Phosphorylation of mouse thymocyte CD4 and CD8: regulation of surface expression.

Murine T cell surface antigens, CD4 and CD8 are phosphorylated in response to phorbol 12-myristate 13-acetate, a protein kinase C activator, but not phosphorylated after concanavalin A, Ca2+ ionophore or dibutyryl-cAMP treatment. We examined the cell surface expression of both antigens and show that surface CD4 on CD4+CD8+ and CD4+CD8- thymocytes is rapidly decreased after PMA treatment, while CD8 expression is unaffected. Prolonged PMA treatment, which down-regulates protein kinase C, allows CD4 reexpression only in the CD4+CD8- population, suggesting that different mechanisms of cell surface antigen expression are operating in the two thymocyte subpopulations.     

Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation

Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.     

CD3Ti+ human thymocyte-derived clones displaying a differential response to activation via CD3Ti and CD2

Previous studies indicated that, unlike peripheral T-cells, freshly isolated thymocytes show little or no proliferation to activation signals via either the antigen/MHC receptor complex (CD3Ti) or the CD2 structure, unless exogenous IL-2 or phorbol esters are added. To investigate these differences in more detail, we have studied the response of clonal populations of mature thymocyte subsets as well as peripheral T-cell clones to activation via either CD3Ti or CD2. Here we report the characterization of three clones belonging to different subsets of mature thymocytes: CD3+ CD4+ (Ti alpha/beta), CD3+ CD8+ (Ti alpha/beta), and CD3+ CD4- CD8- (Ti gamma/delta). All three clones could be induced to proliferate to insolubilized anti-CD3 mAb. In contrast, activating anti-CD2 mAbs, which induced proliferation in all peripheral T-cell clones tested, did not induce an appreciable proliferation of the thymocyte clones. The latter required additional signals provided by the phorbol ester PMA. However, anti-CD2 mAbs were able to induce early activation events such as phosphoinositide turnover and [Ca2+]i increase to an extent similar to the ones elicited by anti-CD3 mAb. These results further support previous findings suggesting that mature thymocytes are not functionally identical to peripheral T-cells.     

Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

1F7 (CD26): a marker of thymic maturation involved in the differential regulation of the CD3 and CD2 pathways of human thymocyte activation.

We have recently shown that solid-phase immobilization of anti-1F7 recognizing the 110-kDa CD26 Ag is comitogenic for human peripheral blood T cell activation via both the CD3 and CD2 pathways. We have also demonstrated that binding of anti-1F7 leads to the disappearance of CD26 surface expression, and this anti-1F7-induced modulation results in an increase in anti-CD3 or anti-CD2-mediated peripheral blood T cell activation. In this report, we extended these findings by examining the expression and functional relationship of 1F7 on the CD3 and CD2 pathways of activation of human thymocytes. We now demonstrated that most of the anti-1F7 reactivity is found on medullary thymocytes, the population of thymocytes expressing high level of CD3 (CD3H). We have also shown that binding of anti-1F7 can induce a decrease in CD26 surface expression, with no detectable effect on the surface expression of CD3 or CD2. Most importantly, we showed that solid-phase immobilization of anti-1F7 has a comitogenic effect on thymocyte activation induced by anti-CD3 but not anti-CD2. In addition, anti-1F7-induced modulation of CD26 results in an enhancement in CD3-mediated but not CD2-mediated human thymocyte activation. The observed functional effect of CD26 on the CD3/TCR pathway of activation is mainly restricted to mature thymocytes as distinguished by high surface expression of CD5, although CD26 is also functionally associated with the CD3/TCR pathway on cells expressing low level of CD5. Demonstrating that CD26 involvement in the regulation of human thymocyte activation is restricted mainly to the CD3 pathway, unlike its involvement with both the CD3 and CD2 pathways of mature peripheral blood T lymphocyte activation, our data hence suggested that CD26 may play a role in thymic differentiation and maturation via the differential engagement of the CD3 pathway.     

Activation of CD3/TCR negative human thymocytes via CD28 molecule.

