T-cell reactivity and tolerance to Mlsa-encoded antigens.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
H R MacDonald
Publications and source records attributed to H R MacDonald.
The cellular basis of T-cell memory.
Explore the source record for details and available documents.
Animals↗
Tolerance to Mlsa by clonal deletion of V beta 6+ T cells in bone marrow and thymus chimeras.
To evaluate the rules of tolerance induction to Mlsa we investigated maturation of T cells in irradiation bone marrow chimeras and thymus transplanted mice expressing the strongly stimulatory Mlsa or the nonstimulatory Mlsb in presence or absence of the permissive MHC class II IE molecule. As shown previously, deletion of V beta 6+, Mlsa reactive T cells required the presence of Mlsa and IE products either on donor cells or on recipient tissue. Additional experiments revealed that tolerance was induced when Mlsa and IE were expressed by distinct cells of the chimera. Similar observations were made in chimeras reconstituted with bone marrow stem cells of two different strains of mice. The presented data confirm earlier evidence that tolerance is induced by cooperation of Mlsa-expressing cells and IE+ Mlsb antigen-presenting cells (APCs). Preliminary results suggest that APCs presenting Mlsa during negative selection of V beta 6+ cells in the thymus are radiosensitive and derived from lymphohemopoietic stem cells.
Animals↗
Fate of potentially self-reactive T cells in neonatal mice: analysis of V beta 6+ T cells in Mlsa mice.
FACS analysis of either CD4 or CD8 depleted thymocytes revealed, that in adult Mlsa mice among CD4+ or CD8+ subpopulation of mature T cells V beta 6+ cells were absent. In contrast neonatal Mlsa mice had CD4+ V beta 6+ cells that rapidly decreased after day 4. On thymus cryosections of neonatal DBA/2 (Mlsa) mice V beta 6+ cells were found both in the cortex and the medulla of the thymus, in addition V beta 6+ cells were also detected in lymph nodes. After day 7-8 V beta 6+ cells disappeared from the medulla and the periphery. In the adult thymus only dull staining V beta 6+ cells were present in the cortex. Comparison of the fluorescence intensities of V beta 6 staining thymocytes from neonatally with Mlsa spleen cells tolerized mice with that of untreated BALB/c controls showed a significant decrease in T cell receptor density. Neonatal V beta 6+ T cells could be shown to be autoreactive against Mlsa, this reactivity declined at about the same time when the stimulatory capacity of Mlsa was established and could be first shown on day 4.
Animals↗
Autoreactivity of hybrids putatively derived from immature CD4+CD8+V beta 6+ thymocytes from an Mlsa mouse.
Explore the source record for details and available documents.
Animals↗
A novel T cell-activating molecule (THAM) highly expressed on CD4-CD8- murine thymocytes.
Recent studies have focused on the potential role of accessory molecules such as CD2, CD28, Thy-1, or TAP in the delivery of activating signals to thymocytes through antigen-independent pathways. To better understand the molecular interactions involved in the expansion of early thymic immigrants, rat mAb were raised against murine thymocyte-surface molecules and screened for their capacity to trigger thymocyte proliferation. One of these mAb (H194-112, IgG2a) was found to recognize a novel heterodimeric thymocyte-activating molecule (THAM) of Mr = 110,000 to 128,000. Flow cytometric analyses and staining patterns on frozen thymus sections subdivided adult thymocytes in three subsets expressing THAM at either low (10%), moderate (80%), or high (5 to 8%) cell-surface density; these cell groups were found to correspond, respectively, to the medullary, the cortical, and the immature CD4-CD8-, J11d+ thymocytes, in which the T cell precursor pool is included. Moreover, most (90%) day 16 fetal thymocytes were also found to upregulate THAM cell-surface expression. The THAMhigh cells were localized in the subcapsular area of the neonatal thymus and scattered throughout the adult organ. Cross-linked mAb H194-112 induced the proliferation of both immature and mature thymocytes in the presence of either PMA or IL-1 and IL-2. The observation that early thymocytes up-regulate THAM along with the IL-2R suggests that this molecule might be involved in an important activation pathway during thymocyte differentiation.
Aging↗
Positive selection of CD4+ thymocytes controlled by MHC class II gene products.
