The cell surface molecule recognized by the erythrocyte receptor of T lymphocytes. Identification and partial characterization using a monoclonal antibody

A monoclonal antibody (mAb) to sheep red blood cells (SRBC), termed L180/1, is described that completely blocks rosette formation between SRBC and human or sheep T lymphocytes. L180/1 precipitated a minor glycoprotein of about approximately 42,000 mol wt from surface-labeled SRBC. This glycoprotein was partially affinity purified and found to block E rosette formation and to compete with anti-T11 mAb for the E receptor. The molecule detected by mAb L180/1 thus appears to be recognized by the E receptor and was given the preliminary name, T11 target structure (T11TS). Since the mAb to sheep T11TS blocks the binding of SRBC to both human and sheep T cells, and mAb to T11 blocks the binding of red cells from human and sheep to the human E receptor, we concluded that analogous receptor-ligand (T11-T11TS) systems exist in man and sheep that are crossreactive over the species barrier. The possibility is discussed that the E receptor, which is known to be involved in T cell activation, and T11TS function as complementary cell interaction molecules in T cell responses.     

Activation of T lymphocytes by immobilized monoclonal antibodies to CD3. Regulatory influences of monoclonal antibodies to additional T cell surface determinants

The effect of soluble or immobilized MAb directed at various additional surface proteins on the proliferation of highly purified T4 cells induced by two immobilized MAb to CD3, OKT3 and 64.1, was examined. High density 64.1 stimulated nearly all T4 cells to enter and progress through the cell cycle. Maximal T4 cell proliferation required stimulation with immobilized 64.1 throughout the length of the incubation and was not effected by any of the additional soluble or immobilized MAb employed. In contrast, low density immobilized 64.1 and all densities of immobilized OKT3 employed stimulated a minority of the cells to enter the cell cycle and proliferate. Immobilized MAb directed at CD2, class I major histocompatibility complex (MHC) encoded gene products or CD11a (LFA-1) dramatically enhanced the response, whereas soluble MAb directed at these determinants did not. Both immobilized and soluble MAb directed at CD5 and CD28 (Tp44) enhanced responses, but they were less effective than immobilized MAb to CD2, LFA-1 or HLA-A,B,C. Soluble anti-CD4 MAb inhibited responses somewhat, whereas immobilized anti-CD4 enhanced responses. Costimulation was observed when MAb to CD3 and class I MHC molecules but not CD2, LFA-1 or CD4 were immobilized to separate surfaces. The data suggest that when anti-CD3 stimulation is suboptimal, responses can be enhanced by MAb to CD5 or CD28 (Tp44) or by immobilized MAb to CD4, CD2, CD11a (LFA-1), or class I MHC encoded gene products. Although crosslinking of CD4, CD2, or CD11a with CD3 may be necessary for costimulation, immobilized MAb to CD3 and class I MHC molecules appear to deliver independent signals that result in enhanced T4 cell activation and proliferation.     

Malignant lymphoma with dual B and T cell markers. Analysis of the neoplastic cells with monoclonal antibodies directed against T cell subsets

In the course of analyzing human lymphoma tissue with conventional surface marker techniques and with monoclonal antibodies directed against T cell subsets, five tumors were encountered with dual B and T cell determinants. All bore on their surface membrane IgM of kappa light chain type, complement receptors, and the Ia-like antigen. In each of the five cases, the neoplastic lymphocytes reacted with a monoclonal antibody that detects the sheep erythrocyte receptor (OKT11); all but one reacted with a monoclonal antibody for peripheral T cells (OKT3); and all but one reacted with a monoclonal antibody specific for either the inducer-helper (OKT4) or the cytotoxic- suppressor (OKT8) T cell subsets. In addition, lymphocytes from two of the five cases formed spontaneous rosettes with sheep erythrocytes (E- rosettes). These tumors with dual B and T surface characteristics were confined to human malignant lymphomas that originate from B lymphocytes of the follicle center.     

