anti-CD3 Epsilon Research Articles

Use of anti-CD3 epsilon F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation.

The use of T cell-specific mAb in vivo for prevention and treatment of graft-vs-host disease (GVHD) and its impact on graft-vs-leukemia (GVL) reactivity was examined in a murine model of MHC-matched bone marrow transplantation (BMT). F(ab')2 fragments of a CD3 epsilon-specific mAb were administered to irradiated AKR (H-2k) hosts after transplantation of BM plus spleen cells from B10.BR donors (BMS chimeras). The effects on GVH and GVL reactivity were Ab dose- and schedule-dependent. A short course of mAb (qe2d, days 0 to 8) prevented clinical evidence of GVHD and mortality. Anti-CD3 F(ab')2 mAb reversed clinical symptoms of acute GVHD when delayed up to 18 days post-transplant. Anti-host (Mls-1a)-specific V beta 6+ cells were absent from the spleens of GVH-negative control mice, but persisted in Ab-treated BMS chimeras despite the absence of GVHD. Leukemic mice given 16.7 micrograms of Ab on days 0, 2, and 4 survived leukemia-free without developing severe GVHD. A longer course of Ab completely prevented GVHD, but led to leukemia relapse in tumor-bearing hosts, despite engraftment of donor T cells. The GVL effect was quantitatively stronger when Ab was used for GVH therapy as compared with GVH prevention. Some Ab-treated, GVH-free chimeras relapsed with lymphomas in unusual sites, suggesting that occult tumor cells may persist in nonlymphoid tissues. These experiments demonstrate that T cell-specific mAb can be used successfully in vivo to avoid severe GVHD, but that excessive or ill-timed administration of Ab may eliminate GVL reactivity.

Regulation of T cell receptor (TCR)-beta locus allelic exclusion and initiation of TCR-alpha locus rearrangement in immature thymocytes by signaling through the CD3 complex.

During thymocyte differentiation, the T cell receptor (TCR)-beta genes are rearranged before the TCR-alpha genes. Immature CD4-8- double-negative thymocytes with a productive rearrangement of the TCR-beta locus are selected to continue maturation to the CD4+8+ double-positive stage, driven by signals through the pre-TCR. The signals through the pre-TCR can be synchronized by injection of mice with anti-CD3 epsilon monoclonal antibody. Using this approach, we demonstrated coordinated induction of a triad of responses in immature thymocytes: arrest of V to DJ rearrangement in the TCR-beta locus, transient down-regulation of rearrangement-activating gene (RAG)-1 and RAG-2 transcripts, and initiation of germ-line transcription of the TCR-alpha locus. These results suggest that the transition from TCR-beta to TCR-alpha locus rearrangement is controlled by signal transduction through the pre-TCR.

Functional analysis of the TCR alpha- beta+ cells that accumulate in the pneumonic lung of influenza virus-infected TCR-alpha-/- mice.

In mice homozygous (-/-) for a targeted TCR-alpha gene disruption, some thymocytes express a cell-surface TCR-beta chain on the cell surface in the absence of a TCR-alpha chain, and a few CD4+CD8- TCR-alpha-beta+ cells accumulate in the peripheral lymphoid organs. We have infected these mutant mice with an influenza A virus to show that large numbers of TCR-beta+ cells (most of which are CD4+) can be retrieved from the pneumonic lung. Both freshly isolated TCR-alpha-beta+ cells and TCR-alpha-beta+ hybridoma cell lines derived from influenza virus-infected mutant mice respond appropriately to stimulation with anti-CD3 epsilon or the Mls-1 superantigen. It thus seems that CD4+ TCR-alpha-beta+ cells in the peripheral lymphoid organs of TCR-alpha mutant mice can signal through their TCR surface complex. However, there are no indications that CD4+ TCR-alpha-beta+ lymphocytes can either recognize a complex between MHC and influenza virus peptide or act as effector or Th cells. The existence and function of such cells in wild-type mice remains to be established.

Resident CD4+ alpha beta T cells of the murine female genital tract: a phenotypically distinct T cell lineage that rapidly proliferates in response to systemic T cell activation stimuli.

