Rescue of thymocytes from glucocorticoid-induced cell death mediated by CD28/CTLA-4 costimulatory interactions with B7-1/B7-2

During the differentiation of thymocytes to mature T cells the processes of positive and negative selection result in signals that either protect thymocytes from cell death, or delete, through apoptosis, thymocytes with self-reactive T cell receptors (TCR). Glucocorticoids have been shown to induce thymocyte apoptosis and are produced within the thymic microenvironment. Furthermore, steroid-induced apoptosis of thymocytes has been suggested as a potential mechanism for removal of nonselected thymocytes. In this report, we demonstrate that thymocytes can be rescued from glucocorticoid-induced apoptosis by incubation with cells that express high levels of B7-1 or B7-2. In addition, the ability to be rescued by B7-1 and/or B7-2 can precede expression of the TCR. We demonstrate that CD3(+)-depleted or CD3+/ TCR-beta(+)-doubly depleted thymocytes can be rescued from glucocorticoid-induced apoptosis through the interaction of CD28 or CTLA-4 on thymocytes with cells bearing high levels of B7-1 or B7-2. Furthermore, these transfected cells are major histocompatibility complex (MHC) class II negative and, while they may express MHC class I, there is no preferential rescue of CD8+ thymocytes in the presence of glucocorticoids. Together, these data suggest that the rescue of thymocytes from glucocorticoids can be independent of the TCR. We also demonstrate that, in addition to CD28, CTLA-4 is expressed on thymocytes, suggesting that rescue from glucocorticoid-induced cell death can be mediated by both CD28 and CTLA-4. A CTLA-4Ig fusion protein which binds to both B7-1 and B7-2 was shown to completely block the rescue of thymocytes from glucocorticoid-induced cell death. Therefore, we conclude that interactions between B7-1/B7-2 and CD28/CTLA-4 are sufficient and necessary for rescue of thymocytes from glucocorticoid-induced cell death.     

Expression of a hypoglycosylated form of CD86 (B7-2) on human T cells with altered binding properties to CD28 and CTLA-4

CD80 (B7-1) and CD86 (B7-2) on APC provide a major costimulatory signal through interactions with CD28 on T cells. Absent from resting human T cells, CD86 is up-regulated early upon T cell activation, whereas CD80 expression appears later. Whereas T cell expression of CD80 has been implicated in costimulation, the functional significance of CD86 expression on T cells is unclear. We now demonstrate that CD86 expressed on human CD4+ T cell clones does not provide a costimulatory signal for other CD4+ T cell clones. Binding studies using CD28-Ig and CTLA-4-Ig fusion proteins demonstrate that CD86 expressed on T cells has significantly reduced binding affinity for CTLA-4 and no detectable binding to CD28. Biochemical analysis demonstrates that post-translational modifications of CD86 in human T cells are different from those of CD86-transfected Chinese hamster ovary cells or EBV-transformed B cells, in that T cells express a hypoglycosylated form of CD86 on the surface membrane. Thus, our results suggest that while CD86 is expressed on a number of different cell types, its costimulatory function and affinity for its ligands may be regulated by cell type-specific post-translational modifications.     

A radiosensitive APC activity dissociates IL-2 secretion and activation-induced cell death by autoreactive T cell hybridomas

T cell hybridomas were generated from a LEW rat T cell line specific for the uveitogenic peptide bov-B1 of bovine retinal S-antigen. Using these autoreactive hybridomas, IL-2 production and activation-induced cell death (AICD) were dissociated as outcomes of activation. The self-reactive hybridomas secrete IL-2 and undergo AICD in response to antigen presented by non-irradiated syngeneic splenocytes, whereas antigen presentation by irradiated splenocytes induced only AICD. IL-2 production by a non-self reactive hybridoma was unaffected by irradiation of the APC. Pretreatment of the APC with phorbol ester or lipopolysaccharide and IL-4 protected their ability to induce IL-2 secretion after gamma-irradiation. Although the co-stimulation-blocking reagent CTLA-4-Ig mimicked the effect of gamma-irradiation by preventing IL-2 secretion but not AICD, B7 expression on the APC was not radiosensitive, nor did co-stimulation, provided 'in trans' with a B7-expressing third-party cell, reconstitute antigen-specific hybridoma IL-2 secretion in response to irradiated APC. In summary, the data show that IL-2 secretion and AICD of a self-reactive T cell hybridoma can be dissociated as consequences of TCR occupancy in the presence of a functional co-stimulatory signal. It is proposed that the signals producing these events are transduced through the TCR-CD3 complex alone and reflect the differential outcomes of high- and low-affinity interactions.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

