Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis.

The Feline Leukemia Virus Subgroup C Receptor 1a (FLVCR1a) is a transmembrane heme exporter essential for embryonic vascular development. However, the exact role of FLVCR1a during blood vessel development remains largely undefined. Here, we show that FLVCR1a is highly expressed in angiogenic endothelial cells (ECs) compared to quiescent ECs. Consistently, ECs lacking FLVCR1a give rise to structurally and functionally abnormal vascular networks in multiple models of developmental and pathologic angiogenesis. Firstly, zebrafish embryos without FLVCR1a displayed defective intersegmental vessels formation. Furthermore, endothelial-specific Flvcr1a targeting in mice led to a reduced radial expansion of the retinal vasculature associated to decreased EC proliferation. Moreover, Flvcr1a null retinas showed defective vascular organization and loose attachment of pericytes. Finally, adult neo-angiogenesis is severely affected in murine models of tumor angiogenesis. Tumor blood vessels lacking Flvcr1a were disorganized and dysfunctional. Collectively, our results demonstrate the critical role of FLVCR1a as a regulator of developmental and pathological angiogenesis identifying FLVCR1a as a potential therapeutic target in human diseases characterized by aberrant neovascularization.

Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma.

Lymphomas express a tumor-specific antigen which can be targeted by cancer vaccination. We evaluated the ability of a new idiotype protein vaccine formulation to eradicate residual t(14;18)+ lymphoma cells in 20 patients in a homogeneous, chemotherapy-induced first clinical complete remission. All 11 patients with detectable translocations in their primary tumors had cells from the malignant clone detectable in their blood by PCR both at diagnosis and after chemotherapy, despite being in complete remission. However, 8 of 11 patients converted to lacking cells in their blood from the malignant clone detectable by PCR after vaccination and sustained their molecular remissions. Tumor-specific cytotoxic CD8+ and CD4+ T cells were uniformly found (19 of 20 patients), whereas antibodies were detected, but apparently were not required for molecular remission. Vaccination was thus associated with clearance of residual tumor cells from blood and long-term disease-free survival. The demonstration of molecular remissions, analysis of cytotoxic T lymphocytes against autologous tumor targets, and addition of granulocyte-monocyte colony-stimulating factor to the vaccine formulation provide principles relevant to the design of future clinical trials of other cancer vaccines administered in a minimal residual disease setting.

Gene complementation. Neither Ir-GLphi gene need be present in the proliferative T cell to generate an immune response to Poly(Glu55Lys36Phe9)n.

The cellular requirements for immune response (Ir) gene expression in a T cell proliferative response under dual Ir gene control were examined with radiation-induced bone marrow chimeras. The response to poly(Glu55Lys36Phe9)n (GLphi) requires two responder alleles that in the [B10.A X B10.A(18R)]F1 map in I-Ab and I-Ek/Cd. Chimeras in which a mixture of the nonresponder B10.A parental cells (which possess only I-Ek/Cd) and the nonresponder B10.A(18R) parental cells (which possess only I-Ab) were allowed to mature in a responder F1 environment did not respond to GLphi, which suggests that at least one cell participating in the response needed to possess both responder alleles to function. When T cells from such A + 18R leads to F1 chimeras were primed in the presence of responder antigen-presenting cells (APC), the chimeric T cells responded to GLphi, which suggests that both responder alleles must be expressed in the APC but not necessarily in the T cell. Interestingly, acutely irradiated F1 animals were found not to be an adequate source of responder APC for priming the proliferating T cell because of the rapid turnover of peripheral APC after irradiation. In adoptive transfer experiments, T cell-depleted bone marrow had to be used as a source of responder APC. When bone marrow cells from (B10.A X B10)F1 responder animals were allowed to mature in a low-responder B10 of B10.A parental environment, neither chimera, F1 leads to A or F1 leads to B, could respond to GLphi. This demonstrated that the presence of high-responder APC, which derive from the donor bone marrow, was not sufficient to generate a GLphi response. It appears that in addition it is essential for the T lymphocytes to mature in a high-responder environment. Finally, B10.A(4R) T cells, which possess neither Ir-GLphi responder allele, could be educated to mount a GLphi-proliferative response provided that they matured in a responder environment and were primed with APC expressing both responder alleles. Therefore, the gene products of the complementing Ir-GLphi responder alleles appear to function as a single restriction element at the level of the APC. T cells that do not possess responder alleles are not intrinsically defective, because they could be made phenotypic responders if they developed in an environment in which responder major histocompatibility complex (MHC) products were learned as self and if antigen was presented to them by APC expressing responder MHC products.