A population of CD4+ cells has been identified in the murine female genital tract (FGT). Phenotypic studies of FGT CD4+ cells demonstrate that they express CD3 and that the majority of these cells are alpha beta TCR+Thy-1+. Most of the Thy-1+CD4+alpha beta TCR+ cells resemble memory T cells based on their expression of CD44, L-selectin and CD45RB antigens. The vast majority of Thy-1+CD4+alpha beta TCR+ FGT cells are CD5+ and all of them are B220-. Systemic stimuli including infection with Trypanosoma brucei brucei, injection with anti-CD3 epsilon, or bacterial superantigens staphylococcal enterotoxin A or B cause a rapid accumulation of CD4+ cells in the FGT exceeding that observed for CD4+ cells in spleen and lymph nodes (LN). Expansion of the FGT CD4+ cells, which are phenotypically distinct from the splenic and LN CD4+ T cells, is due to local proliferation rather than an influx of cells from the circulation. The CD4+ population in the FGT of adult nu/nu mice is dramatically reduced, indicating its thymic dependency. In lpr/lpr mice, FGT CD4 cells do not display changes characteristic of splenic or LN CD4 cells in the same animals. These findings demonstrate that the CD4+ cells of the murine FGT are thymic dependent, but that they constitute a T cell lineage that phenotypically and, probably functionally, is distinct from other peripheral CD4+ T cell populations.

Over-expression of CD3 epsilon transgenes blocks T lymphocyte development.

We have reported previously that mice carrying > 30 copies of the human CD3 epsilon transgene completely lose their T lymphocytes and NK cells (36). Here we demonstrate by immunohistology that in the most severely immunodeficient mouse, tg epsilon 26, the thymus is very small, has sizeable vacuoles and does not contain recognizable T lymphocytes except for a small percentage of Thy-1+ cells and B cells. Cell surface phenotyping and TCR alpha and -beta rearrangement studies confirm that the arrest in T lymphocyte development precedes the arrest in rag-1null, rag-2null and TCR beta nuli mice. Since the T cell progenitors in which the arrest occurred were absent in the transgenic mice, indirect approaches were taken to examine the causes of the block in T cell development. Analyses of 12 independently established mutant mouse lines, generated with five different transgenic constructs, revealed that the severity of the abrogation in T cell development was dependent on the number of copies of transgenes. Since the number of transgene copies generally correlated with the levels of expression of the transgenic CD3 epsilon proteins, we concluded that over-expression of the CD3 epsilon protein was the likely cause of the block in T lymphocyte development. The T cell immunodeficiency was caused by either the human or the murine CD3 epsilon protein. Since transgene coded mRNAs were found in significantly higher quantities than endogenous CD3 epsilon mRNAs in fetal thymi on days 13 and 14 of gestation, over-expression took place very early in development, probably prematurely. Over-expression of the CD3 epsilon transgene in thymocyte precursors may therefore affect T lymphocyte development in the absence of TCR and possibly in the absence of the other CD3 proteins. More importantly, over-expression of the CD3 epsilon protein in thymocytes of mice with a low copy number of transgenes had a significant effect on late thymic development. Over-expression of the CD3 epsilon protein in immature thymocytes mimicked the effects caused by exposure of CD4- CD8- thymocytes to anti-CD3 epsilon treatment: apoptosis and lack of TCR beta expression. We therefore speculate that in the homozygous tg epsilon 26 animals the arrest in T cell development was caused by excessive signal transduction events rather than by a toxic effect of the transgenic protein.

Activation and re-activation potential of T cells responding to staphylococcal enterotoxin B.

To elucidate the parameters that lead to superantigen induced non-responsiveness, an in vitro model for studying primary and secondary responses to the bacterial superantigen staphylococcal enterotoxin B (SEB) was established. Upon re-activation with SEB, in vitro SEB primed T cells show an early proliferative response that 'quenches' in time and is severely impaired 3 days after re-stimulation. Despite their overall impaired proliferative capacity and IL-2 production, these T cells are able to produce IFN-gamma and to up-regulate activation markers CD69 and IL-2R alpha upon re-stimulation with SEB, demonstrating that SEB non-responsiveness is not absolute. Rather, it reflects the inability to mount an ongoing proliferative response upon re-stimulation with SEB. Our results also demonstrate that SEB-induced non-responsiveness is not simply the result of presentation in the absence of co-stimulation, since presentation of SEB on highly purified dendritic cells during the primary response did not prevent the induction of non-responsiveness. As previously shown, SEB induces a Th1 phenotype in responding CD4+ T cells. Skewing towards a Th2 phenotype by adding IL-4 and antibodies to IFN-gamma did not prevent the induction of non-responsiveness by SEB. Interestingly, T cells pretreated with plate-bound anti-CD3 epsilon and anti-V beta 8 were also non-responsive to SEB re-stimulation. Thus, non-responsiveness to SEB (defined here as inability to produce IL-2 and proliferate) seems to reflect an intrinsic inability of previously activated T cells to respond to SEB, probably reflecting differences in signal transduction pathways used by naive versus previously activated T cells.