Fas ligand expression is restricted to nonlymphoid thymic components in situ

The cell surface receptor Fas (Apo-1/CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in activation-induced death of mature T cells and in killing mediated by cytolytic T cells. The role of the Fas pathway in apoptosis associated with thymic selection events is, however, controversial. Although Fas and FasL are known to be expressed in the thymus, the nature and in vivo localization of FasL-expressing cells have not been determined. Using recently developed anti-FasL Abs in combination with in situ hybridization on tissue sections, we show in this work that FasL-expressing cells are present in the thymus, particularly within the medulla. FasL mRNA was detected readily in thymic stromal cell extracts, but not in isolated thymocytes. Moreover, immunohistochemical analysis of serial tissue sections stained with Abs against FasL in conjunction with epithelial and dendritic cell markers indicated that both thymic epithelial and dendritic cells express FasL in situ. The coexistence of FasL-expressing stromal cells and Fas-expressing thymocytes may have important implications for the role of the Fas pathway in apoptosis associated with thymic selection events.     

B70 antigen is a second ligand for CTLA-4 and CD28

The membrane antigen B7/BB1 (refs 1, 2) is expressed on activated B cells, macrophages and dendritic cells, and binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. Discrepancies between results with anti-CD28 and anti-B7 antibodies have suggested the existence of a second ligand for CD28 and CTLA-4 (refs 3, 6-8). We have generated a monoclonal antibody, IT2, that reacts with a 70K glycoprotein (B70). B70 complementary DNA was cloned from a B-lymphoblastoid cell line library and encodes a new protein of the immunoglobulin superfamily with limited homology to B7. B70 is expressed on resting monocytes and dendritic cells and on activated, but not resting, T, NK and B lymphocytes. IT2 substantially inhibited the binding of a CTLA4-immunoglobulin fusion protein to human B-lymphoblastoid cell lines and, together with anti-B7 antibody, completely blocked CTLA-4 binding. Further IT2 efficiently inhibited primary allogeneic mixed lymphocyte responses. These findings indicate that B70 is a second ligand for CD28 and CTLA-4 and may play an important role for costimulation of T cells in a primary immune response.     

CTLA4-Ig inhibits optimal T helper 2 cell development but not protective immunity or memory response to Nippostrongylus brasiliensis

The B7 co-stimulatory pathway is critical to T cell activation, however its role in the generation of Th2 cells in vivo remains controversial. We have studied the role of B7 co-stimulation in the development of a Th2 immune response to the nematode parasite Nippostrongylus brasiliensis. Blockade of B7 co-stimulation with murine CTLA4-Ig (mCTLA4-Ig) resulted in decreased Th2 cell development as determined by IL-4 and IL-5 cytokine production in vitro. It also resulted in lowered Th2 cell effector function in vivo, with marked reductions in IgE production. Blood eosinophilia was variably affected by mCTLA4-Ig treatment, which resulted in both slight and very severe inhibition in different experiments. However, an effective immune response was still evident as demonstrated by the further reduction of cytokine production, IgE titers, and blood eosinophilia in mice treated with a combination of mCTLA4-Ig and anti-CD4 mAb, and by the ability of mCTLA4-Ig-treated mice to expel adult worms. In addition, mCTLA4-Ig treatment did not alter the development of a memory response following secondary infection with N. brasiliensis, with the exception of IgE production. We conclude from these results that B7 co-stimulation is required in this experimental model for optimal Th2 cell development and effector function in vivo but is not necessary for protective immunity.     

B cells directly tolerize CD8(+) T cells

This report investigates the response of CD8(+) T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the Kb-restricted ovalbumin (OVA) determinant OVA257-264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro-activated CD8(+) T cells from the Kb-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8(+) T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.     

