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.     

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.     

Phenotypic analysis of thymocytes that express homing receptors for peripheral lymph nodes

Thymocytes that express high levels of homing receptors for peripheral lymph nodes can be detected with the monoclonal antibody MEL-14. We have shown that in adult mice these rare MEL-14hi thymocytes a) are cortical in location and typically constitute 1 to 3% of the total thymocyte population, b) may be a major source of thymus emigrants, and c) contain a high frequency of precursors of alloreactive cytotoxic T lymphocytes. In this study we have analyzed the phenotype of the MEL-14hi thymocyte subset. Most normal adult MEL-14hi thymocytes are midsize and express the mature phenotype typical of thymus emigrants, medullary thymocytes, and peripheral T cells: they are predominantly PNAlo, H-2K+, Thy-1+, Ly-1hi, and either Lyt-2-/L3T4+ or Lyt-2+/L3T4-. These findings argue strongly for the presence of rare MEL-14hi immunocompetent cortical thymocytes that, aside from their homing receptor expression, are phenotypically indistinguishable from medullary thymocytes. However, a minority (20 to 30%) of MEL-14hi thymocytes are large and phenotypically nonmature: they express intermediate to high levels of PNA binding sites, and are H-2K- to H-2Klo, Thy-1hi, Ly-1+, and either Lyt-2+/L3T4+ or Lyt-2-/L3T4-. Through a technique that selectively labels outer cortical cells, phenotypically nonmature MEL-14hi thymocytes have been shown to be concentrated in the subcapsular blast region of the outer cortex. Although we have no direct evidence of a precursor-product relationship, we consider it likely that the phenotypically nonmature outer cortical MEL-14hi lymphoblasts give rise to phenotypically mature MEL-14hi cells located deeper in the cortex. These results are consistent with our previous proposal that MEL-14hi thymocytes are a major source of thymus emigrants, and indicate that expression of high levels of MEL-14-defined homing receptors may be closely linked to the intrathymic selection process.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

Changes in the expression of the surface antigens of thymocytes during their cultivation with macrophages

The data on changes in expression of H-2 complex and Thy-1 antigens on cell surface of thymocytes resulting from their incubation with peritoneal macrophages has been presented. The process of joint cultivation of thymocytes with macrophages leads to significant decrease in number of cells with Thy-2-antigen and increase in that with H-2 complex antigens. An increase in H-2K+ cells in experimental thymocytes as compared to control ones was observed. No changes in H-2D expression was observed. A significant increase in Ia+ macrophages was observed after interaction with thymocytes as compared with intact mononuclear phagocytes.     

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.     

Phenotypic and functional characterization of c-kit expression during intrathymic T cell development.

We have studied the expression and function of c-kit on subsets of mouse thymocytes. c-kit was primarily expressed on subpopulations of CD4-CD8-CD3- triple negative (TN) cells. The strongest c-kit expression was associated with subsets that represent the least mature TN cells, including CD44+CD25- TN, and a subpopulation of CD25+ TN. These cells were also Thy-1lo, H-2Khi TSA-1hi, HSAlo, B220-, Mac-1-, and Gr-1-. Additionally, the recently described pre-TN thymocyte population (CD4loCD3-CD8-) was also c-kit+. CD25+ TN thymocytes proliferated in the presence of IL-7 and stem cell factor (the ligand for c-kit), and this proliferation was completely inhibited in the presence of anti-c-kit. Furthermore, the addition of anti-c-kit to 2-deoxyguanosine-treated fetal thymic lobes undergoing reconstitution with fetal liver-derived precursor cells inhibited their T cell differentiation potential. These observations indicate an important role for c-kit/stem cell factor interactions during early thymocyte development.     

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes.

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2^k and H-2^d antigens and a similar decrease in the expression of the TL antigen (in TL⁺ strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G_IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.     