Activation specificity of arsonate-reactive T cell clones. Structural requirements for hapten recognition and comparison with monoclonal antibodies

We describe clones of hapten-specific inducer T cells from (BALB/c X A/J)F1 mice that respond to the p-azobenzenearsonate hapten conjugated to carrier proteins or directly conjugated to antigen-presenting cells. Some of the clones are also activated by haptens structurally related to arsonate. All activating analogues are recognized by each clone in association with the same major histocompatibility complex (MHC) protein as is arsonate. Weakly activating and nonactivating analogues are immunogenic in D2.GD amd (BALB/c X A/J)F1 mice, since they can effectively activate primed lymph node cells or long-term hapten-reactive cell lines. Hence the specificities of these clones may reflect their intrinsic recognition of arsonate and its analogues, rather than more efficient presentation of certain analogues than of others by antigen-presenting cells, or differential recognition of associated MHC epitopes by the clones. We compare the activation specificities of the clones with the binding specificities of monoclonal antibodies to arsonate, and discuss structural features of the analogues that may be important for activation and binding. Our results suggest that a site (or subsite) on arsonate-reactive T cell clones may interact directly with hapten, and may be experimentally separable from the site (or subsite) for MHC determinants.     

The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody

An antibody-secreting B cell hybridoma, KJ1-26.1, has been prepared from mice immunized with the T cell hybridoma DO-11.10, which recognizes chicken ovalbumin in association with I-Ad (cOVA/I-Ad). KJ1- 26.1 blocks I-restricted antigen recognition by DO-11.10 and a subclone of this T cell hybridoma, DO-11.10.24, which has the same specificity for cOVA/I-Ad as its parent. KJ1-26.1 does not block I-restricted antigen recognition by any other T cell hybridoma tested, including a number of T cell hybridomas closely related to DO-11.10, with similar, but not identical, specificities for antigen/I. Moreover, KJ1-26.1 binds to DO-11.10 and DO-11.10.24, but not to any other T cell hybridomas tested, including three subclones of DO-11.10 that have lost the ability to recognize cOVA/I-Ad. Thus, in every regard KJ1-26.1 appears to be binding to all or part of the receptors for antigen/I on the T cell hybridoma DO-11.10. KJ1-26.1 appears to bind to approximately 15,000 molecules/cell on the surface of DO-11.10. The antibody precipitates an 80,000 dimer from the cells, which on reduction migrates as 40-44,000 monomers. The receptor(s) for antigen/I on DO-11.10 therefore includes molecules with these properties.     

Identification and characterization of a bile acid receptor in isolated liver surface membranes.

It is generally assumed that hepatic transport of bile acids is a carrier-mediated process. However, the basic mechanisms by which these organic anions are translocated across the liver cell surface membrane are not well understood. Since carrier-mediated transport involved binding of the transported molecule to specific receptor sites, we have investigated the possibility that bile acid receptors are present in liver surface membranes. Isolated liver surface membranes were incubated at 4 degrees C with [14C]cholic acid and [14C]taurocholic acid, and membrane-boudn bile acid was separated from free by a rapid ultrafiltration technique through glass-fiber filters. Specific bile acid binding is rapid and reversible and represents approximately 80% of the total bile acid bound to liver surface membranes. Taurocholic acid binding is independent of the medium pH, while cholic acid binding demonstrates an optimum at pH 6.0. Analysis of equilibrium data for both cholic and taurocholic acid binding indicates that specific binding is saturable and consistent with Michaelis-Menten kinetics, while nonspecific binding is nonsaturable. Apparent maximal binding capacity and dissociation constant values indicate a large capacity system of receptors that have an affinity for bile acids comparable to that of the hepatic transport mechanism. Scatchard analysis of the saturation kinetics as well as inhibition studies suggest that bile acids bind to a single and noninteracting class of anion that competes with bile acids for hepatic uptake, also inhibits cholic acid binding. In contrast, no inhibition was demonstrated with indocyanine green and probenecid. Specific bile acid binding is enriched and primarily located in liver surface membranes and found only in tissues involved in bile acid transport. Specific bile acid binding is independnet of Na+, Ca2+, and Mg2+ and does not require metabolic energy. In addition, thiol groups and disulfide are not required for activity at the binding site. However, specific bile acid binding is markedly decreased by low concentrations of proteolytic enzymes and is also decreased by the action of neuraminidase and phospholipases A and C. These results are consistent with the existence of a homogeneous bile acid receptor protein in liver surface membranes. The primary surface membrane location of this receptor, its binding properties, and its ligand specificity suggest that bile acid binding to this receptor may represent the initial interaction in bile acid transport across liver surface membranes.     

