal cell-associated thymocytes

Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.     

Induction of phenotypic differentiation, interleukin 2 production, and PHA responsiveness of "immature" human thymocytes by interleukin 1 and phorbol ester

Small human thymocytes (ST) representing 70% of the thymocytes were isolated according to size by centrifugal elutriation. Although these ST contained approximately 30% PNA-cells, they failed to respond to lectins, indicating the existence of a PNA-ST subset that can be considered to belong to the "immature" thymocyte population. The ST were induced to proliferate if, in addition to PHA, IL 1-containing supernatants of highly purified monocyte cultures or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were present. The incubation of the ST for 90 hr with TPA or IL 1 in the absence of PHA resulted in a strong reduction in the percentage of cells reacting with the immature thymocyte markers TdT and PNA. In addition, the OKT6+ cells were partially reduced after incubation with IL 1. Concomitantly, an increase in the percentage of cells reacting with the mature T cell markers OKT1 and OKT3 was observed, whereas HLA antigens became strongly expressed on all ST. Although IL 1 or TPA were unable to induce proliferation of the ST, these substances induced IL 2 production by these cells. These shifts to cells with more "mature" phenotypes that are able to produce IL 2 were not observed if the ST were incubated with PHA or culture medium only. The responder capacity of the ST to PHA plus TPA was not significantly affected by the depletion of the more "mature" OKT3+ and OKT1+ cells. In addition, in this situation OKT1+, OKT3+, OKT6- cells were found to be generated from OKT1-, OKT3-, OKT6+ cells. Therefore, it could be excluded that the proliferative responses were due to a selective expansion of a preexisting mature T cell population. Our results indicate that TPA mimics IL 1 in the induction of differentiation of the ST to a stage in which subpopulations of these cells are able to produce IL 2 and to respond to PHA. Because only the proliferating ST were found to react with a monoclonal antibody, which is thought to be directed at the IL 2 receptor (anti-Tac), our data suggest that PHA is required for the induction of expression of receptors for IL 2 in those ST subpopulations that are able to proliferate in the presence of IL 2 generated in situ.     

Studies on thymocyte subpopulations in guinea pigs. VIII. Characterization of a thymocyte population resistant to the cytotoxic effect of normal rabbit serum

Guinea pig thymocytes were incubated with normal rabbit serum, which resulted in the death of a great majority of the cells. The remaining rabbit serum-resistant cells, representing less than 10% of the thymocytes, contained euchromatic DNA and were of intermediate size and low density. Functional tests indicated that they were enriched in immunologically mature cells, which responded to the mitogenic lectins phytohemagglutinin and concanavalin A, and were depleted of immature, spontaneously proliferating cells and in cells responding to the thymocyte growth peptide. The described procedure for enrichment of immunologically mature thymus cells in guinea pigs may become useful since glucocorticoid treatment, used in mice for enrichment of mature thymocytes, cannot be used for this purpose in guinea pigs.     

Natural killer-type lysis of thymocytes from young mice by normal spleen cells in vitro

Normal spleen cells from 6- to 10-week-old mice, enriched for natural killer (NK) cells on a discontinuous polyvinylpyrrolidone-silica (Percoll) gradient, lyse thymocytes of young mice (less than or equal to 19 days old) in a short-term 51Cr release assay. The highest NK-type activity was found in band 3 (density less than or equal to 1.077 g/ml) of a four-step gradient. In some experiments band 2 (density less than or equal to 1.070 g/ml) also showed NK activity. Activity was not unequivocably detectable in cells before separation or in bands 1 and 4. These results also show that the thymocyte sensitivity is dependent on the age of the target cell. Sensitivity of very young thymocytes (less than or equal to 7 days old) was higher than that of thymus cells from 8- to 19-day-old donors. Moreover, it seemed that syngeneic target thymocytes were lysed more effectively than allogeneic. Thus, an NK-type cell population may have the ability to lyse immature thymic target cells at an early stage of their differentiation. This could be of importance as a physiological mechanism for controlling the T cell repertoire and its reactivity.     

Analysis of the human neonatal thymus: evidence for a transient thymic involution

The neonatal period is marked by the impairment of the major components of both innate and adaptive immunity. We report a severe depletion of cortical CD4+CD8+ double-positive thymocytes in the human neonatal thymus. This drastic reduction in immature double-positive cells, largely provoked by an increased rate of cell death, could be observed as early as 1 day after birth, delaying the recovery of the normal proportion of this thymocyte subset until the end of the first month of postnatal life. Serum cortisol levels were not increased in newborn donors, indicating that the neonatal thymic involution is a physiological rather than a stress-associated pathological event occurring in the perinatal period. Newborn thymuses also showed increased proportions of both primitive CD34+CD1- precursor cells and mature TCRalphabetahighCD69-CD1-CD45RO+/RAdull and CD45ROdull/RA+ cells, which presumably correspond to recirculating T lymphocytes into the thymus. A notable reinforcement of the subcapsular epithelial cell layer as well as an increase in the intralobular extracellular matrix network accompanied modifications in the thymocyte population. Additionally neonatal thymic dendritic cells were found to be more effective than dendritic cells isolated from children's thymuses at stimulating proliferative responses in allogeneic T cells. All these findings can account for several alterations affecting the peripheral pool of T lymphocytes in the perinatal period.     

Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.     

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysi? by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.     

Effect of streptozotocin-diabetes on thymus of normal and adrenalectomized rats

The injection of streptozotocin in intact or adrenalectomized rats produced a decrease in the number of thymus lymphoid cells. Bovine serum albumin gradient analyses of thymocytes from control, adrenalectomized, adrenalectomized-diabetic and diabetic animals showed variations in the mature/immature cell ratio. No remarkable changes in the distribution of the different thymocyte subpopulations were observed.     

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.