Reliable method for the simultaneous detection of cytoplasmic and surface CD3ε expression by murine lymphoid cells

During their development, T-cell precursors (pre-T cells) undergo a variety of changes with respect to their expression of specific surface proteins. Among the most critical of the surface markers acquired by developing T cells is the T-cell receptor (TCR)/CD3 complex. Prior to the assembly and transport of complete TCR/CD3 multimeric complexes to the plasma membrane, the individual constituent subunits are expressed in the cytoplasm (ER-Golgi). In order to study the expression of the T-cell receptor TCR/CD3 complex during pre-thymic T-cell differentiation, we have developed a flow cytometric technique for the simultaneous detection of surface (sCD3 epsilon) and cytoplasmic CD3 epsilon (cCD3 epsilon). This technique, which employs fixation in 1% paraformaldehyde and permeabilization with 1% saponin and 0.025% digitonin, features reliable internalization and low nonspecific binding of anti-CD3 epsilon in murine lymphoid cells, as well as tissue culture cell lines. The combination of saponin and digitonin treatment was also compatible with the staining of sCD3 and other lymphocyte surface antigens such as Thy1, CD4, CD8, B220, and IgM. In contrast, permeabilization of cells with the detergents Tween 20 and Triton X-100 was shown to remove surface-bound anti-CD3 epsilon. The present technique permitted the detection of discernible sCD3 epsilon and cCD3 epsilon double and single positive lymphocytes and may prove useful in defining bone marrow-resident pre-T cells.

T cell receptor expression by dendritic epidermal T cells.

Dendritic epidermal T cells (DETC) in murine epidermis express the gamma delta T cell receptor (TCR). The major population of DETC utilize V gamma 5 and V delta 1 without any junctional diversity, corresponding to the earliest fetal thymocytes which express TCR gamma delta. Using PCR, we recently found another population of DETC which express V gamma 1-V delta 6 with junctional diversity in addition to V gamma 5-V delta 1, although they exist in small numbers in normal mice. In athymic nude mice, V gamma 1+ cells also exist. Therefore, this subset of gamma delta T cells is the product of an extrathymic pathway. These V gamma 1+ cells may recognize mycobacterium antigen or heat shock protein (HSP), thus playing an important role in the first defense of the skin. In contrast to normal mice, in nude mice, we could not detect any DETC using anti CD3 epsilon antibody (2C11). In order to solve this puzzle, we examined the components of TCR complex utilizing immunoprecipitation and northern blot analysis. TCR is composed of either alpha beta or gamma delta chains associated with CD3 gamma, delta, epsilon and zeta-zeta chains. By immunoprecipitation of 125I labelled DETC cell lines using anti-CD3 epsilon antibody, we detected gamma delta chains and CD3 gamma and CD3 epsilon chains, but not CD3 delta or CD3 zeta chains. Northern blot analysis showed that these cells express CD3 gamma, epsilon, and zeta chains, but not the CD3 delta chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmentally regulated expression of CD3 components independent of clonotypic T cell antigen receptor complexes on immature thymocytes.

CD3 signal transducing proteins are thought to be expressed on the surface of T cells only as part of clonotypic T cell receptor (TCR) complexes. Contrary to this paradigm, the present study describes surface expression of CD3 proteins independently of clonotypic TCR complexes, but only on immature thymocytes. Such novel clonotype-independent CD3 (CIC) complexes are composed primarily of CD3 gamma epsilon and secondarily of CD3 delta epsilon heterodimers that are independent of one another and are expressed on the cell surface in association with an unknown 90-100 kD protein termed CD3-associated protein (CD3AP). CIC complexes are expressed in normal mice on early thymocytes through the CD4+CD8+ stage of development, but not on mature peripheral T cells. Furthermore, CIC complexes are expressed by both TCR- severe combined immunodeficiency (SCID) thymocytes and thymoma cell lines, in the absence of any clonotypic chains. The isolation and biochemical characterization of surface CIC complexes provides a structural basis for the signaling effects of anti-CD3 epsilon antibody treatment in early thymocyte development.