Differential effects of growth hormone and prolactin on murine T cell development and function.

DW/J dwarf mice have a defect in their anterior pituitary and are deficient in growth hormone (GH) and prolactin (PRL). These mice have been demonstrated previously to have a deficiency in CD4/CD8 double-positive thymocytes, which could be corrected by treatment of these mice with recombinant human GH. Since PRL has been implicated in T cell function and human GH can interact with the PRL receptor, DW/J dwarf mice were treated with either ovine GH (ovGH) (20 micrograms/d) or ovine PRL (ovPRL) (20 micrograms/d). The ovine hormones can only bind their own specific receptors in the mouse. After several weeks of treatment, it was found that these two hormones produced markedly contrasting effects on T cells. Phenotypic analysis of the lymphoid organs was performed by flow cytometry and the functional capability of the peripheral T cells was assessed by immunizing the mice and determining the extent of antigen-specific proliferation of T cells obtained from the draining lymph nodes or by determining splenic mitogen responses. The results indicated that ovGH administration to dwarf mice resulted in significant increases in thymic cellularity yet had little effect on peripheral T cell responses. In contrast, the administration of ovPRL resulted in a further decrease in thymic cellularity when compared with untreated dwarf mice. No thymic effects of either ovGH or ovPRL administration were detected on the normal +/? counterparts. However, ovPRL administration resulted in a significant increase in the number and function of antigen-specific peripheral T cells in both immunized dwarf and +/? mice. The adjuvant effects of PRL occurred even though the mice also received complete Freund's adjuvant. These results suggest that neuroendocrine hormones may act in concert in T cell development. GH appears to promote thymocyte proliferation, while PRL appears to decrease thymus size and yet augment the number and function of antigen-specific T cells in the periphery.

Murine syngeneic mixed lymphocyte response. I. Target antigens are self Ia molecules.

A system has been described that produces a murine syngeneic mixed lymphocyte response (MLR) comparable in magnitude to an allogeneic MLR. The responder cells in these cultures exhibit the classic immunologic characteristics of both memory and specificity. Studies using radiation-induced bone marrow chimeras of F(1) {arrow} parent type indicated that, similar to many other T cell-mediated immune responses, the response of the T lymphocytes in the syngeneic MLR was major histocompatibility complex-restricted and was determined by the environment in which the T cells matured. Using responder T cells from F(1) {arrow} parent chimeras and stimulator cells from H-2 recombinant strains, it was possible to map the genes involved in the stimulation to the K and/or I regions. In addition, blocking studies with monoclonal anti-Ia antibodies suggested that in the B10.A strain the critical molecules were products of both the I-A(k) and I-E(k) subregions. The issue of whether the syngeneic MLR is directed solely at self I-region antigens or whether the response represents proliferation to an unknown antigen in association with self I-region determinants was also addressed. Secondary syngeneic MLR were successfully performed in normal mouse serum and with stimulator cells prepared in the absence of bovine serum albumin to rule out the possibility that xenogeneic serum antigens were involved in the stimulation. The possibility that the syngeneic MLR might represent a secondary response to environmental antigens was eliminated by using germ- free mice as a source of stimulator cells and by demonstrating that spleen cells from unimmunized, fully allogeneic chimeras (B10.A {arrow} B10) could generate a normal syngeneic MLR even though such chimeras could not be primed to respond to any foreign antigens unless supplemented in vivo with a source of antigen-presenting cells syngeneic to the B10 host. The possibility that the syngeneic MLR was a primary response to a foreign antigen was considered unlikely because by using our culture conditions we could not obtain a primary antigen response or a secondary antigen response after in vitro priming to a variety of potent foreign antigens. Finally, the possibility that the syngeneic MLR represents a response to a variety of minor histocompatibility self antigens in association with self Ia molecules was eliminated by showing that the secondary responses to H-2 compatible, non-H-2 different strain (A/J vs. B10.A and C3H, or BALB/c vs. B10.D2 and DBA/2) were comparable to the secondary responses to syngeneic stimulators. Thus, we conclude that the target antigens in the syngeneic MLR are solely determinants on self Ia molecules, although the functionally equivalent possibility of a single, nonpolymorphic, minor self antigen seen in association with self Ia molecules cannot be excluded.