We have observed that the CD28 molecule was present on the cell surface of a large fraction of resting CD3- thymocytes (40 to 100%). Interestingly, the majority (greater than 90%) of surface CD3-CD28-cells reacted in the cytoplasm with anti-CD28 (CK248, 9.3) and anti-CD3 epsilon chain mAbs (Leu4, OKT3). Along this line, we found that CD28 surface expression could be induced within 18 hr on CD3-CD28- thymocytes using very low doses of phorbol-13-myristate-12-acetate (PMA). This event was accompanied by the appearance of CD25 and CD69 activation antigens but not of CD3/TCR complex. These results were further confirmed by immunoprecipitation studies. It is noteworthy that the T-cell activation pathway initiated via the CD28 molecule is functional in resting CD3- thymocytes in the presence of PMA and/or IL2. Finally, stimulation of CD3- immature thymocytes via CD28 gave rise to a large fraction (about one-third) of CD3-CD8+ cells.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

Cytokine production by mature and immature thymocytes.

We have studied the ability of subpopulations of activated thymocytes to produce four cytokines (IL-2, IL-4, IFN-gamma and TNF-alpha) which are believed to play roles in T cell development. Supernatants from various thymocyte subsets activated with calcium ionophore and PMA were tested for these cytokines. All CD3hi thymocyte subsets (CD4+8-, CD4-8- and CD4-8+) produced high titers of these four cytokines except CD3+4-8+ thymocytes, which did not produce IL-4. In contrast, CD4+8+ thymocytes did not produce any detectable cytokines. CD3-4-8- thymocytes produced IL-2, IFN-gamma, and TNF-alpha (but not IL-4) when activated by calcium ionophore + PMA and IL-1. We then separated CD3-4-8- thymocytes into IL-2R+ and IL-2R-. CD3-4-8-IL-2R+ thymocytes only produced small amounts of IL-2 when activated with calcium ionophore + PMA + IL-1, whereas CD3-4-8-IL-2R- thymocytes did not require IL-1 to produce IL-2, IFN-gamma, and TNF-alpha. Finally, CD4-8+3- thymocytes (an immature population believed to be an intermediate between CD3-4-8- and CD4+8+ thymocytes) only produced marginally detectable levels of IL-2 upon stimulation with calcium ionophore, PMA, and the addition of IL-1 did not result in increased levels of cytokine production. These observations indicate discrete patterns of cytokine production by the subsets studied and suggest specific controls of cytokine gene expression during T cell development.     

Triggering of co-mitogenic signals in T cell proliferation by anti-LFA-1 (CD18, CD11a), LFA-3, and CD7 monoclonal antibodies

Proliferative T cell responses were elicited in a comitogenic assay when purified mAb against CD 18, CD11a, LFA-3, and CD7 were immobilized onto solid plastic surfaces together with submitogenic doses of mAb against the CD3 complex. The proliferative response was associated to the production of IL-2 and to the expression of IL-2R. We explored the possibility that a second signal provided by either PMA or a Ca2+ ionofore could replace the anti-CD3 mAb in the comitogenic assay. Interestingly, our data clearly indicate that PMA but not the ionofore was capable of mediating the co-mitogenic effect in conjunction with solid-bound mAb (CDw18, CD11a, LFA-3, and CD7). We also demonstrate that the mAb (anti-CD4 and anti-CD2) which have been previously described as co-mitogenic in combination with anti-CD3 are capable of eliciting this activating signal in the presence of PMA. These data indicate that mAb to certain cell surface differentiation Ag that in soluble form inhibit T cell function such as LFA-1, LFA-3, and CD2 can under appropriate conditions induce co-mitogenic signals on T cells. Our results support the hypothesis that several cell surface differentiation Ag may participate in conjunction with the T3-Ti complex in the transmembrane signal transduction leading to T cell activation.     