Regulation of nuclear factor-kappa B and activator protein-1 activities after stimulation of T cells via glycosylphosphatidylinositol-anchored Ly-6A/E.

Abstract Cross-linking of glycosylphosphatidylinositol-anchored proteins, including mouse Ly-6A/E, leads to IL-2 secretion and T cell activation, whereas engagement of Ly-6A/E uniquely inhibits IL-2 production induced via TCR. However, little is known concerning the molecular mechanism by which glycosylphosphatidylinositol-anchored proteins regulate IL-2 expression. In this study, we have examined the ability of an anti-Ly-6A/E mAb to regulate transcription factors controlling IL-2 expression. Stimulation of EL4J(Ly-6E).A4 cells with anti-CD3 epsilon or anti-Ly6A/E mAbs induced nuclear factor (NF)-kappa B p65-p50 (RelA/p50) and AP-1 (Fos/Jun) binding activities and increased nuclear factor of activated T cells (NF-AT) activity, whereas octamer-binding factor and NF-Y levels were stable. Cyclic AMP response element binding protein and T cell-specific factor-1 (alpha) activities were selectively enhanced by anti-CD3 epsilon, but not by anti-Ly6A/E, which suggests that signaling via the TCR and Ly-6 were not identical. Costimulation of these cells with both mAbs produced substantially reduced levels of AP-1, NF-AT, and, especially, NF-kappa B p65-p50 whereas cyclic AMP response element binding protein and T cell-specific factor-1(alpha) were induced to a level seen after stimulation by anti-CD3 epsilon. The inducibility of the IL-2 enhancer in vivo and the contribution of individual transcription factors for this induction were assessed with use of reporter chloramphenicol acetyltransferase constructs containing the IL-2 enhancer or oligomerized binding sites for transcription factors. These experiments also demonstrated a key role for NF-kappa B and AP-1 in the transcriptional regulation of the IL-2 gene by TCR- and Ly6A/E-mediated signaling. By using the 2B4.11 T cell hybridoma and a mutated variant, were revealed a crucial role for the zeta-chain in Ly6A/E-mediated activation of NF-kappa B.

Regulation of nuclear factor-kappa B and activator protein-1 activities after stimulation of T cells via glycosylphosphatidylinositol-anchored Ly-6A/E.

Cross-linking of glycosylphosphatidylinositol-anchored proteins, including mouse Ly-6A/E, leads to IL-2 secretion and T cell activation, whereas engagement of Ly-6A/E uniquely inhibits IL-2 production induced via TCR. However, little is known concerning the molecular mechanism by which glycosylphosphatidylinositol-anchored proteins regulate IL-2 expression. In this study, we have examined the ability of an anti-Ly-6A/E mAb to regulate transcription factors controlling IL-2 expression. Stimulation of EL4J(Ly-6E).A4 cells with anti-CD3 epsilon or anti-Ly6A/E mAbs induced nuclear factor (NF)-kappa B p65-p50 (RelA/p50) and AP-1 (Fos/Jun) binding activities and increased nuclear factor of activated T cells (NF-AT) activity, whereas octamer-binding factor and NF-Y levels were stable. Cyclic AMP response element binding protein and T cell-specific factor-1 (alpha) activities were selectively enhanced by anti-CD3 epsilon, but not by anti-Ly6A/E, which suggests that signaling via the TCR and Ly-6 were not identical. Costimulation of these cells with both mAbs produced substantially reduced levels of AP-1, NF-AT, and, especially, NF-kappa B p65-p50 whereas cyclic AMP response element binding protein and T cell-specific factor-1(alpha) were induced to a level seen after stimulation by anti-CD3 epsilon. The inducibility of the IL-2 enhancer in vivo and the contribution of individual transcription factors for this induction were assessed with use of reporter chloramphenicol acetyltransferase constructs containing the IL-2 enhancer or oligomerized binding sites for transcription factors. These experiments also demonstrated a key role for NF-kappa B and AP-1 in the transcriptional regulation of the IL-2 gene by TCR- and Ly6A/E-mediated signaling. By using the 2B4.11 T cell hybridoma and a mutated variant, were revealed a crucial role for the zeta-chain in Ly6A/E-mediated activation of NF-kappa B.

