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At least 19 recordsLinked to original sources

PubMed1991

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.

Age Factors↗

PubMed1983

Thymocyte and macrophage interactions: separation of murine thymocyte subsets and enrichment of syngeneic cell-responding thymocytes by adsorption to macrophage monolayers.

The spontaneous binding of murine thymocytes to macrophage monolayers was employed to separate thymocytes into macrophage-unbound and -bound subsets, and the functional reactivities of these two subpopulations were examined. Macrophage-unbound thymocytes were found to be enriched in functional subsets reactive to semi-allogeneic and allogeneic stimulating spleen cells by proliferation in mixed leukocyte culture (MLC). Furthermore, macrophage-unbound thymocytes were frequently found to respond to syngeneic spleen cells. This syngeneic proliferative response showed both memory and specificity upon subsequent restimulation and thus seems to represent a syngeneic mixed leukocyte reaction (SMLR). Syngeneic responding thymocytes were also found to produce interleukin 2 when cultured with syngeneic but not allogeneic stimulator cells. In contrast, macrophage-bound thymocytes showed greatly reduced proliferative responses to allogeneic stimulators and no responses to syngeneic stimulators. The macrophage-bound thymocyte subset was not enriched in detectable suppressive activity; proliferative responses of macrophage-unbound thymocytes to either allogeneic or syngeneic cells were neither suppressed nor enhanced when macrophage-unbound thymocytes were added to the cultures. Thus, the macrophage-unbound subset seems to be enriched in functionally mature thymocytes and the macrophage-bound subset appears to be enriched in functionally immature thymocytes. This functional separation of thymocytes by macrophage adherence also correlated well with thymocyte subpopulations separated by bovine serum albumin density gradients; the low density mature thymocytes showed enhanced responses to both allogeneic and syngeneic stimulators, whereas the high density immature cells were unresponsive. This correlation was supported further by binding studies in which T cell tumor lines derived from C57BL/6 mice were used. ERLD tumor cells, which are similar to cortical immature thymocytes in both enzymatic and surface antigenic markers, were found to bind readily to macrophages, whereas both EL-4 and E male G2 tumor cells, with characteristics of mature thymocytes, bound to macrophages poorly. The binding of thymocytes and ERLD tumor cells to macrophages was not genetically restricted. We speculate that thymocyte binding to macrophages may play a critical role during the functional maturation of thymocytes.

Animals↗

PubMed1981

The role of the maturation stage of thymocytes on the phytohemagglutinin and concanavalin A responses in thymocyte and lymph node lymphocyte cocultures in guinea pig. Effects of pretreatment of thymocytes with thymosin or levamisole.

Guinea pig thymocytes (TH) and lymph node lymphocytes (LNL) synergized optimally in both phytohemagglutinin (PHA) and concanavalin (Con A) responses in mixtures containing 0.3 x 10(6) TH and 0.2 x 10(6) LNL. Using discontinuous albumin gradient centrifugation thymocytes were separated into two subpopulations (F4 and F6) at different stages of maturation. Immature, PHA and Con A unresponsive F6 thymocytes synergized significantly only in the PHA response. More mature, PHA and Con A responsive F4 thymocytes cooperated well in the Con A response, but only a small synergy was observed in the PHA response. Pretreatment of the unfractionated thymocytes with the low concentration (0.05 microgram/ml) of thymosin decreased significantly their capacity to interact with LNL in both PHA and Con A responses. Preincubation of F4 thymocytes with the high concentration (200 microgram/ml) of thymosin increased the synergy in PHA response. All other combinations with thymosin or levamisole and thymocytes were ineffective on the mitogenic responses in the TH and LNL cocultures. Altogether, the results how that the thymocyte populations that induce synergy in PHA, or in (PHA and) Con A responses represent the two subpopulations of thymocytes with different maturation stages. The differential effects of the two concentrations of thymosin on the thymocytes support further the concept that the thymocytes synergizing in responses to both mitogens are more mature than those synergizing only PHA response. Thus, the ability of thymocytes to interact with LNL is dependent on the maturation stage of thymocytes, and can be utilized to study the differentiation of thymocytes.

Animals↗

PubMed1998

Immature thymocyte antigen-1: a novel thymocyte marker discriminating pre- and post-selected thymocytes.