Murine monoclonal anti-s and other anti-glycophorin B antibodies resulting from immunizations with a GPB.s peptide

BACKGROUND: The blood group antigens S and s are defined by amino acids Met or Thr at position 29, respectively, on glycophorin B (GPB). Commercial anti-s reagents are expensive to produce because of the scarcity of human anti-s serum. Our aim was to develop hybridoma cell lines that secrete reagent-grade anti-s monoclonal antibodies (MoAbs) to supplement the supply of human anti-s reagents.
STUDY DESIGN AND METHODS: Mice were immunized with the GPB^s peptide sequence TKSTISSQTNGE T GQLVHRF. Hybridomas were produced by fusing mouse splenocytes with mouse myeloma cells (X63.Ag8.653). Screening for antibody production was done on microtiter plates by hemagglutination. Characterization of the MoAbs was done by hemagglutination, immunoblotting, and epitope mapping.
RESULTS: Eight immunoglobulin G MoAbs were identified. Five antibodies are specific by hemagglutination for s and two MoAbs, when diluted, are anti-S–like, but additional analyses shows a broad range of reactivity for GPB. Typing red blood cells (RBCs) for s from 35 donors was concordant with molecular analyses as were tests on RBCs with a positive direct antiglobulin test (DAT) from 15 patients. The anti-s MoAbs are most reactive with peptides containing the ³¹QLVHRF³⁶ motif, with ²⁹Thr. By Pepscan analyses, the anti-S–like MoAbs reacted within the same regions as did anti-s, but independently of ²⁹Met. One antibody was defined serologically as anti-U; however, its epitope was identified as ²¹ISSQT²⁵, a sequence common for both GPA and GPB.
CONCLUSION: In addition to their value as typing reagents, these MoAbs can be used to phenotype RBCs with a positive DAT without pre-test chemical modification.     

T cell clones specific for hybrid I-A molecules. Discrimination with monoclonal anti-I-A (k) antibodies

