New human leukocyte clusters of differentiation.
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Antigens, CD↗
Biomedical subjects
O Majdic
Publications and source records attributed to O Majdic.
T cell stimulation via CD47: agonistic and antagonistic effects of CD47 monoclonal antibody 1/1A4.
CD47/integrin-associated protein has been extensively studied on various cell types. The function of CD47 on T cells, however, remained poorly understood. We demonstrate here that our CD47 mAb 1/1A4 has both inhibitory as well as costimulatory effects in terms of T cell activation. Soluble, not cross-linked, CD47 mAb 1/1A4 blocks allogeneic MLRs. This effect is predominantly observed with suboptimal numbers of stimulator cells. In contrast, cross-linking of CD47 in the presence of CD28 mAb or phorbol ester induces vigorous T cell proliferation that is sensitive to cyclosporin A. Cross-linking, but not immobilization, of the CD47 mAb 1/1A4 is an essential requirement for the CD28- or phorbol ester-dependent induction of T cell mitogenesis. Furthermore, CD47 mAb 1/1A4 induces T cell proliferation when coimmobilized with a CD3 mAb to the same surface. Ligation with cross-linked 1/1A4 mAb induces an increase in intracellular free calcium levels and leads to tyrosine phosphorylation of various cellular proteins and, in the presence of suboptimal concentrations of plate-bound CD3 mAb, to enhanced IL-2 promotor activity in T cells. Furthermore, we present evidence that upon cross-linking of CD47 with mAb 1/1A4, purified T cells acquire responsiveness for several T cell growth factors. IL-1beta and/or IL-6 dramatically augment this CD47-induced cytokine responsiveness. Our results suggest that the novel activation pathway via CD47 might be critically involved in Ag-dependent and Ag-independent T cell functions.
Antibodies, Monoclonal↗
Efficient retrovirus-mediated gene transfer of dendritic cells generated from CD34+ cord blood cells under serum-free conditions.
A retroviral-vector encoding the low affinity nerve growth factor receptor (LNGFR) was used to transduce dendritic cells (DCs) generated from CD34+ cord blood (CB) progenitor cells under serum-free conditions. Transduction efficiency was monitored by flow cytometry (FACS) using a specific monoclonal antibody. Prior to retroviral infections, CD34+ CB cells were stimulated for 60 h in a serum-free medium containing a DC differentiation inducing cytokine cocktail: stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFalpha), and transforming growth factor beta 1 (TGF-beta1). Addition of flt3-ligand (FL) to the aforementioned growth factors significantly enhanced cell expansion (41.7+/-11.5 fold vs. 22.5+/-4.7 fold without FL) and generation of CD1a+ DCs (mean 45.7+/-9.8% vs. 28+/-6.5% without FL, n = 4,p = 0.01). Furthermore, FL significantly increased the proportion of CD1a+LNGFR+ cells (mean 10%+/-4.4% vs. 6%+/-2.4 without FL n = 4, p = 0.03). When serum-free viral supernatants were used to infect DCs progenitors under entirely serum-free conditions and with the most potent cytokine combination, approximately one-third of the CD1a+ DCs generated co-expressed the LNGFR gene. Moreover, the transduced gene was also identified in more mature CD1a+CD80+ and CD1a+CD86+ DCs after 12-14 days of culture. In addition, transduced CD1a+ DCs maintained their functional properties, stimulating allogeneic T cells with similar efficiency as nontransduced CD1a+ DCs. Thus, the serum-free system described allows efficient generation and transduction of CD1a+ DCs derived from CD34+ progenitor cells and may be very useful for future therapeutic applications of DCs.
Antigens, CD1↗
flt3 ligand in cooperation with transforming growth factor-beta1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions.
Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-beta1 (TGF-beta1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-beta1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor strongly increases both percentages (mean, 36% +/- 5% v 64% +/- 4%; P = .001) and total numbers (4.4- +/- 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-beta1. Upon omission of TGF-beta1, percentages of CD1a+ DC decreased (to mean, 10% +/- 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a- cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-beta1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-beta1- and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% +/- 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-beta1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-beta1 is virtually identical (mean, 41% +/- 6% v 41% +/- 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-beta1 to show this costimulatory effect.