Previously, we described a mAb (1-23) reacting with a novel cell surface antigen expressed on thymocytes at late CD4-CD8- [(double negative (DN)] to early CD4+CD8+ [(double positive (DP)] differentiation stage. Since the expression of this molecule was restricted to immature thymocytes, we designated it as immature thymocyte antigen-1 (IMT-1). In this study, we have investigated the relevance of IMT-1 expression to thymocyte selection using TCR transgenic mice, scid mice or RAG-2-/- mice. The IMT-1+ population in DP thymocytes was decreased in the thymuses of MHC class I-restricted or class II-restricted TCR transgenic mice with a positively selecting MHC background when compared with that of the mice with a non-selecting MHC background. IMT-1+ DP thymocytes were also decreased in TCR transgenic mice in which negative selection occurs. When DP thymocytes in H-Y TCR transgenic mice were stimulated with CD3epsilon mAb in vitro as well as in vivo, the expression of IMT-1 on DP thymocytes was decreased. Furthermore, the expression of IMT-1 on DN thymocytes from RAG-2-/- mice was drastically reduced when CD3epsilon mAb was challenged in vivo. These results suggest that the expression of IMT-1 on DP or DN thymocytes is down-regulated by stimulation through TCR as well as pre-TCR. Taken together, these results show that IMT-1 is a unique surface marker which exquisitely separates pre-selected thymocytes from post-selected thymocytes.

Animals↗

PubMed1992

Influence of ST 789 on murine thymocytes: a flow cytometry study of thymocyte subset distribution and of intracellular free Ca++ increase upon activation. Murine thymocytes and ST 789.

The coordinate expression of CD4 and CD8 antigens defines four major subsets of adult mouse thymocytes (CD4+CD8-, CD4+CD8+, CD4-CD8+ and CD4-CD8-) that represent definite steps in the differentiative pathway of immature T-lymphocytes. Thymocytes from adult C57BL/6 mice were cultured in the presence of ST 789, an L-Arg synthetic derivative of hypoxanthine, (1 to 100 micrograms/ml) or PHA 60.1 to 1 microgram/ml), or both for 48 hours, and then stained with monoclonal antibodies to CD4 and CD8 antigens for dual color flow cytometry analysis. The expression of CD25, was also investigated. ST 789 was ineffective in modifying the distribution of the four thymocyte subsets and did not induce the appearance of CD25 on cortical thymocytes. PHA induced a dramatic dose-dependent decrease of the CD4+CD8+ subset that, however, was neither enhanced nor antagonized by ST 789. We also tested ST 789 for its ability to stimulate intracellular free Ca++ rise in thymocytes. The compound was ineffective in this assay. Conversely, thymocytes promptly responded to PHA stimulation. It is concluded that ST 789 has no effect on normal adult thymocyte differentiation/activation pathway.

Adjuvants, Immunologic↗

PubMed1991

Influence of LTB4 on CD4-, CD8- thymocytes. Evidence that LTB4 plus IL-2 generate CD8+ suppressor thymocytes involved in tolerance to self. Effect of LTB4 and IL-2 on double negative thymocytes.

Leukotriene B4 (LTB4) is produced by a large variety of cells involved in immune response and it has been demonstrated that this arachidonic acid metabolite acts as an immunomodulator. Because LTB4 and IL-2 both influence the physiology of immature cells we studied the effects of the leukotriene on double negative thymocytes. For that purpose C57 Bl/6 double negative thymocytes were treated by LTB4 plus IL-2 in the presence of either autologous or allogenic red blood cells (RBC). Then, preincubated CD4- CD8- thymocytes were cocultured with red blood cells stimulated fresh splenocytes. We observed that fresh splenocytes responding to autologous RBC were CD4+ cells and that the proliferative response of spleen lymphocytes driven by RBC was inhibited by preincubated double negative thymocytes. On the other hand a majority of double negative thymocytes overnight preincubated in vitro in the presence of both IL-2 and LTB4 give rise to CD8+ CD4- cells. Therefore we speculate that LTB4 plus IL-2 generate CD8+ suppressor thymocytes among double negative thymocytes and that these suppressive T cells are involved in tolerance to self.

Animals↗

PubMed1975

Correlation between mature and immature thymocytes. Immature thymocyte potentiating factor (IMPF) produced by mature thymocytes.