Alloreactive and soluble antigen-reactive, I-A-restricted T cell clones were examined for their ability to recognize hybrid I-A antigens. Several clones that recognized hybrid I-A(b)/I-A(k) molecules on (C57BL/6 x A/J)F(1) [(B6A)F(1)] spleen cells were studied. We were able to distinguish clones that recognized hybrid I-A molecules of the A(b)(a)A(k)(β) type from those that recognized A(k)(a)A(b)(β) molecules. We reached this conclusion by considering data from three independent types of experiments. (a) Monoclonal antibodies were used to inhibit T cell stimulation. Antibodies 10.2.16 and H116.32 distinguished two mutually exclusive “families” of T cell clones. One group of clones was inhibited by 10-2.16 and not H116.32, the other group exhibited reciprocal inhibition. (b) T cell proliferation was assayed using antigen-presenting cells from B6.C-H-2(bml2) (bml2) and [bml2 × B10.A(4R)]F(1) mice. Because the bml2 strain has a mutation that results in an altered A(b)(β) polypeptide chain (A(bm12)(β)), we reasoned that clones that could recognize the [bm12 × B 10.A(4R)]F(1) cells were recognizing A(b)(a)A(k)(β) molecules. Alternatively, clones not recognizing [bml2 × B10.A(4R)]F(1) cells had specificity for A(k)(a)A(b)(β) molecules. (c) I-A molecules immunoprecipitated from radiolabeled (B6A)F(1) splenocyte extracts were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These experiments confirmed an earlier report that antibody 10.2.16 recognized determinants on the A(k)(β) chain (12). Antibody H116.32 immunoprecipitated products consistent with recognition of A(k)(a) determinants. Taken together, these three types of results offer conclusive evidence that T cell clones recognizing “hybrid” I-A molecules use either A(b(k)A(k)(β) or A(k)(a)A(b)(β) molecules as recognition or restriction sites. Clones whose proliferation was supported by [bm 12 x B10.A(4R)]F(1) cells and blocked by anti-I-A(k) antibody 10-2.16 recognized A(b)(a)A(k)(β) B molecules. Clones that were blocked by antibody H116.32 and did not recognize [bml2 X B10.A(4R)]F(1) cells use a recognition site(s) on A(b)(a)A(k)(β) molecules. Thus, we can demonstrate both functionally and biochemically that hybrid F(1) I-A molecules of the structure A(k)(a)A(b)(β) and A(b)(a)A(k)(β) both exist on (B6A)F(1) splenocytes and that both configurations are used in immune recognition phenomena.     

Identification of a human T lymphocyte surface protein associated with the E-rosette receptor

We describe a new monoclonal murine antibody that reacts with a 50,000- mol wt polypeptide that appears to be present on all E-rosetting cells. We conclude that this antigen is either identical to or closely associated with the E receptor because of (a) the high degree of concordance between E-rosette formation and 9.6 antigen expression, (b) the inhibition of rosette formation by preincubation of cells with 9.6 antibody, and (c) the observed failure of cells lysostripped of 9.6 antigen to form E-rosettes. This last finding suggests cocapping of 9.6 antigen and the E receptor.     

Homologies between T cell receptor junctional sequences unique to multiple sclerosis and T cells mediating experimental allergic encephalomyelitis.

The selection of T cell clones with mutations in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene has been used to isolate T cells reactive to myelin basic protein (MBP) in patients with multiple sclerosis (MS). These T cell clones are activated in vivo, and are not found in healthy individuals. The third complementarity determining regions (CDR3) of the T cell receptor (TCR) alpha and beta chains are the putative contact sites for peptide fragments of MBP bound in the groove of the HLA molecule. The TCR V gene usage and CDR3s of these MBP-reactive hprt-T cell clones are homologous to TCRs from other T cells relevant to MS, including T cells causing experimental allergic encephalomyelitis (EAE) and T cells found in brain lesions and in the cerebrospinal fluid (CSF) of MS patients. In vivo activated MBP-reactive T cells in MS patients may be critical in the pathogenesis of MS.     

Diagnosis of cutaneous T cell lymphoma by use of monoclonal antibodies reactive with tumor-associated antigens.