Antigens, CD1↗
Expression of Wiskott-Aldrich syndrome protein (WASP) gene during hematopoietic differentiation.
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder described as a clinical triad of thrombocytopenia, eczema, and immunodeficiency. The gene responsible for WAS encodes a 502-amino acid proline-rich protein (WASp) that is likely to play a role in the cytoskeleton reorganization and/or in signal transduction of hematopoietic cells. However, the function and the regulation of the WAS gene (WASP) have not yet been clearly defined. We have studied WASP expression at the transcriptional level in freshly isolated mature peripheral blood cells and during hematopoietic development. For this purpose, we have isolated CD34+ hematopoietic precursor cells from cord blood. These cells were cultured in vitro with various growth factors to generate committed or mature cells belonging to different hematopoietic differentiation pathways, such as granulocytic (CD15+) cells, monocytic (CD14+) cells, dendritic (CD1a+) cells, erythroid lineage (glycophorin A+) cells, and megakaryocytic cells (CD41+). We have shown by reverse transcriptase polymerase chain reaction analysis that the WASP transcript is ubiquitously detectable throughout differentiation from early hematopoietic progenitors, including CD34+CD45RA- and CD34+CD45RA+ cells, to cells belonging to different hematopoietic lineages, including erythroid-committed and dendritic cells. In addition, Northern blot analysis showed that peripheral blood circulating lymphocytes (CD3+ and CD19+ cells) and monocytes express WASP mRNA. Several hematopoietic cell lines were tested and higher levels of expression were consistently detected in myelomonocytic cell types. By contrast, primary nonhematopoietic cells, including fibroblasts, endothelial cells, and keratinocytes, were consistently negative for WASP mRNA.
Animals↗
Molecular and functional characterization of the urokinase receptor on human mast cells.
The urokinase receptor system is involved in several biological processes including extracellular proteolysis, cell invasion, and chemotaxis. Mast cells are multifunctional perivascular cells that play an important role in the regulation of microenvironmental events. We report that primary human mast cells and the human mast cell line HMC-1 express the receptor for urokinase. As assessed by Northern blotting and reverse transcription polymerase chain reaction technique, purified human lung mast cells and HMC-1 cells expressed urokinase receptor mRNA in a constitutive manner. Using a toluidine blue/immunofluorescence double staining technique and monoclonal antibodies, surface expression of urokinase receptor was demonstrable in lung, skin, uterus, heart, and tonsil mast cells, whereas the low density lipoprotein receptor-related protein was not detectable. Binding of monoclonal antibody VIM5 (recognizing the urokinase binding domain of urokinase receptor) to HMC-1 could be blocked by high molecular weight but not low molecular weight urokinase. Binding analyses performed with 123I-urokinase revealed expression of 271,000 +/- 55,000 high affinity urokinase binding sites per HMC-1 cell, with a calculated dissociation constant of 1. 29 +/- 0.3 nM. Purified urokinase induced dose-dependent migration of primary mast cells and HMC-1 in a chemotaxis assay without inducing release of histamine. The mast cell agonist stem cell factor also induced migration of HMC-1 and caused up-regulation of expression of urokinase receptor mRNA. Together, our data show that human mast cells express functional receptors for urokinase. Expression of urokinase receptors on mast cells may have implications for mast cell-dependent microvascular processes associated with fibrinolysis, migration, or local tissue repair.
Binding Sites↗
MUC18/MCAM (CD146), an activation antigen of human T lymphocytes.