Low-dense mature thymocytes (MT) and high-dense immature thymocytes (IMT) were investigated. Immature thymocyte-potentiating factor (IMPF) was found in the supernatant from MT stimulated by Con A. On the other hand, suppressing activity was present in the supernatant from the unstimulated IMT. IMPF was different from Con A itself. Sephadex G-100 gel chromatography of IMPF revealed that the activity was recovered at the position between bovine serum albumin and ovalbumin.

Animals↗

PubMed1993

Thymic nurse cell clone supports the differentiation of CD4-8- thymocytes into CD4+8+ thymocytes in vitro.

A previously reported thymic nurse cell clone, TNC-R3.1 could form a unique complex with isolated adult mouse CD4-8- (DN) thymocytes and greatly sustained the cell viability of DN thymocytes in suspension culture. In addition, the TNC-R3.1 clone supported the differentiation of DN thymocytes into CD4+8+ (DP) thymocytes in a short-term culture. Addition of IL-7 into the coculture markedly enhanced DN thymocyte-TNC interaction and induced the proliferation and differentiation of DN thymocytes, though IL-7 alone did not induce the differentiation of DN thymocytes. Separation of DN thymocytes from TNC-R3.1 monolayer using a Millicell caused a great inhibition of the DN thymocyte differentiation, suggesting that direct contact between TNC-R3.1 cells and immature thymocytes was required for the differentiation of DN thymocytes. The kinetics study demonstrated that DN thymocytes started to differentiate into DP thymocytes through CD3-CD4+J11d+ intermediate cells 8-12 h after the initiation of the culture with TNC-R3.1 plus IL-7. The generation of DP thymocytes became maximal 20 h after coculture and gradually decreased thereafter. Furthermore, we demonstrated that TNC-R3.1 could support the differentiation of CD3+CD4+CD8- or CD3+CD4-CD8+ thymocytes from CD3-CD4-CD8- thymocytes in the presence of IL-7 and IL-2. These data indicate that our established in vitro culture system mimics the early stage of the intrathymic T cell developing pathway.

Animals↗

PubMed1989

Triggering of thymocyte function by IL-2 as the only exogenous stimulus; analysis of two distinct modes of IL-2-induced thymocyte proliferation and IL-3 secretion in vitro.

Addition of recombinant interleukin-2 (rIL-2) to normal adult murine thymocytes in vitro as the only exogenous stimulus leads to a dose-dependent mitogenic response characterized by two distinct dosage kinetic components. The high-affinity IL-2 thymocyte response is mounted by in vivo-activated (IL-2 receptor light chain positive) thymocytes, while the low-affinity IL-2 response, of larger amplitude, is carried out by resting thymocytes. Addition of IL-2 to thymocytes also triggers intense IL-3 secretory responses with both high and low IL-2 affinity components. Addition of high IL-2 dosages to thymocyte bulk cultures results in a dramatic increase in IL-2 responsiveness for both proliferation and IL-3 secretion on a per viable cell basis and with tightly coupled temporal kinetics. The low-affinity component of IL-2-proliferative and IL-3-secreting responses is carried out by resting mature CD4+ thymocytes, as assessed by negative selection with monoclonal antibodies (mAb) plus complement. The mechanism of resting thymocyte activation by high doses of IL-2 is partially characterized. Depletion of endogenous thymus-adherent cells abolished both proliferation and IL-3 secretion, and addition of splenic accessory cells or peritoneal macrophages to depleted thymocytes restored IL-2 responsiveness. Mature CD4+ thymocytes spontaneously form rosettes with adherent accessory cells, while CD8+ thymocytes do so with much less efficiency. Rosette formation of CD4+, but not of CD8+ thymocytes, can be blocked by anti-CD4 mAb GK1.5. At the same dosage as it prevents rosette formation, mAb GK1.5 also blocks the low-affinity thymocyte response to IL-2. The high-affinity IL-2 response is completely resistant to the action of cyclosporin A (CsA), but the low-affinity IL-2 response, although of much larger amplitude, can be almost completely suppressed by CsA. Together, these results demonstrate that resting CD4+ thymocytes can be induced to proliferation and lymphokine secretion by IL-2 alone in a process that is dependent on interaction with accessory cells, involves CD4 adhesion molecules and triggers activation through a CsA-sensitive pathway. In addition, the results demonstrate that IL-2 alone is able to enhance thymocyte IL-2 responsiveness and IL-3 secretory responses in vitro. The ability of IL-2 to induce and maintain thymocyte function is discussed in the light of these results.