Aggregation of Thy-1 Glycoprotein Induces Thymocyte Apoptosis through Activation of CPP32-like Proteases

Mouse thymocytes are known to undergo apoptosis by ligating some unique anti-Thy-1 monoclonal antibodies (mAbs), G7 and KT16. However, the precise mechanisms of Thy-1-mediated apoptosis are as yet unclear. We investigated Thy-1-mediated apoptosis using our previously generated anti-Thy-1 mAb, MCS-34, which was similar to G7 because both antibodies recognized both Thy-1.1 and Thy-1.2 and bound Thy-1A epitope. Unlike G7, MCS-34 alone could not induce apoptosis in thymocytes; however, it could induce apoptosis when it was cross-linked with second antibodies. Thus, MCS-34 could not aggregate by itself, but G7 could. In the course of investigating the apoptosis-related molecules that were involved in the thymocyte apoptosis induced by cross-linking of MCS-34 or by G7 ligation, we found that CPP32-like proteases were activated during the apoptosis. Furthermore, the expression of bcl-2 and bcl-X_Lproteins was decreased in these apoptosis processes. Whereas the ligation of MCS-34 alone could not generate apoptosis signals that led to the activation of CPP32-like proteases and the decrease in bcl-2 and bcl-X_Lexpression, the aggregation of Thy-1 glycoprotein might be crucial to signal thymocyte apoptosis. These results indicate that MCS-34 is a useful anti-Thy-1 mAb for analyzing the Thy-1-mediated signals since MCS-34 can control the level of apoptosis by using second antibodies.     

The expression, function, and ontogeny of a novel T cell-activating protein, TAP, in the thymus

The TAP molecule is an allelic 12,000 m.w. membrane protein that participates in T cell activation. This report analyzes the expression, function, and ontogeny of this molecule in the thymus. TAP is expressed on a small subset (10 to 20%) of thymocytes which is distinct from its expression on a majority (70%) of peripheral T lymphocytes. In the adult thymus, the majority of the TAP-bearing thymocytes are cortisone-resistant, Thy-1+, TL-, J11D-, and PNA-, which localizes TAP expression to medullary thymocytes. Cortical thymocytes do not bear this determinant. Parallel functional studies demonstrated that TAP+ thymocytes are required for Con A and MLR responsiveness. Anti-TAP MAb plus PMA specifically induces proliferation of mature thymocytes comparable in magnitude to the Con A response. These results demonstrate that TAP expression defines the immunocompetent thymocyte compartment and, further, that this molecule is functional on these cells. The ontogeny of TAP expression was also analyzed. TAP is expressed early in fetal thymic development at a time when most T cell markers (except Thy-1 and the iL2-R) are absent. The small sub-population of adult L3T4- and Lyt-2- thymocytes, which resemble early fetal thymocytes, also express TAP. These early thymocytes are capable of being activated through the TAP molecule. The implications of these findings for T cell development and, in particular, the relationship of TAP to T cell receptor expression and acquisition of immunocompetence are discussed.     

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.     

Leukemic transformation in hrs mice occurs in an immature-nonactivated thymocyte subpopulation

The murine leukemic strain HRS/J has an autosomal-recessive, mutant gene, hr, with homozygotes (hr/hr) having a 72% incidence of thymic leukemia at 18 months of age compared to 20% in heterozygotes (hr/+). This study was done to (a) determine if expression of thymocyte differentiation and murine leukemia virus (MuLV) antigens during leukemic transformation were different in hr/hr compared to hr/+ mice, (b) define the subpopulations that were targets for leukemic transformation, and (c) compare the results to reports in other leukemic strains. Flow cytometry analysis of thymus cell suspensions was done with anti-T-cell and anti-H-2 monoclonal antibodies, peanut agglutinin (PNA), and heteroantisera to MuLV antigens. Thymocytes of 1- to 3-month-old HRS/J mice were Thy 1.2+, Lyt 1+2+, H-2Kk-, and MuLV- with an immature-nonactivated phenotype, i.e., PNA+, and Iak-. Preleukemic and leukemic thymocytes showed diversity in expression of Thy 1.2 and Ly antigens with increased H-2Kk and MuLV expression. No differences in phenotype patterns were noted between hr/+ and hr/hr mice during the time course of leukemogenesis. Persistently high PNA/low Iak expression of preleukemic and leukemic thymocytes indicated that the target for HRS leukemic transformation was an immature-nonactivated thymocyte subpopulation in contrast to AKR/J mice in which leukemic transformation involves a mature-activated thymocyte subpopulation. These findings suggest that spontaneously generated leukemogenic viruses in HRS mice have tropism for thymocytes of an immature-nonactivated phenotype.     