Ecologic studies of rodent reservoirs: their relevance for human health

To gain insight into the immunomodulatory effects of vitamin E (VE), immune cell population analyses were conducted using thymus and spleen from male broilers fed diets with various levels of VE supplementation (0, 17, 46, and 87 mg dl-alpha-tocopherol acetate/kg of feed). At 2 and 7 wk of age, the percentages of B cells, macrophages, and T cell subsets, delineated by the expression of CD4, CD8, and T cell receptor (TCR) isotype, in thymus and spleen were determined by flow cytometry. The percentages of thymic and splenic B cells and macrophages from 2- and 7-wk-old chickens, as well as the percentage of thymic T cells in 2-wk-old chickens, were unaffected by VE treatment. However, 7-wk-old broilers maintained on 87 mg VE/kg feed had a higher percentage of CD4+CD8- thymocytes, a higher CD4+CD8- to CD4-CD8+ thymocyte ratio, and a lower percentage of CD4+CD8+ thymocytes than chickens receiving no dietary VE supplementation. The VE-induced increase in the percentage of CD4+CD8- thymocytes was due to an increase in the TCR2+CD4+CD8- thymocyte subset, whereas the decrease in the percentage of CD4+CD8+ thymocytes involved all TCR defined T cell subsets. In the spleen, the percentage of CD4+CD8- T cells was lower in 2-wk-old chickens and higher in 7-wk-old chickens maintained on 87 mg/kg feed than in chickens receiving no dietary VE supplementation. The decrease in CD4+CD8- splenocytes at 2 wk of age was due to a decline in the percentage of TCR2+CD4+CD8- splenocytes, whereas the increase in CD4+CD8- splenocytes in 7-wk-old chicks was due to an increase in the percentages of all TCR defined CD4+CD8- T cell subsets. These data support an immunomodulatory effect of VE on CD4+CD8- T cells.     

Cardiac risk in surgery. A review and guidelines for risk evaluation and reduction of cardiac risk in general surgery

We report herein the phenotypic and functional analysis of human bone marrow and thymus derived early T cells. Commitment to T cell lineage is acquired during CD7 antigen expression by CD34+ precursors in human bone marrow and before thymus colonization. Early thymocytes show similar phenotypic characteristics as bone marrow T cells. They rapidly acquire CD4 before the dual expression of CD4 and CD8. Their expansion and differentiation is regulated by two major factors: thymic stroma and cytokines produced by these stroma cells or by thymocytes themselves. Among cytokines, IL1 and sCD23 produced by thymic epithelial cells support in vitro early T cell development.     

Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection

Analysis of antigen-induced negative selection of thymocytes in T cell receptor (TCR)-transgenic mice is complicated by the presence of an antigen-responsive peripheral T cell compartment. Our experiments address the question of whether and how peripheral T cell activation can affect immature thymocytes. Following three daily injections of peptide antigen into mice expressing a peptide-specific transgenic TCR and deficient for TAP1, we and others have found profound deletion of the CD4+CD8+ (DP) thymocyte subset. However, our work shows that even though mature CD8+ T cells are inefficiently selected in TAP1-deficient mice, there was a striking degree of peripheral expansion and activation of CD8+ peripheral T cells. Furthermore, when cells from TCR-transgenic mice were adoptively transferred, we found that deletion of nontransgenic DP thymocytes occurred in Thy-1-congenic and even more efficiently in TAP1-deficient recipients after repeated peptide injection resulting in peripheral T cell activation. In the adoptive transfer experiments the degree of deletion of immature bystander thymocytes was decreased upon blocking of TNF. These data show that deletion of DP thymocytes can result from excessive peripheral T cell activation and identify TNF as an important effector molecule for this process. When steps are taken to avoid peripheral T cell activation, peptide antigen can induce TCR-mediated thymocyte deletion, presumably in the thymus cortex, since injection of TAP1-deficient TCR-transgenic mice resulted in deletion of immature DP thymocytes prior to detectable peripheral T cell expansion and activation. This effect was not blocked by inhibiting tumor necrosis factor activity. In addition, DP depletion was seen in the absence of peripheral T cell activation when antibody-mediated depletion of CD8+ T cells was performed. Our work clearly shows that two mechanisms for deletion of DP thymocytes exist: deletion induced by antigen presentation in the thymus and deletion as a consequence of repeated stimulation of mature peripheral T cells.     

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.     

CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma

Generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. CTLA-4 is a second B7 receptor expressed by T cells upon activation that, unlike CD28, appears to deliver an inhibitory signal to T cells. Recently, we and others demonstrated that administration of an anti-CTLA-4 antibody was sufficient to promote regression of several murine tumors. However, certain tumors, such as the SM1 mammary carcinoma, remain refractory to this type of immunotherapy. In the present study, we report that the combination of both CTLA-4 blockade and a vaccine consisting of granulocyte-macrophage colony-stimulating factor-expressing SM1 cells resulted in regression of parental SM1 tumors, despite the ineffectiveness of either treatment alone. This synergistic therapy resulted in long-lasting immunity to SM1 and depended on both CD4(+) and CD8(+) T cells. Interestingly, synergy was not observed between CTLA-4 and a B7-expressing SM1 vaccine. Given that granulocyte-macrophage colony-stimulating factor promotes differentiation and activation of dendritic cells as well as enhances cross-priming of T cells to tumor-derived antigens and that SM1 is major histocompatibility complex class II-negative, our findings suggest that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell. In addition, these results also support the idea that the most effective and synergistic vaccine strategy targets treatments that enhance T cell priming at the level of host-derived antigen-presenting cells.     