Murine cytomegalovirus-associated pneumonitis in the lungs free of the virus.

At 4 wk after intraperitoneal inoculation of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable only in the salivary glands. When T cells of these mice were activated by a single injection of anti-CD3 epsilon monoclonal antibody, mice died of interstitial pneumonitis at 24-48 h after injection, accompanied by elevation of serum levels of TNF-alpha and IFN-gamma. However, MCMV remained undetectable in the lungs during the period. Simultaneous injection of cyclosporin A reduced such effects of anti-CD3. In conclusion, although the presence of MCMV in the host may be required, MCMV-associated pneumonitis is not mediated by virus in the lung but probably by the cytokines released from T cells, of which responsiveness to stimulation via CD3 molecule has been presumably modified by MCMV infection.

Virus-specific CD8+ T-cell responses in mice transgenic for a T-cell receptor beta chain selected at random

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.

Aged T cells are hyporesponsive to costimulation mediated by CD28.

The ability of T cells to proliferate in response to a number of stimuli is impaired in healthy, aged individuals. T cells from young (2 to 4 mo) and aged (20 to 26 mo) mice were stimulated with immobilized anti-CD3 epsilon-chain mAb and soluble anti-CD28 mAb. T cells from young and aged mice proliferated comparably in response to anti-CD3 epsilon mAb alone. However, aged T cells were significantly less responsive to costimulation by anti-CD28 mAb. This decreased response of aged T cells was seen at all dosages tested of anti-CD3 epsilon and anti-CD28 mAbs and in the presence and absence of APC. Similar results were observed when costimulation was provided by B7-transfected L cell fibroblasts. T cells from young and aged mice had comparable expression of CD28 by flow cytometry, both before and after stimulation. The defect in response to CD28 was seen both in CD4+ and CD8+ T cells and in CD44lo (naive) and CD44hi (memory) T cells. The impaired response to costimulation mediated by CD28 on T cells from aged mice may be an important factor in reduced T cell responses associated with aging.

Maturational changes in CD4+ cell subsets and lymphokine production in BXSB mice.

CD4+ cells are thought to play a significant role in the development of lupus-like disease in a variety of autoimmune disease-prone mouse strains. In one such strain, BXSB/MpJScR, male mice develop severe lupus-like symptoms early in life but females do not. In this study, splenic CD4+ cells from male and female BXSB mice were evaluated for age-related changes in: 1) membrane expression of CD4+ cell subset markers (1, 2, and 4 mo) and activation Ags (4 mo) and 2) the capacity to proliferate and produce cytokines (4 mo) in response to polyclonal stimuli. CD4+ cells from females of all age groups and from younger males were predominantly CD44lo, CD45RBhi, MEL-14hi, and 3G11hi (phenotypes associated with naive T cells). In contrast, 4-mo-old males were predominantly CD44hi, CD45RBlo, MEL-14lo, and 3G11lo (phenotypes associated with activated/memory T cells). Furthermore, an increased constitutive expression of the activation Ags RL388, IL-2R, and TfR was observed in CD4+ cells of 4-mo-old male BXSB mice in comparison with age-matched females. In 3-day cultures, purified CD4+ cells from 4-mo-old males proliferated significantly less than cells from age-matched females in response to plate-bound anti-CD3 epsilon (2C11i). The reduced proliferation was restored in large part by PMA and ionomycin. CD4+ cells from older males generally produced increased amounts of IFN-gamma and IL-4 and significantly less IL-2 than age-matched females in response to either stimulus (IL-2 mRNA was also decreased in response to 2C11i). Taken together, these studies suggest that profound phenotypic and functional changes occur with age in the CD4+ cells of male BXSB mice that are indicative of an activated state.

In vivo or in vitro anti-CD3 epsilon chain monoclonal antibody therapy for the prevention of lethal murine graft-versus-host disease across the major histocompatibility barrier in mice.