Animals↗

PubMed1993

PNA lectin-based separation of thymocytes into mature and immature subpopulations: CD4-8- double negative cells display characteristics of PNAlo mature thymocytes.

Cortical (immature) thymocytes are widely reported to express intermediate to high levels of receptors for the lectin, peanut agglutinin (PNA). Light-scatter studies of murine fetal thymocytes stained with PNA or anti-mouse CD4 and CD8 monoclonal antibodies indicated, however, that the most immature CD4-8- (DN) thymocyte subpopulation binds levels of PNA commonly described as PNAlo. Evaluation of the PNA binding characteristics of fetal thymocytes negative for the CD8 antigen confirmed the existence of a major population (approximately 20% of total cells) of CD4-8- PNAlo fetal thymocytes. The majority of these DN thymocytes were subsequently found to bind sub-agglutinating levels of PNA, similar to mature CD4+ or CD8+ single positive (SP) thymocytes. Given this information, an immunomodulating compound (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) known to produce a maturational delay in murine thymocytes was tested for a possible concurrent effect on thymocyte PNA lectin binding. A TCDD-induced increase in DN thymocytes was found to be paralleled by an increase of equal magnitude in PNAlo thymocytes. Taken together, these data provide evidence that acquisition of the PNA receptor may be a maturational event occurring during the DN stage of thymocyte ontogeny. Further, these results suggest that separation of thymocytes into mature (medullary) and immature (cortical) subpopulations by PNA agglutination may result in contamination of medullary cells by the most immature (DN) subpopulation of thymocytes.

Animals↗

PubMed1990

Growth-promoting activity of IL-1 alpha, IL-6, and tumor necrosis factor-alpha in combination with IL-2, IL-4, or IL-7 on murine thymocytes. Differential effects on CD4/CD8 subsets and on CD3+/CD3- double-negative thymocytes.

Many cytokines (including IL-1, IL-2, IL-4, IL-6, and TNF-alpha) have been shown to induce thymocyte proliferation in the presence of PHA. In this report, we demonstrate that certain cytokine combinations induce thymocyte proliferation in the absence of artificial comitogens. IL-1 alpha, IL-6, and TNF-alpha enhanced the proliferation of whole unseparated thymocytes in the presence of IL-2, whereas none of them induced thymocyte proliferation alone. In contrast, of these three enhancing cytokines, only IL-6 enhanced IL-4-induced proliferation. We also separated thymocytes into four groups based on their expression of CD4 and CD8, and investigated their responses to various cytokines. The results indicate that each cytokine combination affects different thymocyte subsets; thus, IL-1 alpha enhanced the proliferation of CD4-CD8- double negative (DN) thymocytes more efficiently than IL-6 in the presence of IL-2, whereas IL-6 enhanced the responses of CD4+CD8- and CD4-CD8+ single positive (SP) thymocytes to IL-2 or IL-4 better than IL-1 alpha. TNF-alpha enhanced the proliferation of both DN and both SP subsets in the presence of IL-2 and/or IL-7. None of these combinations induced the proliferation of CD4+CD8+ double positive thymocytes. Finally, DN were separated into CD3+ and CD3- populations and their responsiveness was investigated, because recent reports strongly suggest that CD3+ DN thymocytes are a mature subset of different lineage rather than precursors of SP thymocytes. CD3+ DN proliferated in response to IL-7, TNF-alpha + IL-2, and IL-1 + IL-2. CD3- DN did not respond to IL-7 or to IL-1 + IL-2, but did respond to TNF-alpha + IL-2. Finally, we detected TNF-alpha production by a cloned line of thymic macrophages, as well as by DN adult thymocytes. These results suggest that cytokines alone are capable of potent growth stimuli for thymocytes, and indicate that different combinations of these molecules act selectively on thymocytes at different developmental stages.

Animals↗

PubMed1988

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.

Animals↗

PubMed1994

Cytotoxicity of fresh NK1.1+ T cell receptor alpha/beta+ thymocytes against a CD4+8+ thymocyte population associated with intact Fas antigen expression on the target.