Two murine monoclonal antibodies (BE1 and BE2), produced by using leukemic helper T cells from a patient with cutaneous T-cell lymphoma (CTCL) as immunogens, reacted selectively with CTCL lymphocytes and some transformed cultured lymphocytes, as determined by radioimmunoassay (RIA) and indirect immunofluorescence (IIF). BE1 reacted significantly (P less than or equal to 0.001) with leukemic CTCL lymphocytes and with CTCL cells from infiltrated lymph nodes (RIA, mean +/- SD = 776 +/- 275 cpm), as compared with background counts (263 +/- 68). BE1 binding to normal blood mononuclear cells (RIA, mean +/- SD = 283 +/- 58 cpm) was indistinguishable from background. BE1 also reacted with Epstein-Barr virus (EBV)-transformed B-cell lines (RIA, mean +/- SD = 794 +/- 230) and some long-term T-cell lines. BE1 did not react with the majority of lymphoid cell lines or tumor cell lines tested. BE1 also did not react with any normal tissues screened by IIF. BE1 precipitated a molecule from CTCL cells that, under reducing conditions, has two components with molecular mass of 27,200 and 25,800 D. BE2 also reacted significantly (P less than or equal to 0.001) with CTCL cells from two of four patients (RIA, mean +/- SD = 519 +/- 113 cpm). The binding of BE2 to normal mononuclear cells was indistinguishable from background (309 +/- 38 cpm). BE2 also reacted with an antigen present on EBV-B-cell lines (RIA, mean +/- SD = 654 +/- 194) and MOLT 3 and HUT 78 T-cell lines. BE2 reacted with an antigen expressed on a subpopulation of lymphocytes from five of eight patients with B-cell CLL studied by IIF (mean +/- SD = 18 +/- 6). Other long-term T-cell lines and tumor cell lines studied by IIF were unreactive with BE2. BE2 did not react with any of the normal tissues studied. BE2 precipitated a molecule (78,000 D) from CTCL cells and EBV-B cells with a single component under reducing conditions. Immunoperoxidase-labeled BE1 and BE2 reacted with CTCL cells in frozen sections of infiltrated lymph nodes and skin. In addition, BE1 and BE2 reacted with blood lymphocytes from 16 of 21 patients whose CTCL had otherwise been considered localized to skin. These two monoclonal antibodies react with tumor antigens associated with CTCL and appear to be useful in the diagnosis of this disorder.     

Distinct molecular forms of human T cell receptor gamma/delta detected on viable T cells by a monoclonal antibody

A second type of TCR molecule has been identified on human and murine T lymphocytes, which involves the protein products of the gamma and delta genes. T lymphocytes bearing this receptor may constitute a separate cell lineage with a distinct immune function. We have produced an mAb, which specifically detects human TCR-gamma/delta in native as well as denatured states, this in contrast to previously used anti-gamma chain peptide sera, which only reacted with denatured protein. The receptor occurs in different molecular forms, with or without interchain disulphide bonds, in which a delta chain may or may not be detected by cell surface iodination. The mAb is reactive with all these receptor forms. Therefore, this antibody could be used to determine the expression of TCR-gamma/delta on viable human T lymphocytes. In normal individuals, TCR-gamma/delta was found on a subset composing 2-7% of CD3+ lymphocytes in peripheral blood and 0.1-1.0% in thymus. The majority of these cells do not express the CD4 or CD8 antigens, although a significant percentage of CD8+ cells was found. TCR-gamma/delta+ cells in peripheral blood are resting lymphocytes, as judged by ultrastructural analysis. T cell clones with different receptor types can display MHC-nonrestricted cytolytic activity, which is shown to be induced by the culture conditions, most likely by growth factors such as IL-2. This strongly suggests that TCR-gamma/delta does not play a role in target cell recognition in MHC-nonrestricted cytotoxicity. The anti-TCR-gamma/delta antibody can specifically induce cytotoxic activity in clones expressing the receptor, but in addition inhibit growth factor induced cytotoxicity, which indicates a regulatory role of the TCR-gamma/delta/CD3 complex in MHC-nonrestricted cytotoxicity.     

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. Delivery of ribosome-inactivating proteins to spleen and lymph node T cells.

The selective delivery in vivo of a T lymphocyte-specific monoclonal antibody and immunotoxin conjugates to T cells in lymph node and spleen was assessed in rhesus monkeys. A transient coating of all T lymphocytes in the lymph nodes and spleens of healthy rhesus monkeys could be achieved after infusion of unconjugated anti-T11. Because derivatized antibody is cleared more rapidly than unconjugated antibody, it was necessary to infuse a higher dose of immunotoxin than antibody alone to achieve saturation of the lymphocyte binding sites with anti-T11. When sufficient antibody-toxin conjugate was infused, toxin was readily demonstrable on lymph node and spleen T cells by 16 h after infusion. This demonstration that toxins can be successfully delivered with specificity to target T cell populations in the monkey suggests that killing of restricted cell populations in vivo should be feasible.     