We explored the use of a single in vivo dose of an anti-CD3 epsilon mAb for graft-vs-host disease (GVHD) prevention in mice. Anti-CD3 epsilon (145-2C11) given on the day of bone marrow (BM) transplant was highly effective in preventing GVHD in a dose-dependent manner. However, mice experienced acute toxicity as measured by early post-BM transplantation mortality, severe weight loss, and cachexia, which is likely due to T cell-dependent lymphokine production. Donor T cells were partly responsible because toxicity still occurred in mice injected with an allelic specific anti-CD3 epsilon mAb that only reacted with the donor strain. Toxicity was also dependent on the number of radioresistant host T cells present at the time of transplant, because toxicity still occurred when recipients were depleted of mature T cells in vivo with anti-CD4+ anti-CD8 mAbs, and were given T cell-depleted BM from SCID mice. Toxicity was eliminated by treating BM with anti-CD3 epsilon mAb in vitro before BM transplant instead of administering the mAb in vivo. However, GVHD protection was not complete. Direct comparison of the in vivo and in vitro approaches revealed that the in vivo administration of anti-CD3 epsilon mAb was more effective for GVHD protection and that a combination of the approaches was not beneficial. Flow cytometry studies were performed to assess the degree of CD3 epsilon chain modulation and T cell depletion obtained with in vitro vs in vivo anti-CD3 epsilon chain mAb. In mice given syngeneic BM transplants to avoid the confounding effects of GVHD, we found that in vivo anti-CD3 epsilon mAb administration induced a more prolonged period of CD3 epsilon chain modulation and/or T cell depletion than either the in vitro approach or a third approach that involved anti-CD3 epsilon F(ab')2 fragments administered in vivo. These data indicate that targeting the Ag-specific TCR, especially in such a way as to avoid toxicity associated with T cell activation, may provide an important key for developing future GVHD therapies.

CD3 epsilon-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR beta chain expression.

Recent studies have shown that TCR beta chain expression can effect the differentiation of CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) thymocytes. The TCR beta chain is expressed on the surface of DP thymocytes in association with CD3 gamma, delta and epsilon chains, suggesting a potential role for CD3 components in this signaling process. We now report detection of a very low level of surface expression of CD3 epsilon on adult DN RAG-2-/- thymocytes. This surface CD3 epsilon was associated with CD3 gamma and delta chains, as detected by anti-CD3 epsilon immunoprecipitation analyses. Significantly, injection of anti-CD3 epsilon mAb into RAG-2-/- mice led to the accumulation of an IL-2R alpha- CD2+ DP cell population and a nearly 100-fold increase in thymic cellularity to essentially normal levels. Together, these data strongly indicate that TCR beta chain-mediated developmental signals are transduced by CD3 components and provide potential insights into mechanisms by which TCR beta chain expression may effect this process.

Regulation of thymocyte development through CD3. II. Expression of T cell receptor beta CD3 epsilon and maturation to the CD4+8+ stage are highly correlated in individual thymocytes.

Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.

Restoration of early thymocyte differentiation in T-cell receptor beta-chain-deficient mutant mice by transmembrane signaling through CD3 epsilon.

Thymic repertoire selection requires the expression of the alpha beta CD3 T-cell receptor (TCR) together with the coreceptors CD4 and CD8. The appearance of CD4 and CD8 on thymocytes is the hallmark of a complex maturation step, accompanied by downregulation of the interleukin 2 receptor (IL-2R) alpha chain, arrest of rearrangement (i.e., allelic exclusion) of the TCR beta-chain locus, a burst of cell divisions, and reduction in cell size. This maturation step is inhibited in TCR beta-chain-deficient mouse strains and may depend on surface expression of an immature TCR complex containing CD3 and TCR beta chains but no TCR alpha chain. Here we show that the CD4+8+ double-positive (DP) stage can be induced by treatment of fetal thymic organ cultures with anti-CD3 epsilon monoclonal antibodies in several TCR beta-chain-deficient mouse strains: severe combined immunodeficient (scid) mice, mice carrying a mutation in the recombination activating gene 1 (Rag-1), or mice carrying a deletion in the TCR beta-chain locus itself. These findings suggest that CD3 epsilon is expressed on the thymocyte surface independent of and prior to the TCR beta chain. The data are consistent with the notion that in wild-type mice the DP stage is induced by transmembrane signaling through an immature CD3-TCR beta-chain complex, which can be bypassed by crosslinking of CD3 epsilon alone.

CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration.

The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti-CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi-specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post-translational modification of CD4.