The mature T-cell antigen receptor repertoire is characterized by lack of reactivity to self-components as well as by preferential reactivity to foreign antigens in the context of polymorphic self-proteins encoded within the major histocompatibility complex. Whereas the former characteristic (referred to as negative selection or tolerance) is associated with intrathymic deletion of T cells expressing T-cell antigen receptor beta-chain variable (V beta) domains, which confer a preferential reactivity to self antigens, the existence of the latter (referred to as positive selection or MHC restriction) has so far only been inferred indirectly from functional studies. We show here that intrathymic deletion of V+beta 6 T cells (reactive with a self-antigen encoded by the Mlsa locus) is controlled by polymorphic MHC class II determinants. Furthermore, in mice lacking expression of Mlsa, the same class II MHC loci control the frequency of occurrence of V+beta 6 cells among mature CD4+ T lymphocytes. These data are direct evidence for positive selection by MHC determinants in the thymus in unmanipulated animals.
Animals↗
Deletion of self-reactive T cells before entry into the thymus medulla.
The thymus is important in the differentiation of bone marrow-derived precursor cells into functional T cells; humoral factors, as well as physical interactions with nurse cells, dendritic cells and epithelial cells, are thought to be instrumental in this process. Thymic lymphocytes mature during their migration from the cortical to the medullary region of the thymus, when they undergo phenotypic changes that include the acquisitions of T-cell antigen receptors, hormone receptors and differentiation antigens. Cortical T cells are thus mostly CD4+CD8+, whereas medullary T cells are either CD4+CD8- or CD4-CD8+. During this period T cells are subjected to two types of repertoire selection: all T cells recognizing self-MHC with low affinity may be preferentially amplified (positive selection), and in a second step T cells with high-affinity receptors for self-MHC determinants plus self antigens are eliminated (negative selection). We have described two monoclonal antibodies specific for the V beta 6 gene segment of the alpha/beta heterodimeric T-cell antigen receptor and have shown that most CD4+/V beta 6+ T cell recognize the Mlsa antigenic determinant but not Mlsb; similar results have been reported for V beta 8.1 and Mlsa. In both situations, tolerance to Mlsa correlated in an MHC-dependent fashion with absence of V beta 6 or V beta 8.1 T-cell antigen receptor expressing T cells in the periphery. We show here by immunostaining of thymus cryosections and cytofluorometric analysis that V beta 6-expressing cortical T cells are present at high density in both Mlsa and Mlsb mice, but do not enter the medullary region of Mlsa animals.
Animals↗
Intrathymic deletion of self-reactive cells prevented by neonatal anti-CD4 antibody treatment.
T-cell differentiation in the thymus involves the coordinate expression of genes encoding the alpha and beta chains of the major histocompatibility complex-restricted heterodimeric antigen receptor (TCR) complex, as well as other functionally important molecules such as CD4 and CD8. The repertoire of TCR expressed by T cells is generally thought to be influenced by positive and/or negative selection events occurring when TCRs on developing T cells interact with self-antigens and major histocompatibility complex components. Using a model system in which specific antigen-reactive cells can be monitored by virtue of their preferential expression of certain TCR beta-chain variable (V beta) domains, it has been shown that self-reactive T cells are clonally deleted during development. We report here that clonal deletion of V+ beta 6 cells in Mlsa mice can be prevented by in vivo neonatal administration of monoclonal antibodies directed against CD4. Furthermore, as anti-CD4 monoclonal antibody treatment resulted in the reappearance of V+ beta 6 cells in the mature CD8+ T-cell subset, it is likely that clonal deletion acts on the CD4+CD8+ thymocyte subset and that this subset is an intermediate stage in the differentiation pathway of both CD4+ and CD8+ T-cell lineages.
Animals↗
cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily.
Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.
Amino Acid Sequence↗
Intrathymic elimination of Mlsa-reactive (V beta 6+) cells during neonatal tolerance induction to Mlsa-encoded antigens.
The cellular basis of neonatally induced T cell tolerance has been investigated in a model system in which usage of a particular TCR V beta segment (V beta 6) is strongly correlated with reactivity to antigens encoded by the Mlsa genetic locus. Expression of V beta 6 by peripheral T cells was virtually abolished in BALB/c (H-2d, Mlsb) mice rendered neonatally tolerant to DBA/2 (H-2d, Mlsa) lymphoid cells, whereas control V beta 8-bearing T cells remained at near normal levels. Further analysis revealed that elimination of V beta 6+ T cells occurred in the thymus of neonatally tolerant mice and could not be explained by receptor modulation or T cell chimerism. These data thus support the clonal deletion model of tolerance induction.