Extravasation and tissue infiltration of leukocytes and metastatic tumor cells require the regulated expression and function of adhesive and pro-proteolytic surface molecules. We demonstrate here that human T cells, upon activation, neo-express the melanoma metastasis-associated surface molecule MUC18/melanoma cell adhesion molecule (MCAM). Expression of MUC18/MCAM (CD146) on T cells could be identified with two mAbs (541-10B2 and 541-2E5) obtained after immunization with HUT102 T cells and found to react with activated T cells. The specificity of our mAbs for MUC18/MCAM (CD146) was revealed by 1) definition of the appropriate molecular mass of approximately 110 kDa unreduced and 120 kDa reduced, 2) reactivity of mAbs with MUC18/MCAM (CD146) cDNA-transfected mouse L cells, 3) conclusive crosswise immunoblotting experiments with MUC18/MCAM (CD146)-specific mAbs, and 4) N-terminal amino acid sequencing of precipitated protein. In vitro activation by PHA caused neo-expression of MUC18/MCAM (CD146) on peripheral blood T cells within 1 day of stimulation, reaching a maximum on day 3. In vivo expression of MUC18/MCAM (CD146) was confirmed on CD3+ T cells infiltrating delayed-type hypersensitivity lesions of the skin, on synovial fluid T cells of rheumatoid arthritis patients, and on distinct T leukemia cells. MUC18/MCAM (CD146) cell surface expression on activated T cells is mirrored by the presence of specific mRNA. Leukocytes of healthy donors do not show significant MUC18/MCAM (CD146) expression. The finding that MUC18/MCAM (CD146) is also expressed on activated T cells might suggest that this adhesion molecule is involved in the extravasation and/or homing of activated T cells.
Antibodies, Monoclonal↗
TGF-beta 1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis.
Our previous studies demonstrated that TGF-beta 1 is required for efficient in vitro generation of dendritic cells (DC) from CD34+ progenitor cells under serum-free conditions. Here we show that TGF-beta 1 promotes the growth and differentiation of DC primarily by protecting the viability of DC precursors and not by enhancing their proliferative response. Addition of TGF-beta 1 to TNF-alpha, granulocyte-macrophage CSF, and stem cell factor-supplemented cultures had no significant effect on the proportions of cycling cells. Already at 72 h of culture, however, the proportion of apoptotic cells was reduced by more than 60% in the presence of TGF-beta 1. This early protective effect of TGF-beta 1 correlates with the outgrowth of higher numbers and proportions of CD1a+ DC at day 7 of culture. It also correlates with a significantly reduced Fas/APO-1 expression on TGF-beta 1 cultured cells. In contrast, granulomonocytic cells, also arising under these culture conditions, are not affected to such an extent. They are found at equal proportions, both in the presence and absence of TGF-beta 1. The striking DC growth-promoting effect of TGF-beta 1 could only be observed when both TGF-beta 1 and TNF-alpha were present in the cultures. TGF-beta 1, in the absence of TNF-alpha, rather inhibited than enhanced cell expansion. Thus, for optimal in vitro DC development to occur, all four cytokines must obviously act in concert and the combination of TGF-beta 1 with TNF-alpha seems to be particularly critical.
Apoptosis↗
Analysis of the requirement for beta 2-microglobulin for expression and formation of human CD1 antigens.
Human CD1 form a group of nonpolymorphic leukocyte surface molecules with homology to major histocompatibility complex (MHC) proteins. Recent findings in human and in mouse demonstrate the capacity of CD1 molecules to present nonpeptide components like lipids or lipoglycans as well as peptides. We studied the involvement of beta 2-microglobulin (beta 2m) in expression of the classic human CD1 proteins CD1a, CD1b, and CD1c. The beta 2m-deficient human melanoma cell line FO-1 was transiently transfected with either CD1a, CD1b, or CD1c DNA alone, or in combination with beta 2m using the adenovirus-enhanced receptor-mediated transfer infection system. Only co-transfection of FO-1 cells with CD1+ beta 2m resulted in the detection of CD1 Ag by monoclonal antibodies (mAb). This indicated that CD1 mAb recognized determinants are dependent on beta 2m and raised the question whether beta 2m-free forms of CD1 can be expressed. Therefore, to visualize CD1 molecule expression independently of beta 2m, we expressed tagged recombinant forms. A full-length CD1b construct tagged at the very C terminus with a small peptide was transported to the plasma membrane only when beta 2m was co-transfected. beta 2m involvement in the transport of CD1 was confirmed by expression of soluble forms of CD1a, CD1b, and CD1c in three different cell types. Analogous to tagged full-length CD1b, secretion of the soluble CD1 constructs was strictly dependent on beta 2m. The soluble CD1 chimeras were secreted as complexes with endogenous beta 2m. Thus, similar to its role for MHC class I expression, beta 2m is essential for processing and surface transport of the classic human CD1 molecules CD1a, CD1b, and CD1c.
Antibodies, Monoclonal↗
TGF-beta 1 dependent generation of LAG+ dendritic cells from CD34+ progenitors in serum-free medium.