B lymphocyte immune response gene phenotype is genetically determined.

We examined the effects of the developmental milieu on the capacity of B cells to undergo immune response gene-controlled, T cell-dependent polyclonal proliferation. Although I-Aq poly(Glu60 Ala30 Tyr10)n (GAT)-nonresponder T cells developing in a responder environment become phenotypic GAT-responders, I-Aq B cells remain unresponsive to GAT, even after maturation in a GAT-responder animal. Conversely, (B10.A x B10.Q)F1 ([GAT responder x GAT nonresponder]F1) T cells developing in a B10.Q GAT nonresponder host fail to respond to GAT, but F1 B cells from the same F1 leads to parent chimeras make excellent proliferative responses in the presence of GAT and responder T cells. Thus, by this assay, B cell immune response gene function is genetically determined and is not affected by the developmental milieu.

Preferential differentiation of T cell receptor specificities based on the MHC glycoproteins encountered during development. Evidence for positive selection.

T cells recognize foreign antigens together with those MHC glycoproteins they have encountered during their development in the thymus. How the repertoire of antigen-specific TCRs is selected has not yet been fully defined. We have investigated the T cell repertoire specificities of CD4-CD8+ cytotoxic T cells developing under conditions where one of the class I MHC-encoded molecules is blocked, while other class I-MHC glycoproteins are still expressed. We show that antigen-specific T cells restricted to the blocked class I fail to develop, while generation of other class I-specific T cell proceeds undisturbed. This highly selective perturbation of the T cell receptor repertoire demonstrates that development of CD4-CD8+ T cells with a certain TCR specificity requires expression of particular alleles of class I MHC. Thus, TCR-MHC interactions provide signals essential to the differentiation of precursor T cells.

Selection of antigen-specific, idiotype-positive B cells in transgenic mice expressing a rearranged M167-mu heavy chain gene.

Flow cytometric analysis of antigen-specific, idiotype-positive (id+), B cell development in transgenic mice expressing a rearranged M167-mu gene shows that large numbers of phosphocholine (PC)-specific, M167-id+ B cells develop in the spleen and bone marrow of these mice. Random rearrangement of endogenous V kappa genes, in the absence of a subsequent receptor-driven selection, should give rise to equal numbers of T15- and M167-id+ B cells. The observed 100-500-fold amplification of M167-id+ B cells expressing an endogenous encoded V kappa 24]kappa 5 light chain in association with the M167 VH1-id transgene product appears to be an antigen driven, receptor-mediated process, since no amplification of non-PC-binding M167 VH1/V kappa 22, T15-id+ B cells occurs in these mu-only transgenic mice. The selection and amplification of antigen-specific, M167-id+ B cells requires surface expression of the mu transgene product; thus, no enhancement of M167-id+ B cells occurs in the M167 mu delta mem-transgenic mice, which cannot insert the mu transgene product into the B cell membrane. Surprisingly, no selection of PC-specific B cells occurs in M167-kappa-transgenic mice although large numbers of B cells expressing a crossreactive M167-id are present in the spleen and bone marrow of these mice. The failure to develop detectable numbers of M167-id+, PC-specific B cells in M167-kappa-transgenic mice may be due to a very low frequency of M167-VH-region formation during endogenous rearrangement of VH1 to D-JH segments. The somatic generation of the M167 version of a rearranged VH1 gene may occur in less than one of every 10(5) bone marrow B cells, and a 500-fold amplification of this M167-Id+ B cell would not be detectable by flow cytometry even though the anti-PC antibody produced by these B cells is detectable in the serum of M167-kappa-transgenic mice after immunization with PC.

Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fas ligand transcription.

Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

CD8 is required during positive selection of CD4-/CD8+ T cells.

Interactions between self-MHC molecules and T cells are necessary for the proper development of mature T cells, in part due to an absolute requirement for self-MHC-TCR interactions. Recently, we showed that CD4-mediated interactions also participate in shaping the T cell repertoire during thymic maturation. We now examine the possible role of the CD8 molecule during in vivo T cell development. Our results demonstrate that perinatal thymi treated with intact anti-CD8 mAb fail to generate CD8 single-positive T cells, while the generation of the other main phenotypes remains unchanged. Most importantly, the use of F(ab')2 anti-CD8 mAb fragments gave identical results, i.e., lack of generation of CD4-/CD8+ cells, with no effect on the generation of CD4+/CD8+. Furthermore, selective blocking of one CD8 allele with F(ab')2 mAbs in F1 mice expressing both CD8 alleles did not interfere with the development of CD4-/CD8+ cells, demonstrating that the absence of CD8+ T cells in homozygous mice is not due to depletion, but rather is caused by a lack of positive selection. This is most likely attributable to a deficient CD8-MHC class I interaction. Our findings strongly advocate that CD8 molecules are vital to the selection process that leads to the development of mature single-positive CD8 T cells.

Essential role of the interleukin 2-interleukin 2 receptor pathway in thymocyte maturation in vivo.

The role of the IL-2-IL-2-R pathway in thymocyte differentiation in vivo is unknown. We have examined fetal thymocyte development in vivo, under conditions where all IL-2-R were saturated from day 13 of gestation with anti-IL-2-R mAbs that were previously shown to render mature T cells unable to respond to IL-2. This produced a dramatic change in the composition of developing T cells: thymocytes from day 1 neonatal mice born to anti-IL-2-R-treated mothers did not contain CD4+ or CD8+ single-positive cell populations. In addition, no generation of surface TCR beta chain-expressing T cells or antigen-reactive functional T cells occurred in treated mice. These data suggest that IL-2-IL-2-R interactions provide signals crucial to in vivo intrathymic development of mature T cells.

Self-recognition specificity expressed by T cells from nude mice. Absence of detectable Ia-restricted T cells in nude mice that do exhibit self-K/D-restricted T cell responses.

The presence in athymic nude mice of precursor T cells with self-recognition specificity for either H-2 K/D or H-2 I region determinants was investigated. Chimeras were constructed of lethally irradiated parental mice receiving a mixture of F1 nude mouse (6-8 wk old) spleen and bone marrow cells. The donor inoculum was deliberately not subjected to any T cell depletion procedure, so that any potential major histocompatibility complex-committed precursor T cells were allowed to differentiate and expand in the normal parental recipients. 3 mo after reconstitution, the chimeras were immunized with several protein antigens in complete Freund's adjuvant in the footpads and their purified draining lymph node T cells tested 10 d later for ability to recognize antigen on antigen-presenting cells of either parental haplotype. Also, their spleen and lymph node cells were tested for ability to generate a cytotoxic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified stimulator cells of either parental haplotype. It was demonstrated that T cell proliferative responses of these F1(nude)----parent chimeras were restricted solely to recognizing parental host I region determinants as self and expressed the Ir gene phenotype of the host. In contrast, CTL responses could be generated (in the presence of interleukin 2) to TNP-modified stimulator cells of either parental haplotype. Thus these results indicate that nude mice which do have CTL with self-specificity for K/D region determinants lack proliferating T cells with self-specificity for I region determinants. These results provide evidence for the concepts that development of the I region-restricted T cell repertoire is strictly an intrathymically determined event and that young nude mice lack the unique thymic elements responsible for education of I region-restricted T cells.