Recent studies have revealed that 10-20% of CD4+8- or CD4-8- thymocyte populations contain NK1.1+ T cell receptor (TCR)-alpha/beta+ cells. This subpopulation shows characteristics that are different from NK1.1- CD4+ or NK1.1- CD8+ T cells and seems to have developed in a manner different from NK1.1- T cells. Although extensive studies have been performed on the NK1.1+ TCR-alpha/beta+ thymocytes, the physiological role of the NK1.1+ TCR-alpha/beta+ thymocytes has been totally unclear. In the present study, we found that freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes, but neither whole thymocytes nor lymph node T cells, directly killed CD4+8+ thymocytes from normal syngeneic or allogeneic mice by using a long-term cytotoxic assay in which flow cytometry was used to detect the cytotoxicity. However, only weak cytotoxicity was detected against thymocytes from lpr mice on which the Fas antigen that transduces signals for apoptosis into the cells is not expressed. Furthermore, the NK1.1+ TCR-alpha/beta+ thymocytes exhibited high cytotoxicity against T lymphoma targets transfected with fas genes as compared with the parental T lymphoma targets or target cells transfected with mutated fas genes, which lack the function of transducing signals. On the other hand, NK1.1+ effector thymocytes from gld mice that carry a point mutation in Fas ligand did not kill thymocyte targets from normal mice. The present findings, thus, consistently suggest that the NK1.1+ TCR-alpha/beta+ thymocytes kill a subpopulation among CD4+8+ thymocytes via Fas antigen and in this way regulate generation of T lineage cells in the thymus.

Animals↗

PubMed1996

Physical and functional association of CD45 and CD3-TCR complex on CD1+ human thymocytes. Evidence that the engagement of CD45 molecules can prevent CD1+ thymocytes from apoptosis.

In this study the effects of CD45 engagement on CD3-TCR-driven stimulation of CD1+ human immature thymocytes have been analyzed. Simultaneous cross-linking of CD45 and CD3 antigens on highly purified CD1+ thymocytes reduced the number of cells undergoing apoptosis after 16 h of in vitro culture. This cell population might represent immature thymocytes committed in vivo to die by programmed cell death (PCD). CD45 engagement could also increase the number of cycling CD1+ thymocytes; of note, the large majority (> 95%) of dividing cells expressed the CD1 molecule at the cell surface, indicating that proliferating cells were actually represented by immature thymocytes. These data suggest that the CD45 molecule might play a role in the rescue of immature thymocytes from PCD during differentiation. Along this line, we found that activation of CD1+ thymocytes via the CD3-TCR complex could be enhanced by CD45, both in terms of transcription and surface expression of IL-2R. These effects might be explained by the finding that the CD45 molecule (but not its isoforms CD45RO and RA) was physically associated with the CD3-TCR complex at the cell surface of CD1+ human thymocytes, as shown by co-precipitation and co-capping experiments. Finally, cross-linking of CD45 and CD3 antigens led to the expansion of CD3+ thymocytes co-expressing CD4 and CD8, indicating that simultaneous engagement of CD45 and CD3 molecules can block CD1+ cells at the double-positive (CD3+CD4+CD8+) differentiation stage. On the other hand, stimulation through CD3 resulted in the expansion of thymocytes showing a mature phenotype (CD3+CD4+ or CD3+CD8+). Altogether, these findings suggest that the CD45 molecule is involved both in early activation and in the regulation of CD1+ thymocyte differentiation.

Antibodies, Monoclonal↗

PubMed1990

Thymocyte LFA-1 and thymic epithelial cell ICAM-1 molecules mediate binding of activated human thymocytes to thymic epithelial cells.