Animals↗
CD3-associated alpha/beta and gamma/delta heterodimeric receptors are expressed by distinct populations of CD4- CD8- thymocytes.
Immature double negative (DN) CD4-8- thymocytes expressing the CD3 molecular complex can be subdivided into two distinct subsets based on expression of the B2A2 antigen. Thus, B2A2+ CD3+ DN thymocytes express CD3-associated 35 kDa and 45 kDa polypeptide chains characteristic of a gamma/delta T cell receptor, whereas B2A2- DN thymocytes express predominantly 38 to 43 kDa CD3-associated structures typical of alpha/beta TCR. At the mRNA level, B2A2+ DN thymocytes express full length gamma and delta transcripts but no alpha message and only short (1 kb) B transcripts. In contrast, the B2A2- DN subset expresses full length alpha and beta transcripts and minimal levels of delta transcripts. Interestingly, B2A2- DN thymocytes, unlike mature T cells, also express abundant levels of full length TCR gamma mRNA, perhaps suggesting that these cells represent an early stage after divergence of the gamma/delta and alpha/beta T cell lineages.
Animals↗
T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.
T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.
Animals↗
Heterogeneity of immature (Lyt-2-/L3T4-) thymocytes. Identification of four major phenotypically distinct subsets differing in cell cycle status and in vitro activation requirements.
Thymocytes that bear neither Lyt-2 nor L3T4 differentiation Ag (2-4- thymocytes) contain the precursors of mature Lyt-2+ and L3T4+ T cells. In the present study, we have identified four major subpopulations of 2-4- cells in adult C57BL/6 mice that differ in surface phenotype and in situ proliferative status. Two-color immunofluorescence analysis with RL-73 (a mAb recognizing an as yet unidentified activation Ag) and PC-61 (an anti-IL-2R mAb) revealed three distinct subsets of 2-4-thymocytes: RL-73+ IL-2R- (30%), RL-73+/-IL-2R+ (45%), and RL-73- IL-2R- (25%). The RL-73+ IL-2R- subset had the highest percentage of large blasts and cycling cells, whereas the RL-73+/- IL-2R+ and RL-73- IL-2R- subsets had intermediate and low percentages, respectively, indicating that in situ proliferation correlated better with RL-73 intensity than with IL-2R expression. An additional marker, heat-stable Ag (HSA), was found to further subdivide the RL-73- population into RL-73- HSA- (10% of total 2-4-) and RL-73- HSA+ (15%) fractions. The two latter (RL-73-) subsets appeared to be more "mature" than the former since they expressed high levels of Lyt-1 and appeared later during fetal thymus ontogeny. In parallel with the phenotypic analysis, we compared the in vitro activation requirements of each of the four purified 2-4- subsets. All four populations proliferated well to the combination of phorbol ester (PMA), ionomycin, and IL-2. In response to PMA and ionomycin (without added IL-2), only RL-73- HSA-cells proliferated and this proliferation was correlated with IL-2 production. However, if IL-1 was included with PMA and ionomycin then all four populations responded. Finally, a proliferative response to Con A or mitogenic anti-Thy-1 mAb was observed only for RL-73- HSA+ and (to a lesser extent) RL-73- HSA-cells. These data indicate that each of the four phenotypically distinct subpopulations of immature thymocytes can also be distinguished on the basis of their in vitro activation requirements.
Animals↗
Regulation of expression of T cell gamma chain, L3T4 and Ly-2 messages in Abelson/Moloney virus-transformed T cell lines.
Recent results have suggested that T cells may exist in two distinct pathways, one expressing alpha and beta chain of the T cell receptor genes with either or both of the cell surface markers CD4 and CD8, while the other is negative for these cell surface markers and expresses the T cell-specific gamma chain genes. The relationship between these two pathways is not known. In this study, we have examined a series of either Abelson virus or Moloney virus-derived T cell lines for their expression of these T cell receptor and cell surface marker genes. Results indicate that the Abelson T cell lines do not express the cell surface markers CD4 and CD8, but express relatively high levels of gamma chain transcripts. After culture of these cell lines with the phorbol ester phorbol myristate acetate and interleukin 2, a down-regulation of these gamma chain transcripts can be observed. More interestingly, we found that the Moloney virus-derived T cell lines, which express the cell surface markers CD4 and/CD8, contain high levels of alpha and beta chain T cell receptor transcripts but little or no gamma transcripts even though they have rearranged these latter genes. The gamma transcripts, however, can be induced to high levels after culture with phorbol myristate acetate and interleukin 2. In the process, the cell surface markers CD4 and CD8 and their transcripts were dramatically down-regulated resulting in cells with high levels of gamma chain transcripts and a CD4-CD8- phenotype. The regulation of expression of these genes is reversible. Taken together, these results indicate that the T cell receptor gamma chain genes and those of the cell surface markers CD4 CD8 can be regulated in vitro by external factors and it opens up the possibility of studying the regulatory sequences associated with these genes.