The induction of transplantation tolerance by intrathymic (i.t.) delivery of alloantigen: a critical relationship between i.t. deletion, thymic export of new T cells and the timing of transplantation

Intrathymic (i.t.) injection of donor alloantigens has proved to be an effective strategy for the induction of tolerance. However, the mechanisms by which tolerance is induced and maintained after transplantation remain unclear. In this report we show that tolerance to donor cardiac allografts can be induced across a MHC class I difference by i.t. injection of donor splenocytes and transient T cell depletion. Furthermore, using H-2K(b)-specific TCR transgenic mice (BM3), we demonstrate that prolonged deletion of donor-reactive thymocytes was essential to induce tolerance by i.t. injection and this was dependent upon donor cells persisting in the thymus. Examination of the kinetics of thymic export following i.t. injection revealed that prolonged deletion of thymocytes was required to delay export of new T cells to the periphery until the time of transplantation. Importantly, after transplantation donor cell persistence in the thymus and i.t. deletion were no longer necessary to maintain tolerance. The graft itself or cells from the graft was responsible for maintaining tolerance at this stage. These findings reveal that multiple mechanisms are responsible for the induction and maintenance phases of tolerance to alloantigens in vivo after i.t. delivery, and that a complex inter-relationship between donor cell persistence in the thymus, i.t. deletion, thymic export of T cells and the timing of transplantation is involved.     

Fas-independent death of activated CD4(+) T lymphocytes induced by CTLA-4 crosslinking

The CTLA-4 receptor is a critical inhibitory regulator of T cell proliferation and effector function. However, the mechanisms through which CTLA-4 modulates the activation of T cells remain uncertain. Initial studies, using activated human T cells, have suggested that CTLA-4 crosslinking may induce apoptosis. However, more recent experiments have demonstrated that crosslinking of the CTLA-4 receptor on the surface of resting murine T cells blocks cell cycle progression without inducing apoptosis. Here we provide evidence that CTLA-4 crosslinking on the surface of activated murine CD4(+) T lymphocytes leads to death of a substantial fraction of the cells whereas in resting CD4(+) T cells the same stimulation conditions induce cell cycle arrest without apoptosis. Cell death induced by CTLA-4 stimulation occurs independently of Fas and therefore may involve a novel pathway. CTLA-4-mediated apoptosis may be a means of terminating the function of previously stimulated T cells. Exploitation of this mechanism also may provide a therapeutic strategy to eliminate alloreactive or autoreactive T cells.     

T-cell apoptosis detected in situ during positive and negative selection in the thymus

Because of positive and negative selection to molecules of the major histocompatibility complex (MHC), only a small proportion of the massive numbers of T cells generated in the thymus are selected for export. Immature thymocytes have a rapid turnover, and it has long been assumed that most thymocytes die in situ, presumably from apoptosis. This has yet to be proved, however, and conventional staining techniques have shown only minimal evidence of cell death in the normal thymus. Using a method for detecting cells with DNA strand breaks, we now present direct evidence for apoptosis in the normal thymus. In sections of thymus from adult mice, apoptotic cells are scattered throughout the cortex and are engulfed locally by F4/80+ macrophages. Apoptosis in the thymic cortex is not reduced in MHC-deficient mice, which suggests that T-cell death is primarily a reflection of lack of positive selection rather than negative selection. Direct evidence for apoptosis due to negative selection was obtained by crossing a V beta 5 transgenic line to I-E+ and I-E- mice: I-E+ mice are known to eliminate V beta 5+ T cells in the thymus whereas I-E- mice do not. In marked contrast to I-E- mice, the medulla of I-E+ V beta 5 transgenic mice contains dense aggregates of apoptotic cells; these cells are engulfed by a distinct population of F4/80- MAC-3+ macrophages. Negative selection of V beta 5+ cells is thus restricted to the medulla.     