Thy-2: A murine thymocyte-brain alloantigen controlled by a gene linked to the major histocompatibility complex

We describe a new murine cell-surface alloantigen, provisionally designated Thy-2, which is expressed primarily on thymocytes and brain tissue. Although Thy-2 is also expressed at lower levels on bone-marrow and spleen cells, this antigen does not appear to be present on lymph-node, liver, or red blood cells. Immunoprecipitation of surface-labeled thymocyte extracts from a variety of inbred strains reveals this antigen to be a single polypeptide of 150 000 daltons. Quantitative membrane immunofluorescence demonstrates that Thy-2 is a minor cell-surface component which is present on the majority of thymocytes. Mice heterozygous at theThy-2 locus express approximately 50 percent as much antigen as positive homozygotes. Expression of the Thy-2 alloantigen is controlled by a single semidominant gene located approximately 3 cM to the right of theH-2K locus on chromosome 17.     

Analysis of intrathymic differentiation patterns during the course of AKR leukemogenesis

Thymocytes from AKR mice in different stages of leukemia development were analyzed with the fluorescence-activated cell sorter (FACS) using monoclonal antisera to Lyt-1, Lyt-2, Thy-1.1, H-2K^k, and Ia^k. In addition, the number of cells bearing receptors for peanut agglutinin (PNA) was assessed. The results were correlated with the expression of murine leukemia virus (MuLV) antigen. Thymocytes from late preleukemic and leukemic stages were found to have a phenotype characteristic of a more mature cell population in that there was an increase in the expression of determinants encoded within the K end of H-2^k and Ia^k. This was associated with a decrease in the number of thymocytes bearing receptors for PNA during the leukemic stage. Simultaneously, a shift from a Lyt-1⁺ 2⁺ thymocyte population to cells with varying expressions of Ly antigens was observed. Analysis of Lyt determinants on thymomas indicated that they could arise from cells bearing any of the different possible combinations of Ly phenotypes. The cell surface antigen changes occurred in temporal correlation with an increased expression of MuLV antigens.     

Bone marrow-derived cells are essential for intrathymic deletion of self-reactive T cells in both the host- and donor-derived thymocytes of fully allogeneic bone marrow chimeras

The fate of self-reactive T cells was examined in both the host- and donor-derived thymocytes of fully allogeneic bone marrow (BM) chimeras of two strain combinations of AKR/J (H-2k, IE+, Thy-1.1, Mls-1a2b) and C57BL/6 (H-2b, IE-, Thy-1.2, Mls-1b2b). Sequential appearance of host- and donor-derived T cells occurred in the thymus of both AKR----B6 and B6----AKR chimeras in which 5 x 10(6) of T cell-depleted BM cells were used to reconstitute recipients lethally irradiated with 950 rad. Thymocytes bearing V beta 6 high, which recognize MHC class II IE-binding Ag encoded by Mls-1a allele, were detected in neither host- nor donor-derived thymocytes of AKR-B6 chimeras in which Mls-1a and IE were expressed only by the BM-derived cells. Thymocytes bearing V beta 11high capable of recognizing IE were also deleted in the host- and donor-derived thymocytes of the AKR----B6 chimeras. One million of BM cells were inadequate to deletion of the B beta 6high and V beta 11high T cells in the host-derived thymocytes of these chimeras. On the other hand, significant number of V beta 6high and V beta 11high thymocytes were detected in both the host- and donor-derived thymocytes in B6----AKR chimeras where sufficient dose of IE- stem cells were used to reconstitute irradiated Mls-1aIE+ recipients. These results suggest that clonal deletion of the host- and donor-reactive T cells in both the host- and donor-derived thymocytes is an important mechanism for the induction of transplantation tolerance in allogeneic BM chimeras and that BM-derived APC may be essential for the intrathymic elimination of both the host- and donor-reactive T cells in the BM chimeras.     