We have investigated the binding in vitro of activated thymocytes to thymic epithelial (TE) cells, and studied the effect of up-regulation of TE cell surface intracellular adhesion molecule 1 (ICAM-1) and HLA-DR by IFN-gamma on the ability of TE cells to bind to both resting and activated human thymocytes. TE cell binding to activated and resting thymocytes was studied by using our previously described suspension assay of TE-thymocyte conjugate formation. We found that activated mature and immature thymocytes bound maximally at 37 degrees C to IFN-gamma-treated ICAM-1+ and HLA-DR+ TE cells and this TE-activated thymocyte binding was inhibited by antibodies to LFA-1 alpha-chain (CD11a) (68.1 +/- 5.6% inhibition, p less than 0.01) and ICAM-1 (73.9 +/- 7.7% inhibition, p less than 0.05). Neither anti-HLA-DR antibody L243 nor anti-MHC class I antibody 3F10 inhibited IFN-gamma-treated TE binding to activated thymocytes. As with antibodies to LFA-3 and CD2, antibodies to LFA-1 and ICAM-1 also inhibited PHA-induced mature thymocyte activation when accessory signals were provided by TE cells in vitro. Finally, LFA-1 and ICAM-1 were expressed early on in human thymic fetal ontogeny in patterns similar to those seen in postnatal thymus. Taken together, these data suggest that resting mature and immature thymocytes bind to TE cells via the CD2/LFA-3 ligand pair, whereas activated thymocytes bind via both CD2/LFA-3 and LFA-1/ICAM-1 ligand systems. We postulate that IFN-gamma produced intrathymically may regulate TE expression of ICAM-1 and therefore potentially may regulate TE cell binding to activated thymocytes beginning in the earliest stages of human thymic development.

Antibodies, Monoclonal↗

PubMed1989

IL-7 promotes thymocyte proliferation and maintains immunocompetent thymocytes bearing alpha beta or gamma delta T-cell receptors in vitro: synergism with IL-2.

IL-7 induced the proliferation of normal thymocytes and the effect was synergistically potentiated by a small dose of IL-2, which by itself hardly affected thymocyte proliferation. No synergism was observed between IL-7 and any one of the other lymphokines including IL-1, IL-3, and IL-4. The thymocyte culture stimulated with IL-7 and IL-2 consisted of single positive (CD4+CD8- and CD4-CD8+) and double negative (CD4-CD8-) populations, and double positive (CD4+CD8+) cells were completely deleted. Both single positive and double negative thymocytes expressed CD3, but only the former exhibited V beta 8 and V beta 6 in an expected proportion (approximately 30% in BALB/c mice) and the latter none at all. Immunoprecipitation of the cultured thymocytes by anti-TCR gamma antibody, on the other hand, revealed the presence of a TCR gamma chain. Taken together, these results indicated that the thymocyte cultured with IL-7 and IL-2 consisted of mature T cells bearing alpha beta or gamma delta TCR. Experiments using preselected thymocyte subpopulations indicated that double negative cells responded to both IL-7 and IL-2 with positive synergism when combined, while thymocytes enriched for single positive cells preferentially responded to IL-7 with little response to IL-2 and no detectable synergism. Double positive thymocytes showed no proliferation in response to IL-7 and IL-2. In contrast to single positive thymocytes, splenic T cells hardly responded to IL-7, although significant proliferation was induced in the presence of a low dose of IL-2. Thymocytes cultured with IL-7 and IL-2 showed little nonspecific cytotoxic activity, but responded to Con A or alloantigen, whereas those stimulated with a high dose of IL-2 alone exhibited potent cytotoxic activity. These results indicated that IL-7 was involved in the generation of immunocompetent T cells in the thymus in concert with IL-2.

Animals↗

PubMed1984

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.

Animals↗

PubMed1995

Enhancement of VLA integrin receptor function on thymocytes by cAMP is dependent on the maturation stage of the thymocytes.

A class of adhesion molecules, the VLA integrins, are expressed on thymocytes and have been shown to affect immature thymocyte differentiation in vitro. This study examines the ability of cAMP to regulate VLA receptor function in thymocytes. Pharmacologic agents that raise intracellular cAMP enhanced the binding of immature CD4- CD8- and CD4+ CD8+ thymocytes to fibronectin while having no effect on the binding of the more mature JIId- thymocytes. PGE2, a hormone produced by thymic epithelial cells and known to raise intracellular cAMP levels in thymocytes, also increased the binding of immature thymocytes to fibronectin. In contrast, activation of protein kinase C via PMA enhanced the binding of all three thymocyte subsets. The cAMP-induced binding was blocked by mAbs to the VLA integrin chains alpha 4 and alpha 5 and by the protein kinase A (PKA) inhibitor, (Rp)-cAMPS, indicating that activation of PKA enhances VLA-4 and VLA-5 receptor function. Activation of PKA was induced in all three thymocyte subsets following addition of cAMPa or forskolin, indicating that the inability of cAMP to enhance the binding of JIId- thymocytes was not due to an inability to activate PKA. Thus, cAMP enhances integrin function in thymocytes in a maturation stage-specific manner.

Animals↗