CD5 costimulation up-regulates the signaling to extracellular signal-regulated kinase activation in CD4+CD8+ thymocytes and supports their differentiation to the CD4 lineage

CD5 positively costimulates TCR-stimulated mature T cells, whereas this molecule has been suggested to negatively regulate the activation of TCR-triggered thymocytes. We investigated the effect of CD5 costimulation on the differentiation of CD4+CD8+ thymocytes. Coligation of thymocytes with anti-CD3 and anti-CD5 induced enhanced tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase C-gamma (PLC-gamma) compared with ligation with anti-CD3 alone. Despite increased phosphorylation of PLC-gamma, this treatment down-regulated Ca2+ influx. In contrast, the phosphorylation of LAT and enhanced association with Grb2 led to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. When CD3 and CD5 on CD4+CD8+ thymocytes in culture were coligated, they lost CD8, down-regulated CD4 expression, and induced CD69 expression, yielding a CD4+(dull)CD8-CD69+ population. An ERK inhibitor, PD98059, inhibited the generation of this population. The reduction of generation of CD4+CD8- cells resulted from decreased survival of these differentiating thymocytes. Consistent with this, PD98059 inhibited the anti-CD3/CD5-mediated Bcl-2 induction. These results indicate that CD5 down-regulates a branch of TCR signaling, whereas this molecule functions to support the differentiation of CD4+CD8+ thymocytes by up-regulating another branch of TCR signaling that leads to ERK activation.     

Subpopulations of fetal thymocytes defined by expression of T cell receptor/CD3 and IL-2 receptor. CD3 and IL-2 receptor alpha-chain are expressed on reciprocal cell populations

The expression of the TCR/CD3 complex and the IL-2R alpha chain (p55) on fetal thymocytes has been analyzed by flow cytometry (FCM). Two-parameter immunofluorescence identified three subpopulations which were respectively IL-2R alpha-/CD3+, IL-2R alpha+/CD3-, or IL-2R alpha-/CD3-; no detectable population of IL-2R alpha+/CD3+ cells was found in unstimulated fetal thymocytes. Fractionation by "panning" and by sterile flow cytometric separation was used to characterize the functional responsiveness of these three subpopulations to a variety of stimuli. All three populations proliferated in response to PMA + ionomycin + rIL-2. In contrast, stimulation with anti-CD3 + IL-2 induced proliferation in IL-2R alpha-/CD3+ and IL-2R alpha-/CD3- but not in IL-2R alpha+/CD3- thymocytes. IL-2R alpha- cells, including sorted IL-2R alpha-/CD3- thymocytes, underwent a phenotypic change in response to in vitro stimulation with anti-CD3 + IL-2, resulting in the appearance of an IL-2R alpha+/CD3+ population that was not detected in freshly isolated thymocytes. The ability of fractionated fetal thymocytes to produce lymphokine in response to PMA + ionomycin was also evaluated. Only the IL-2R alpha-/CD3- fraction generated detectable IL-2. These findings demonstrate for the first time that CD3 and IL-2R alpha are expressed in a mutually exclusive fashion in fetal thymocytes and define three subpopulations of thymocytes that differ significantly in their proliferative and differentiative responses to TCR-mediated, IL-2R-mediated, and pharmacologic stimulation.     

Expression of CD1 and class I MHC antigens by human thymocytes

The acquisition of surface class I MHC molecules is associated with the maturation of thymocytes. Here, surface expression of class I MHC and CD1, which represents a family of MHC-related molecules, was analyzed on various human immature and mature thymocyte subpopulations. Class I expression was inversely related to the expression of CD1. The majority of CD4+ CD8+ cortical type thymocytes expressed low levels of class I MHC Ag, the previously described CD4+ CD8+ thymocyte subpopulation with low CD8 expression exhibited intermediate levels of class I MHC, whereas most of the single positive CD4 and CD8 thymocytes displayed high levels of class I MHC. Biochemical comparison of CD1 and class I showed that thymic class I molecules were post-translationally modified by phosphorylation, whereas CD1 was not phosphorylated. Furthermore, our studies suggested that in addition to CD1/CD8 complexes, thymocytes bear CD8/class I complexes. Chemical cross-linking and peptide mapping studies clearly identified the CD8-associated protein on thymic clones as the class I MHC molecule.     