Recombinant transforming growth factor beta 1 and beta 2 protect mice from acutely lethal doses of 5-fluorouracil and doxorubicin.

Transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2 can reversibly inhibit the proliferation of hematopoietic progenitor cells in vivo, leading us to hypothesize that such quiescent progenitors might be more resistant to high doses of cell cycle active chemotherapeutic drugs, thereby allowing dose intensification of such agents. Initial studies showed that whereas administration of TGF-beta 1 or TGF-beta 2 did not prevent death in normal mice treated with high doses of 5-fluorouracil (5-FU), those mice that received TGF-beta 2 did exhibit the beginning of a hematologic recovery by day 11 after administration of 5-FU, and were preferentially rescued by a suboptimal number of transplanted bone marrow cells. Subsequently, it was found that the administration of TGF-beta 2 protected recovering progenitor cells from high concentrations of 5-FU in vitro. This protection coincided with the finding that significantly more progenitors for colony-forming unit-culture (CFU-c) and CFU-granulocyte, erythroid, megakaryocyte, macrophage (GEMM) were removed from S-phase by TGF-beta in mice undergoing hematopoietic recovery than in normal mice. Further studies showed that the administration of TGF-beta protected up to 90% of these mice undergoing hematologic recovery from a rechallenge in vivo with high dose 5-FU, while survival in mice not given TGF-beta was < 40%. Pretreatment of mice with TGF-beta 1 or TGF-beta 2 also protected 70-80% of mice from lethal doses of the noncycle active chemotherapeutic drug, doxorubicin hydrochloride (DXR). These results demonstrate that TGF-beta can protect mice from both the lethal hematopoietic toxicity of 5-FU, as well as the nonhematopoietic toxicity of DXR. This report thus shows that a negative regulator of hematopoiesis can be successfully used systemically to mediate chemoprotection in vivo.

Absence of the Lyt-2-,L3T4+ lineage of T cells in mice treated neonatally with anti-I-A correlates with absence of intrathymic I-A-bearing antigen-presenting cell function.

In an effort to elucidate the role of intrathymic Ia-bearing antigen-presenting cells (APC) on the development of the class II-restricted T cell repertoire, we examined the effect of neonatal anti-I-A treatment on both intrathymic and splenic APC function; on the generation of Lyt-2-,L3T4+, Lyt-2+,L3T4-, and Lyt-2+,L3T4+ T cells; and on the development of class I- and class II-specific T cell functions. Both the thymus and the spleen are completely devoid of Lyt-2-,L3T4+ T cells in young mice treated from birth with anti-I-A, and also lack functions associated with this subset, i.e., alloantigen-specific interleukin 2 production (present report), allo-class II-specific and self-class II-restricted T cell proliferative responses, and helper cell function for the generation of cytotoxic T lymphocyte responses (18). Development of the Lyt-2+,L3T4- subset proceeds undisturbed in these mice, in accord with the previously reported normal levels of cytotoxic T lymphocyte precursors (18). The thymus contains normal numbers of the immature cortical Lyt-2+,L3T4+ cells, indicating that acquisition of the L3T4 marker, in and of itself, is not influenced by anti-I-A treatment. This striking absence of the lineage of T cells responsible for class II-specific T cell functions is correlated with absence of thymic APC function for class II-restricted T cell clones. When anti-I-A-treated mice are allowed to recover from the antibody treatment, splenic and thymic APC function return to normal in 2-3 wk, and thymic Lyt-2-,L3T4+ T cell numbers and functions reappear before such cells are detectable in the spleen. Collectively, these findings suggest that development of the Lyt-2-,L3T4+ lineage of class II-specific T cells is entirely dependent on functional I-A-bearing APC cells in the thymus. In addition, the presence of normal levels of Lyt-2+,L3T4-T cells argues that generation of the two major subsets of T cells (i.e., Lyt-2+,L3T4- and Lyt-2-,L3T4+) occurs through separate events, involving unique sites of interactions between precursor T cells and nonlymphoid major histocompatibility complex-bearing thymus cells.