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Antigens, CD34↗
Leukosialin (CD43)-major histocompatibility class I molecule interactions involved in spontaneous T cell conjugate formation.
Resting T cells spontaneously adhere in a selective manner to potent accessory cells, such as dendritic cells (DC) and lymphoblastoid B blasts (LCL). Here we demonstrate that leukosialin (CD43) and major histocompatibility complex class I molecules (MHC-I) might play a critical role in this process. T cell conjugate formation with monocyte-derived DC (md-DC) and LCL could be strongly inhibited by either preincubating T cells with Fab fragments of CD43 monoclonal antibody (mAb) 6F5 or by preincubating md-DC or LCL with MHC-I mAb W6/32. Intact CD43 mAb 6F5, in contrast to monovalent Fab fragments, enhanced T cell adhesiveness by transactivating CD2 binding to CD58 molecules. Interestingly, induction of this proadhesive signal via CD43 with intact 6F5 mAb was found to revert mAb W6/32-mediated inhibition of T cell conjugate formation. These observations indicated that CD43 cross-linkage mimics and monovalent mAb 6F5 inhibits interaction of T cell CD43 with a stimulatory ligand on opposing cells, presumably MHC-I. For the demonstration of direct physical interaction between CD43 on T cells and MHC-I-coated beads it was necessary, however, to ligate CD2 on T cells with a stimulatory pair of CD2 mAbs (VIT13 plus TS2/18). This suggests that CD2 ligation crosswise upregulates CD43 binding avidity for MHC-I and that both adhesion molecule pairs (CD43/MHC-I and CD2/CD58) act in concert to induce and mediate T cell conjugate formation with certain cell types.
Antigen-Presenting Cells↗
Molecular and functional characteristics of dendritic cells generated from highly purified CD14+ peripheral blood monocytes.
Dendritic cells (DC) are the most potent APCs within the immune system. We show here that highly purified CD14(bright) peripheral blood monocytes supplemented with granulocyte-monocyte (GM)-CSF plus IL-4 develop with high efficacy (>95% of input cells) into DC. They neo-expressed CD1a, CD1b, CD1c, CD80, and CD5; they massively up-regulated CD40 (109-fold) and HLA-DQ and DP (125- and 87-fold); and significantly (>5-fold) up-regulated HLA-DR, CD4, CD11b, CD11c, CD43, CD45, CD45R0, CD54, CD58, and CD59. CD14, CD15s, CD64, and CDw65 molecules were down-regulated to background levels, and no major changes were observed for HLA class I, CD11a, CD32, CD33, CD48, CD50, CD86, CDw92, CD93, or CD97. Monocytes cultured in parallel with GM-CSF plus TNF-alpha were more heterogeneous in expression densities but otherwise similar in their surface molecule repertoire. They clearly differed, however, in their accessory cell capacity. Only GM-CSF plus IL-4-cultured cells were found to be potent stimulators in allogeneic and autologous MLR and they presented tetanus toxoid 100- to 1000-fold more efficiently than other cell populations tested. Furthermore, only cytokine-treated monocytes formed clusters with resting T cells. At variance from all these similarities between in vitro-generated monocyte-derived DC and in vivo-developing DC, the DC populations generated by us contained significant amounts of myeloperoxidase and also expressed lysozyme. At least in this respect they, thus, differ from "classical" DC types.
Animals↗
Mac-1 (CD11b/CD18) and the urokinase receptor (CD87) form a functional unit on monocytic cells.