Differential in vitro activation of CD8-CD4+ and CD4-CD8+ T lymphocytes by combinations of anti-CD2 and anti-CD3 antibodies

Purified peripheral blood T lymphocytes and the CD8-CD4+ and CD4-CD8+ T cell subsets, exhaustively depleted of APC have been studied for their capacity to respond to mAb directed against the CD3-Ti Ag-specific TCR complex and against the CD2 SRBCR. It is demonstrated that high affinity IL-2R can be readily induced by either anti-CD3 and/or anti-CD2 stimulation. However, IL-2 production can be observed only in the CD4+CD8- T cell subset. These results clearly contrast data obtained with purified CD4-CD8+ T cells, which are able to proliferate when the CD3-Ti complex is activated in the presence of APC. The data presented in the present study demonstrate that a simplified model for T cell activation and clonal expansion of the two major T cell subsets involve only the CD3-Ti complex and the CD2 Ag. Under conditions where the activation signals for the T cells are restricted only to the activation of CD3-Ti and CD2, the CD4+CD8- T cells respond with IL-2 production and expression of high affinity IL-2R, whereas the CD4-CD8+ T cell subset depends on exogenous IL-2 provided by the CD4+CD8- cells. These data do not, however, exclude an involvement of other cell-surface signals for regulation and control of T cell activation and T cell effector functions.     

Distinct patterns of lymphokine requirement for the proliferation of various subpopulations of activated thymocytes in a single cell assay

The frequency and capacity for clonal expansion of several murine thymocyte subpopulations responsive to various IL (fetal day 15, and adult CD4-8-, CD4+8- and CD4-8+) were investigated using a single-cell limiting-dilution cell culture system without filler cells. This assay requires the presence of PMA and ionomycin. The main conclusions of these studies are the following: 1) IL-4 is a better growth factor than IL-2 for immature thymocytes (fetal day 15 or adult CD4-8-). 2) IL-2 is a better growth factor than IL-4 for mature phenotype thymocytes (CD4+8- and CD4-8+). 3) IL-4 is a relatively poor growth factor for adult CD4-CD8- thymocytes and CD4+CD8- thymocytes, while it induced strong responses in fetal day 15 and CD4-8+ thymocytes. 4) IL-6 enhanced the response of CD4+8- thymocytes to either IL-2 or IL-4. 5) Cortisone-resistant thymocytes grown initially with IL-4 and then switched to IL-2 showed a significant decrease in cloning efficiency. No inhibitory effect was observed when cells were cultured first with IL-2 and then switched to IL-4. 6) Finally, supernatant from Con-A stimulated rat spleen cells induced maximal growth of all adult thymocyte populations tested, suggesting that unidentified thymocyte growth factor(s) remain to be characterized. These results indicate that the maturational stage of thymocytes determines their requirements for activation and proliferation.     

Interaction of CD2 with its ligand, LFA-3, in human T cell proliferation

Recently, it has been demonstrated that lymphocyte function-associated Ag (LFA-3) is a natural ligand for CD2 and that this receptor-ligand interaction functions in cell-cell adhesion. In this report, we demonstrate that LFA-3 plays a role in T cell activation. L cells were transfected with human genomic DNA and sorted for expression of LFA-3. We demonstrate that LFA-3+ L cells, together with anti-CD3 mAb or with suboptimal doses of PHA, stimulate proliferation of human peripheral blood T cells. Furthermore, thymocyte proliferation was induced by LFA-3+ L cells and suboptimal doses of PHA. Proliferation was inhibited by mAb directed against either CD2 or LFA-3. Stimulation of thymocytes by the combination of PHA and LFA-3+ L cells resulted in the increased expression of the IL-2R, as well as of the surface Ag 4F2, transferrin receptor, and HLA-DR. These data support the conclusion that LFA-3 plays a role in CD2-dependent T cell activation. LFA-3 is widely distributed and is expressed on all APC and target cells. Thus, the ability of the CD2/LFA-3 interaction to costimulate with an anti-CD3 mAb suggests that the CD2/LFA-3 interaction may be involved not only in an Ag-independent alternate pathway of T cell activation but also in Ag-specific T cell activation.     

Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development

To delineate the cellular targets and mechanisms by which glucocorticoids (GCs) exert their actions, we generated mice in which a green fluorescent protein (GFP)-GC receptor (GR) fusion gene is knocked into the GR locus. In these mice, the GFP-GR protein, which is functionally indistinguishable from endogenous GR, allows the tracking and quantitation of GR expression in single living cells. In GFP-GR thymus, GR expression is uniform among embryonic thymocyte subpopulations but gradually matures over a 3-wk period after birth. In the adult, GR is specifically induced to high levels in CD25(+)CD4(-)CD8(-) thymocytes and returns to basal levels in CD4(+)CD8(+) thymocytes of wild-type and positively selecting female HY TCR-transgenic mice, but not negatively selecting male HY TCR-transgenic mice. In GFP-GR/recombinase-activating gene 2(-/-) thymocytes, GR expression is down-regulated by pre-TCR complex stimulation. Additionally, relative GR expression is dissociated from GC-induced apoptosis in vivo. Results from these studies define differential GR expression throughout ontogeny, suggest pre-TCR activation as a specific mechanism of GR down-regulation, define immature CD8(+) thymocytes as novel apoptosis-sensitive GC targets, and separate receptor abundance from susceptibility to apoptosis across thymocyte populations.     

Thymic shared antigen-2: a novel cell surface marker associated with T cell differentiation and activation

Thymic shared Ag-2 (TSA-2) is a 28-kDa, glycophosphatidylinitosol-linked cell surface molecule expressed on various T cell and thymic stromal cell subsets. It is expressed on most CD3-CD4-CD8-, CD4+CD8+, and CD3highCD4-CD8+ thymocytes but is down-regulated on approximately 40% of CD3highCD4+CD8- thymocytes. Expression on peripheral TCR-alphabeta+ T cells is similar to that of CD3+ thymocytes, although a transient down-regulation occurs with cell activation. Consistent with the recent hypothesis that emigration from the thymus is an active process, recent thymic emigrants are primarily TSA-2-/low. TSA-2 expression reveals heterogeneity among subpopulations of CD3highCD4+CD8- thymocytes and TCR-gamma delta+ T cell previously regarded as homogenous. The functional importance of TSA-2 was illustrated by the severe block in T cell differentiation caused by adding purified anti-TSA-2 mAb to reconstituted fetal thymic organ culture. While each CD25/CD44-defined triple-negative subset was present, differentiation beyond the TN stage was essentially absent, and cell numbers of all subsets were significantly below those of control cultures. Cross-linking TSA-2 on thymocytes caused a significant Ca2+ influx but no increase in apoptosis, unless anti-TSA-2 was used in conjunction with suboptimal anti-CD3 mAb. Similar treatment of mature TSA-2+ T cells had no effect on cell survival or proliferation. This study reveals TSA-2 to be a functionally important molecule in T cell development and a novel indicator of heterogeneity among a variety of developing and mature T cell populations.     

CD4 and CD7 molecules as targets for drug delivery from antibody bearing liposomes.

We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation of d-[3H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4-targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly.     

Purified lymphocyte function-associated antigen-3 (LFA-3) activates human thymocytes via the CD2 pathway

Defining the cellular and molecular mechanisms of interaction of developing thymocytes with nonlymphoid cells of the thymic microenvironment is critical for understanding normal thymus function. We have previously shown that the CD2/LFA-3 adhesion pathway is important in the interaction of thymocytes with a variety of LFA-3+ nonlymphoid thymic microenvironment cell types. Moreover, T cell activation via the CD2 (alternative, Ag independent) pathway is considered an important mechanism for intrathymic T cell proliferation. To study the relevance of CD2/LFA-3 interactions to human thymocyte activation, we have used purified LFA-3 Ag in several in vitro assays of thymocyte proliferation. Whereas LFA-3 Ag alone did not induce thymocyte proliferation, LFA-3 Ag in combination with the anti-CD2 antibody, CD2.1, and rIL-2 induced marked thymocyte proliferation. Additionally, the anti-CD28 antibody, Kolt2, could substitute for rIL-2, resulting in thymocyte activation induced by LFA-3 Ag in combination with antibodies CD2.1 and Kolt2. In both triggering systems, LFA-3 induced thymocyte activation was dependent upon the concentration of LFA-3 Ag. LFA-3 Ag-dependent thymocyte activation was directed primarily toward CD1-, mature thymocytes. Finally, intact SRBC that express the sheep homolog of LFA-3, T11TS, in combination with antibody CD2.1 and rIL-2 could also induce thymocyte activation. These data suggest that interaction of LFA-3 molecules with thymocyte CD2 molecules may provide a component of the stimulus for normal intrathymic thymocyte activation leading to thymocyte proliferation.