Human T cell leukemia/lymphoma virus I infection and subsequent cloning of normal human B cells. Direct responsiveness of cloned cells to recombinant interleukin 2 by differentiation in the absence of enhanced proliferation.

A human T cell leukemia/lymphoma virus (HTLV)-I-infected B cell clone expressed Tac antigen on its cell surface and responded to recombinant interleukin 2 (IL-2) by increased production of IgM without any increase in proliferation. Anti-Tac antibody completely inhibited the IL-2-induced differentiation of this HTLV-I-infected B cell clone. This study demonstrates that HTLV-I can directly infect normal mature human B cells, and that the Tac antigen, which may be induced by infection with HTLV-I, is the functional receptor for IL-2-induced B cell differentiation. The availability of such cell lines and clones should provide useful tools to delineate precisely the differentiation step in the human B cell cycle.

Interleukin 2 receptors on human B cells. Implications for the role of interleukin 2 in human B cell function.

In the present study, we examined the expression of interleukin 2 (IL-2) receptors on normal human B cells as well as established B cell lines. Anti-Tac monoclonal antibody did not bind to freshly separated normal human B cells. Unexpectedly, with the appropriate activation of the normal B cells by anti-mu antibody, phorbol myristate acetate, or Staphylococcus aureus Cowan I (SAC), Tac antigen was induced on the activated B cells. Anti-Tac antibody showed consistent reactivity with two B cell lines that were infected by human T cell leukemia virus (HTLV) and some reactivity with two out of eight Epstein-Barr virus-transformed B cell lines established from normal adult donors. Immunoprecipitation analysis revealed that antigens of similar size with a molecular weight of 50,000-60,000 can be precipitated with anti-Tac antibody from phytohemagglutinin-stimulated normal T cell blasts and normal activated B cells, as well as a cloned B cell line. Binding assays of IL-2 on normal activated B cells and on the cloned B cell (HS1) revealed that B cells have significantly fewer sites and lower-affinity IL-2 receptors compared with phytohemagglutinin-stimulated normal T cell blasts. Finally, biological properties of the IL-2 receptor on B cells were examined by incubating B cells with recombinant IL-2. It was found that moderate concentrations of IL-2 induce significant enhancement of proliferation and differentiation in SAC-activated normal B cells. These results suggest that normal B cells may express functional IL-2 receptors or closely related proteins and thus IL-2 may play a significant role in the modulation of B cell function.

Role of CD4 in thymocyte selection and maturation.

We examined the possible role of CD4 molecules during in vivo and in vitro fetal thymic development. Our results show that fetal thymi treated with intact anti-CD4 mAbs fail to generate CD4 single-positive T cells, while the generation of the other phenotypes remains unchanged. Most importantly, the use of F(ab')2 and Fab anti-CD4 mAb gave identical results, i.e., failure to generate CD4+/CD8- T cells, with no effect on the generation of CD4+/CD8+ T cells. Since F(ab')2 and Fab anti-CD4 fail to deplete CD4+/CD8- in adult mice, these results strongly argue that the absence of CD4+/CD8- T cells is not due to depletion, but rather, is caused by a lack of positive selection, attributable to an obstructed CD4-MHC class II interaction. Furthermore, we also observed an increase in TCR/CD3 expression after anti-CD4 (divalent or monovalent) mAb treatment. The TCR/CD3 upregulation occurs in the double-positive population, and may result from CD4 signaling after mAb engagement, or may be a consequence of the blocked CD4-class II interactions. One proposed model argues that the CD3 upregulation occurs in an effort to compensate for the reduction in avidity or signaling that is normally provided by the interaction of the CD4 accessory molecule and its ligand. As a whole, our findings advocate that CD4 molecules play a decisive role in the differentiation of thymocytes.