Murine Peyer''s patch T cell clones. Characterization of antigen- specific helper T cells for immunoglobulin A responses

We successfully cloned antigen-specific T cells from murine gut-associated lymphoreticular tissue, i.e., Peyer's patches, which are dependent upon T cell growth factor and independent of antigen for continuous growth. These clones exhibit helper activity for IgA responses to sheep erythrocytes (SRBC) and have been designated T helper (Th) A. Two broad categories of Th A clones have been maintained in continuous culture. The first group supports IgM and largely IgA anti-SRBC plaque-forming cell (PFC) responses in both normal and SRBC-primed splenic B cell cultures, whereas the second group supports low IgM, IgG1, and IgG2 and high IgA PFC responses. Subclones derived from single cells maintain the parent helper properties when propagated in culture for long periods (greater than 7 mo). Cloned Th A cells are antigen specific and do not support polyclonal or immune responses to other thymus dependent antigens in normal B cell cultures. Th A cells require full histocompatibility for helper functions because addition of cloned Th A cells to B cell cultures from other H-2 types does not result in IgA responses. Cloned Th A cells are Thy-1.2+ and Lyt-1+ and Lyt-2-, Ig-, and I-A-. Th A cells bear Fc receptors for IgA and do not possess receptors for IgM or IgG isotypes. Thus, T cells that primarily promote IgA isotype responses have been isolated in high frequency from murine PP, an anatomical site of major importance for induction and regulation of the IgA response.     

Expansion and characterization of T cells transduced with a chimeric receptor against ovarian cancer

Adoptive immunotherapy with genetically modified T lymphocytes is being utilized in clinical trials for the treatment of a broad range of diseases including cancer and HIV infection. To improve on these treatments, and to better understand their mechanisms of action, it is necessary to develop techniques to generate large numbers of cells and characterize the functional heterogeneity of the cells produced. In this study, patient peripheral blood lymphocytes were transduced with a chimeric antigen receptor (MOv-gamma) derived from a mouse monoclonal antibody against folate-binding protein, which is overexpressed on many ovarian cancers. Thus, irrespective of their original specificity, normal human T lymphocytes were redirected to react against ovarian cancer cells. Lymphocytes from five patients were transduced and grown to large numbers, with a median expansion of more than 7000-fold. When proliferation was inadequate, the cells were expanded by stimulation utilizing anti-CD3, IL-2, and irradiated allogeneic PBMCs. The cells maintained their functional ability to recognize ovarian cancer over several months. Cloning of transduced cells was undertaken to determine the level of gene expression and function of individual cells making up the bulk population. Transduced CD4(+) and CD8(+) cell clones were isolated from the bulk and demonstrated antitumor activity. These clones had a diverse repertoire with respect to secretion of cytokines, and individual clones maintained their cytokine profile on subsequent expansion. These studies establish the feasibility of consistently generating large numbers of gene-modified tumor-reactive lymphocytes, with a stable and diverse cytokine repertoire, that could be utilized for patient treatment.     

Anti-interleukin 2 receptor monoclonal antibodies spare phenotypically distinct T suppressor cells in vivo and exert synergistic biological effects