Evidence that induction of tolerance in vivo involves active signaling via a B7 ligand-dependent mechanism: CTLA4-Ig protects V beta 8+ T cells from tolerance induction by the superantigen staphylococcal enterotoxin B

Co-stimulation through CD28 is thought to be necessary for the activation of unprimed CD4+ T cells, which are otherwise rendered tolerant. However, we previously found that CD4+ T cell priming was normal or augmented in mice which overexpressed a soluble form of CTLA4 where co-stimulation through CD28 was abrogated. To investigate this CD4+ T cell response, we exploited the capacity of the superantigen staphylococcal enterotoxin B to stimulate T lymphocytes bearing V beta 8+, which represent approximately 30% of all CD4+ T cells. In litter-mate controls of CTLA4-Ig transgenic mice, immunization with staphylococcal enterotoxin B leads to expansion, followed by deletion of V beta 8+ T cells, and the remaining cells are tolerant when stimulated in vitro. Comparable expansion and deletion of V beta 8 T cells occurs in CTLA4-Ig transgenic mice. However, in contrast to normal mice, the remaining V beta 8+ T cells from CTLA4-Ig transgenic mice are not anergic and remain responsive to superantigen in vitro.     

Endovascular treatment of ruptured posterior circulation aneurysms using electrolytically detachable coils

The murine interleukin-7 (IL-7) gene was disrupted to examine the role of IL-7 in the lymphoid system. Expansion of lymphoid cells is sharply curtailed in IL-7-deficient mice. This is evident in a dramatic reduction but not elimination of lymphoid cells in the thymus, bone marrow and spleen. The few thymocytes present express CD4 and/or CD8 markers associated with T-cell maturation. Similarly, a limited number of B cells detected in the bone marrow rearrange and express immunoglobulin genes. Small but distinct populations of B and T cells are found in the spleens of IL-7-deficient mice. Thus the signal transmitted by IL-7 plays a central role in the expansion of lymphocytes while it is not absolutely required for their maturation. A transgene that directs expression of IL-7 to lymphoid cells was found to restore the numbers of thymocytes, bone marrow B-cell progenitors and splenic lymphocytes of IL-7-deficient mice to approximately normal levels. This genetic complementation confirms that the lymphoid defect is specifically due to the absence of IL-7 and demonstrates that the expansion of lymphoid cells in an organism is regulated by their exposure to IL-7.     

Interactive effects of cocaine and gender on thymocytes: a study of in vivo repeated cocaine exposure

This study used a mouse model including both sexes to assess the impact of repeat cocaine exposure on the differentiation and function of T cell in thymus. Cocaine hydrochloride in 0.9% saline, 5 mg or 40 mg/kg, was administrated by i.p. injection to C57BL/6 mice for 10 days. Thymocytes were obtained 24 h after the 10th injection. Repeat in vivo cocaine exposure inhibited the proliferation of T lymphocytes in response to Con-A and Con-A plus anti-CD28. The proliferation induced by IL-2 in the Con-A stimulated T blasts was attenuated in cocaine treated mice. These effects were seen at a lower cocaine dose in female mice. The total number of thymocytes was reduced. Although the percentage of mature thymocytes (CD4+ CD8- and CD4- CD8+ cells) was not altered, the absolute cell numbers were attenuated. Both percentage and absolute cell number of immature thymocytes (CD4+ CD8+) decreased and the pre-mature (CD4- CD8-) cells increased. CD28 and CD25 expression were attenuated in Con-A stimulated thymocytes of mice treated with cocaine at 40 mg/kg. Interleukin 2 production was not significantly altered, however, gamma-IFN production was decreased by cocaine exposure at 40 mg/kg. In conclusion, cocaine exerts inhibitory effects on the function of mature thymocytes, and on the differentiation of thymocytes. A gender difference in response to cocaine was noted in that female mice were more sensitive to lower dose of cocaine exposure.     

The BB1 monoclonal antibody recognizes both cell surface CD74 (MHC class II-associated invariant chain) as well as B7-1 (CD80), resolving the question regarding a third CD28/CTLA-4 counterreceptor

The identification of all CD28/CTLA-4 counterreceptors is critical to our understanding of this pivotal pathway of T cell activation. Clouding our understanding has been the reported discrepancies in expression and function of the B7-1 (CD80) molecule based upon the use of the BB1 vs other anti-B7-1 mAbs. To resolve this issue, we have cloned a BB1-binding molecule from the BB1+B7-1(-) NALM-6 pre-B cell line. Here, we demonstrate that this BB1-binding molecule is identical to the cell surface form of CD74 (MHC class II-associated invariant chain). CD74-transfected cells bound the BB1 mAb but not other anti-CD80 mAbs, CD28-Ig, or CTLA4Ig. Absorption and blocking experiments confirmed the reactivity of BB1 mAb with CD74. A region of weak homology was identified between CD74 and the region of B7-1 encoding the BB1 epitope. Therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Many of the puzzling observations in the literature concerning the expression of human B7-1 are resolved by an understanding that BB1 staining is the summation of CD74 plus B7-1 expression. This observation requires the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.