Abelson murine leukemia virus↗
A CD3- subset of CD4-8+ thymocytes: a rapidly cycling intermediate in the generation of CD4+8+ cells.
T lymphocytes with the surface phenotype CD4+8- and CD4-8+ are considered to be representative of functionally mature cells. We show here that adult murine thymus contains a subpopulation of CD4-8+ cells that differ from CD4-8+ cells found in the periphery in that they do not express the T cell receptor-associated CD3 molecular complex. Such CD3-4-8+ thymocytes are cortisone sensitive and rapidly cycling in situ. Furthermore, in contrast to mature T cells, most CD3-4-8+ thymocytes express low levels of CD5 and high levels of the B2A2 antigen. CD3-4-8+ thymocytes fail to respond to a variety of mitogenic stimuli in vitro but do give rise upon short-term culture to CD4+8+ cells. It is suggested that CD3-4-8+ thymocytes represent a transitional stage of thymus differentiation between the CD4-8- and CD4+8+ compartments.
Animals↗
The glioblastoma-derived T cell suppressor factor/transforming growth factor-beta 2 inhibits T cell growth without affecting the interaction of interleukin 2 with its receptor.
Human glioblastoma cells secrete a peptide termed glioblastoma-derived T cell suppressor factor (G-TsF) which inhibits T cell activation. Recently, purification and cloning of G-TsF revealed that G-TsF is identical to transforming growth factor-beta 2. As shown here, G-TsF suppresses the growth of an ovalbumin-specific mouse T helper cell clone (OVA-7T) independently of the stimulus used being either (a) antigen in the presence of antigen-presenting cells, or (b) interleukin 2 (IL2) or (c) phorbol ester and calcium ionophore. Furthermore, in the presence of antibodies against IL2 receptors, G-TsF was able to suppress the residual proliferation still observed when OVA-7T were stimulated with phorbol ester/ionophore. G-TsF failed to inhibit the release of IL3 from OVA-7T activated with IL2. Taken together, the data provide evidence that G-TsF does not directly interfere with interactions of IL2 with its receptor but rather inhibits T cell activation by interfering with an as yet unidentified pathway used by both IL2 and phorbol ester/ionophore. When analyzing different monokines and lymphokines for its effect on G-TsF-induced suppression of T cell growth the only factor found to partially neutralize the effect of G-TsF was tumor necrosis factor-alpha.
Animals↗
Gene transfer of the Ly-3 chain gene of the mouse CD8 molecular complex: co-transfer with the Ly-2 polypeptide gene results in detectable cell surface expression of the Ly-3 antigenic determinants.
The CD8 molecule is a glycoprotein expressed on a subset of mature T lymphocytes. It has been postulated to be a receptor for class I major histocompatibility complex molecules. In the mouse, CD8 is a heterodimer composed of Ly-2 and Ly-3 chains. We have isolated and analyzed cDNA and cosmid clones corresponding to the Ly-3 subunit. One of the isolated, cosmid clones was subsequently transfected, alone or in combination with the Ly-2 gene, into mouse Ltk- cells. Analysis of the Ly-2,3 molecules expressed at the surface of the double transfectants indicated that they are serologically and biochemically indistinguishable from their normal counterparts expressed on lymphoid cells. Ltk- cells transfected with the Ly-2 gene alone were shown to react with a subset of anti-CD8 monoclonal antibodies whereas Ly-3 transfectants did not stain with any of the anti-Ly-3 antibodies employed in this study. Since at least one of these antibodies (53-5.8) has been previously shown to recognize an epitope which is retained on the Ly-3 subunit after dissociation of the heterodimeric Ly-2,3 complex, these observations suggest that the expression of the Ly-2 polypeptide is required to permit the detectable cell surface expression of the antigenic determinants carried by the Ly-3 subunit.
Amino Acid Sequence↗