The leukocyte integrin Mac-1 (CD11b/CD18) and the urokinase receptor (uPAR, CD87) mediate complementary functions in myelomonocytic cells. Both receptors promote degradation of fibrin(ogen) and also confer adhesive properties on cells because Mac-1 and uPAR bind fibrin and vitronectin, respectively. Staining of lung biopsy specimens from patients with acute lung injury indicated that fibrin and vitronectin colocalize at exudative sites in which macrophages bearing these receptors accumulate. Because of the parallel roles and physical proximity of Mac-1 and uPAR, the capacity of these receptors to functionally interact was explored. Induction of Mac-1 and uPAR expression on monocytic cell lines by transforming growth factor- beta 1 and 1.25-(OH)2 vitamin D3 conferred urokinase and uPAR-dependent adhesion to vitronectin, which was further promoted by engagement of Mac-1. Vitronectin attachment promoted subsequent Mac-1-mediated fibrinogen degradation threefold to fourfold. In contrast, enhancement of uPAR occupancy by exogenous urokinase or receptor binding fragments thereof inhibited Mac-1 function. Addition of urokinase progressively inhibited Mac-1-mediated fibrinogen binding and degradation (maximal inhibition, 91% +/- 14% and 72% +/- 15%, respectively). Saturation of uPAR with urokinase also inhibited binding of the procoagulant Mac-1 ligand, Factor X. These inhibitory effects of uPAR were reproduced in fresh monocytes, cultured monocytic cells, and in Chinese hamster ovary (CHO) cells transfected with both human Mac-1 and human uPAR. These data show that the procoagulant and fibrinolytic potential of monocytic cells is co-ordinately regulated by ligand binding to both Mac-1 and uPAR and identify uPAR as a regulator of integrin function. Vitronectin-enhanced fibrin(ogen) turnover by Mac-1 may operate as a salvage pathway in the setting of urokinase and plasmin inhibitors to promote clearance of the provisional matrix and subsequent healing.
Animals↗
TGF-beta 1 promotes in vitro development of dendritic cells from CD34+ hemopoietic progenitors.
Several studies have demonstrated that dendritic cells (DC) can be generated in vitro from CD34+ hemopoietic progenitor cells. The growth requirements for these cells are poorly characterized, however. In particular, undefined serum/plasma components seem to significantly contribute to in vitro DC development. We report here that the cytokine combination granulocyte-macrophage CSF (GM-CSF) plus TNF-alpha and stem cell factor (SCF) commonly used for the in vitro generation of DC in serum/plasma-supplemented medium is, in the absence of serum supplementation, very inefficient in inducing DC development. We further demonstrate that supplementation with TGF-beta 1 is required for substantial DC development to occur in the absence of serum. Culture of CD34+ cells under serum-free conditions with TGF-beta 1 plus GM-CSF, TNF-alpha, and SCF strongly induces DC differentiation. This culture condition is even more efficient than culturing CD34+ cells with GM-CSF plus TNF-alpha and SCF in the presence of cord blood plasma. The proportions and total yields of cells with typical DC morphology and CD1a molecule expression are higher. The allostimulatory capacity of DC from TGF-beta 1-supplemented, cultures exceeds allostimulation by cells grown in plasma-containing medium. Substantial numbers (21 +/- 7%) of cells grown in TGF-beta 1-supplemented, but not plasma-supplemented, cultures express the Birbeck granule marker molecule Lag and display numerous Birbeck granules. Cells with distinct monocytic features are less frequently observed in TGF-beta 1-supplemented serum-free cultures. The addition of neutralizing anti-TGF-beta 1 Ab abrogates the observed TGF-beta 1 effects.
Animals↗
Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation.
The two-signal model of T-cell activation postulates that T lymphocytes require at least two distinct signals for activation. This model has been established with bulk cultures of T cells in which T-cell-T-cell interaction can occur, possibly delivering further unrecognized costimulatory signals. The signal requirements of single T cells for the induction of clonal cell growth or the transcription of cytokines would best be studied in a cell cloning system in the absence of feeder cells; however, such an experimental system has not been reported so far. In this study, we report the long-term cloning of human resting peripheral blood CD4+CD45RO- T cells under feeder cell-free conditions in response to CD3 and CD28 stimulation in the presence of exogenous interleukin-2 (IL-2). Cloning efficiency ranged from 40% to 60% depending on the presence of additional cytokines IL-1 and IL-6. Single-call polymerase chain reaction showed that transcription of IL-2 occurred in cells stimulated through CD3 plus CD28 alone. T cells grown in response to CD3 plus CD28 plus IL-2 stimulation produced both IL-4 and interferon-gamma (IFN-gamma) on restimulation (Th0 cells) and could be functionally differentiated into Th1- or Th2-type cells by the addition of IFN-gamma or IL-4, respectively, during cell cloning. These data show on the single-cell level a two-signal model of T- cell activation for the transcription of IL-2. In addition, these experiments show that IFN-gamma and IL-4 exert their T-cell-differentiating effects directly on the T cell without any further need for antigen-presenting cells. Together, our experiments show the feasability of a defined long-term clonal cell culture system to study the growth and differentiation of human T lymphocytes.