The therapeutic efficacies of ART-18, ART-65, and OX-39, mouse antibodies of IgG1 isotype recognizing distinct epitopes of the p55 beta chain of the rat IL-2-R molecule, were probed in LEW rat recipients of (LEW X BN)F1 heterotopic cardiac allografts (acute rejection in untreated hosts occurs within 8 d). A 10-d course with ART-18 prolongs graft survival to approximately 21 d (p less than 0.001). Therapy with ART-65, but not with OX-39, was effective (graft survival approximately 16 and 8 d, respectively). Anti-IL-2-R mAb treatment selectively spared T cells with donor-specific suppressor functions; the CD8+ (OX8+ W3/25-) fraction from ART-18-modified recipients, and primarily the CD4+ (W3/25+ OX8-) subset from ART-65-treated hosts conferred unresponsiveness to naive syngeneic rats after adoptive transfer, increasing test graft survival to approximately 16 and 45 d, respectively. Concomitant administration of ART-18 and ART-65 to recipient animals in relatively low doses exerted a strikingly synergistic effect, with 30% of the transplants surviving indefinitely and 50% undergoing late rejection over 50 d. These studies provide evidence that anti-IL-2-R mAbs selectively spare phenotypically distinct T cells with suppressor functions. The data also suggest that in vivo targeting of functionally different IL-2-R epitopes may produce synergistic biological effects.     

T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.     

Monoclonal antibodies with specificity for hairy cell leukemia cells.

Hairy cell leukemia is a well described clinical entity, but the cell of origin for this leukemic cell and its function are still unknown. There are no totally specific markers for this cell, although tartrate-resistant acid phosphatase staining has been used extensively as a diagnostic test. This study describes three monoclonal murine antibodies with variable specificity for hairy cells. Antibody 1 was highly specific for hairy cells and was not found to react with normal or leukemic cells in this limited study. It did not react with the cells of all patients. It also did not react with all of the hairy cells of some of the positive cases. Antibodies 2 and 3 reacted with virtually all hairy cells but not with normal peripheral blood cells. However, reactions were obtained with certain leukemic myelomonoblasts and some activated B cells. The most obvious use for these three antibodies is for diagnostic purposes. They should also be helpful reagents to investigate the origin of the leukemic hairy cell. The possibility that antibody 1 detects a tumor-specific antigen is discussed.     

Development, characterization, and use of monoclonal antibodies made to antigens expressed on the surface of fetal nucleated red blood cells.

BACKGROUND: Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small (0.5-1.0%) but definite risk of miscarriage. An attractive alternative would be isolation of fetal cells from peripheral maternal blood using antibodies with high specificity and avidity. METHODS: To generate antibodies, we purified nucleated red blood cells (NRBCs) from fetal livers and used them as the immunogen to generate monoclonal antibodies (mAbs) directed against surface antigens. RESULTS: The four antibodies recognized at least two conformationally sensitive epitopes of the transferrin receptor. Isolation of NRBCs from 252 maternal blood samples using these antibodies in magnetic activated cell sorting after an initial density gradient centrifugation yielded 0-419 NRBCs per 25 mL of maternal blood. One antibody, 2B7.4, not only isolated the highest number of NRBCs (>10 in 90% of the samples) but also isolated these NRBCs in 78 consecutive maternal samples. CONCLUSION: Antibody 2B7.4 shows promise for the isolation of NRBCs from maternal blood and should allow studies concerning the source of these cells, fetal vs maternal, and the factors controlling their prevalence.     

Triggering a second T cell receptor on diabetogenic T cells can prevent induction of diabetes.

In this paper, we test the hypothesis that triggering of a second T cell receptor (TCR) expressed on diabetogenic T cells might initiate the onset of diabetes. A cross between two TCR-transgenic strains, the BDC2.5 strain that carries diabetogenic TCRs and the A18 strain that carries receptors specific for C5, was set up to monitor development of diabetes after activation through the C5 TCR. F1 BDC2. 5 x A18 mice developed diabetes spontaneously beyond 3-4 mo of age. Although their T cells express both TCRs constitutively, the A18 receptor is expressed at extremely low levels. In vitro activation of dual TCR T cells followed by adoptive transfer into neonatal or adult F1 mice resulted in diabetes onset and death within 10 d after transfer. In contrast, in vivo immunization of F1 mice with different forms of C5 antigen not only failed to induce diabetes but protected mice from the spontaneous onset of diabetes. We propose that antigenic stimulation of cells with low levels of TCR produces signals inadequate for full activation, resulting instead in anergy.