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.     

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.     

Characterization of thymocyte phenotypic alterations induced by long-lasting β-adrenoceptor blockade in vivo and its effects on thymocyte proliferation and apoptosis

Adult male Wistar rats were subjected to propranolol (P, 0.40 mg/100 g/day) or saline (S) administration (controls) over 14 days. The expression of major differentiation molecules on thymocytes and Thy-1 (CD90) molecules, which are shown to adjust thymocyte sensitivity to TCRαβ signaling, was studied. In addition, the sensitivity of thymocytes to induction of apoptosis and concanavalin A (Con A) signaling was estimated. The thymocytes from P-treated (PT) rats exhibited an increased sensitivity to induction of apoptosis, as well as to Con A stimulation. Furthermore, P treatment produced changes in the distribution of thymocyte subsets suggesting that more cells passed positive selection and further differentiated into mature CD4+ or CD8+ single positive (SP) TCRαβ^high cells. These changes may, at least partly, be related to the markedly increased density of Thy-1 surface expression on TCRαβ^low thymocytes from these rats. The increased frequency of cells expressing the CD4+25+ phenotype, which has been shown to be characteristic for regulatory cells in the thymus, may also indicate alterations in thymocyte selection following P treatment. Inasmuch as positive and negative selections play an important role in continuously reshaping the T-cell repertoire and maintaining tolerance, the hereby presented study suggests that pharmacological manipulations with β-AR signaling, or chemically evoked alterations in catecholamine release, may interfere with the regulation of thymocyte selection, and consequently with the immune response. (Mol Cell Biochem xxx: 1–13, 2005)     

A Thy-1.1-specific monoclonal alloantibody activates both mouse and rat T cells

In an attempt to further evaluate the role of Thy-1 in the antigen-independent triggering of mouse T cells, we have examined the activating properties of two Thy-1.1-specific mouse monoclonal antibodies (mAb). These reagents were established from an (A.TH X A.TL)F1 hybrid mouse (Thy-1b) immunized with IL-2 producing (BALB/c (Thy-1b) X BW5147 (Thy-1a)) T hybridoma cells. Although both mAb recognized the same Thy-1.1 determinant, one mAb of the gamma 3,kappa class (H171-146) was found to induce several T hybridoma cells to produce IL-2, and AKR thymocytes or cloned helper T cells to proliferate, whereas another mAb of the gamma 1,kappa class (H171-112) failed to do so even in the presence of phorbol myristic acetate (PMA). Increased IL-2 responses of T hybridoma cells were observed when the cell bound Thy-1.1-specific mAb were crosslinked by goat anti-mouse Ig (GaMIg) antibodies. Both a T-cell activating rat anti-Thy-1.2 mAb and the anti-Thy-1.1 mAb H171-146, although directed at distinct cell surface molecules, synergistically stimulated IL-2 production by T hybridoma cells. In addition, the mouse mAb H171-146 was found to stimulate LOU/M rat thymocytes to proliferate in the presence of exogenous IL-2. These data demonstrate that T cells can use Thy-1 as a signal-transducing molecule in both mouse and rat species, and support the notion that the activating properties of Thy-1.1-specific mAb are influenced by their heavy chain isotypes.