Antibodies, Monoclonal↗
Fas- and activation-induced apoptosis are reduced in human T cells preactivated in the presence of TGF-beta 1.
The elimination of activated but not resting T cells involves apoptosis induced either by restimulation via the TCR/CD3 complex, CD2, or by signaling through the Fas Ag. The factors regulating the shift of an apoptosis-resistant to an apoptosis-sensitive phenotype and vice versa have not so far been clarified. Here we report that TGF-beta 1, when present during a PHA activation course, significantly increases viability of human T cells upon reculture in medium alone, following restimulation via CD2, CD3, or after triggering the Fas Ag. Using DNA gel electrophoresis and an in situ nick translation technique we further show that activation-induced and Fas-mediated apoptosis are reduced in T cells that were prestimulated with PHA plus TGF-beta 1, compared with control cells prestimulated with PHA alone. Moreover, when PHA-preactivated T cells are further expanded in IL-2, inclusion of TGF-beta 1 results in higher cell yields at any timepoint from day 30 to 75 of cell culture compared with control cultures without TGF-beta 1. However, no differences in Fas or bcl-2 protein expression are found between cells stimulated in the absence or presence of TGF-beta 1. Together, our data identify TGF-beta 1, when present during an activation course, as an important viability factor possibly of importance for the generation of effector and/or long-lived memory T cells.
Apoptosis↗
CD4 monoclonal antibody VIT4 in human alloimmune response in vitro and in vivo.
In the present report the immunosuppressive effects of the murine anti-human CD4 monoclonal antibody (mAb) VIT4 on human alloimmune response in vitro were analyzed. Moreover, the antibody was tested for its activity to prolong allograft survival in seven patients with steroid-refractory allograft rejection. VIT4 inhibited the proliferative response to alloantigens in the mixed lymphocyte reaction (MLR) in a dose-dependent manner. At concentrations of 1 and 10 micrograms/ml VIT4 blocked MLR by 55 +/- 11% and 77 +/- 1%, respectively. Also alloantigen-specific proliferation of in vitro- generated memory T cells was dose-dependently reduced to 23 +/- 1% at a VIT4 concentration of 100 micrograms/ml. Furthermore, at the same dose level VIT4 blocked proliferation of antigen-specific short-term alloreactive CD4+ cell lines and significantly inhibited the in vitro generation of cytotoxic T lymphocytes (CTL). In a pilot study VIT4 (5 mg/d i.v.) was administered to 7 patients with steroid-refractory allograft rejection for 14 days. In 4 of 7 patients graft function transiently improved and graft survival in all patients was prolonged to a mean of 694 days (range 128-2163) from the beginning of the VIT4 treatment. In the light of our in vitro results and the preliminary clinical data, further clinical trials using higher antibody doses are greatly warranted to assess the efficacy of anti-CD4 mAb VIT4 in the treatment of allograft rejection.
Animals↗
Expression of beta 2-microglobulin-free HLA class I alpha-chains on activated T cells requires internalization of HLA class I heterodimers.
HLA class I molecules on activated T cells are expressed as heterodimers associated with beta 2-microglobulin (beta 2-m) and also beta 2-m-free HLA class I alpha-chains. Mechanisms leading to the expression of the activation associated beta 2-m-free HLA class I alpha-chains are poorly defined, however. Upon enzymatical removal of HLA class I alpha-chains on activated T cells, re-expression is observed within minutes upon reculture, reaching half-maximal levels within 1 hr. This process is independent of de novo protein synthesis and of export of newly synthesized proteins. Inhibition of the formation of coated pits by potassium depletion of cells abrogated the re-expression of HLA class I alpha-chains, suggesting that recycling events of HLA class I heterodimers via endosomal compartments are required for the generation of monoclonal antibody LA45-reactive alpha-chains. Furthermore, the rate of alpha-chain generation seems to be governed by the amount of cell surface-expressed HLA class I heterodimers. Taken together these findings suggest that beta 2-m-free HLA class I alpha-chains are generated during the process of class I heterodimer recycling.
Antibodies, Monoclonal↗