Expression and role of p75 interleukin 2 receptor on human monocytes.

We investigated the expression of IL-2R subunits in human monocytes using the TU27 mAb, which recognizes the p75 chain, and anti-Tac mAb, which recognizes the p55 moiety of the IL-2R. We found that p75 but not p55 is constitutively expressed in more than 90% of fresh human monocytes. Antibody to p75, but not to p55, inhibited the activation of monocytes to a cytotoxic stage induced by IL-2 but did not block IFN-gamma-induced cytotoxicity. Our data demonstrate that the p75 chain is expressed on human monocytes and is involved in the activation of monocytes by IL-2.

p52-Independent nuclear translocation of RelB promotes LPS-induced attachment.

The NF-kappaB signaling pathways have a critical role in the development and progression of various cancers. In this study, we demonstrated that the small cell lung cancer cell line (SCLC) H69 expressed a unique NF-kappaB profile as compared to other cancer cell lines. The p105/p50, p100/p52, c-Rel, and RelB protein and mRNA transcripts were absent in H69 cells but these cells expressed RelA/p65. The activation of H69 cells by lipopolysaccharide (LPS) resulted in the induction of RelB and p100 expression. The treatment also induced the nuclear translocation of RelB without the processing of p100 to p52. Furthermore, LPS-induced beta1 integrin expression and cellular attachment through an NF-kappaB-dependent mechanism. Blocking RelB expression prevented the increase in the expression of beta1 integrin and the attachment of H69. Taken together, the results suggest that RelB was responsible for the LPS-mediated attachment and may play an important role in the progression of some cancers.

Regulation of intracellular actin polymerization by prenylated cellular proteins.

Posttranslational modification by covalent attachment of polyisoprene intermediates to a carboxyterminal CAAX-box motif is required for the biologic function of proteins such as p21ras, the supergene family of ras-related proteins, nuclear lamins, and subunits of heterotrimeric G-proteins. Cells grown in the presence of lovastatin, which inhibits HMG-CoA reductase and prevents synthesis of intermediates required for protein prenylation, develop a round, refractile morphology. Our data indicate that this is due to the selective loss of actin cables without gross changes in the microtubular lattice or intermediate filament structure. Microinjection of a competitive peptide inhibitor of protein prenyltransferases into the cytoplasm of cells induces an identical change in morphology with loss of actin cables. Mevalonate (MVA) reverses the lovastatin-induced morphologic change by inducing a rapid repolymerization of actin cables with coincident reversion to the flat morphology. Furthermore, microinjection of farnesyl-pyrophosphate or geranylgeranyl-pyrophosphate into lovastatin-treated cells also results in rapid morphologic reversion. The morphologic reversion induced by MVA requires the presence of serum, and is independent of extracellular calcium. The addition of cycloheximide to the growth medium prevents lovastatin-induced loss of actin cables, and causes morphologic reversion of lovastatin-treated cells by a mechanism that is independent of MVA. A1F4- induces morphologic reversion in a manner indistinguishable from MVA. These data indicate that prenylated protein(s) play a critical role in regulating the state of intracellular actin, and that GGPP can rescue the lovastatin-induced morphologic phenotype in the absence of upstream intermediates of cholesterol biosynthesis. We have begun to dissect the signaling events that mediate this pathway.