New differentiation antigens associated with the growth and maturation of B lymphocytes

Monoclonal antibodies (mAb) NDA3 and NDA4 were generated by hyperimmunizing mice with an alloreactive human T cell clone and fusing the splenocytes with the NS1 myeloma. Immunofluorescence studies indicated that these mAb react with activated, but not with resting T and B lymphocytes or with other types of cells. Immunoprecipitation studies with 125-I-labeled extracts from T and B cell lines demonstrated that the m.w. of the antigen recognized by mAb NDA3 is 36,000 and that of NDA4 is 46,000. Studies on the effect of mAb NDA3 and NDA4 on Epstein Barr Virus-transformed lymphoblastoid B cell lines (LBCL) demonstrated that these antibodies stimulate B cell proliferation and Ig synthesis. The level of expression of NDA3 and NDA4 on the membrane of LBCL is significantly augmented when cells are grown in the presence of recombinant human interferon-beta and gamma and is decreased in the presence of 12-O-tetradecanoyl-phorbol-13-acetate. These observations, in conjunction with the stimulatory effect of mAb NDA3 and NDA4 on the growth and differentiation of LBCL, suggest that these new differentiation antigens, NDA3 and NDA4, may serve as growth factor receptors.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. II. Requisite role of the monoclonal antibody-defined antigen systems in activation and proliferation of human and rat lymphocytes

A murine monoclonal antibody (MoAb) B3 to rat cells and MoAb HBJ127 and HBJ98 to human cells were found previously to recognize the homologous antigen systems (gp130 in the rat and gp125 in the human) which are predominantly distributed on the cell surface of proliferating cells of the respective species, and the expression of the antigen systems in lymphocytes were indicated previously to correlate closely with the activation and proliferation of the lymphocytes. In this respect, the in vitro effects of these MoAb on the nucleic acid synthesis, cell cycles, or proliferation of stimulated rat and human lymphocytes were examined by use of T cell-enriched and B cell-enriched cell populations. The addition of B3 MoAb to cultures diminished Con A-induced or allogeneic mixed lymphocyte culture-induced rat T cell proliferation and lipopolysaccharide-induced rat B cell proliferation, whereas B31 MoAb, which is unreactive with the gp130 antigen, did not inhibit these lymphocyte responses. Similarly, both HBJ127 and HBJ98 MoAb could inhibit the human lymphocyte proliferation in vitro, although HBJ127 MoAb showed about eight times greater inhibitory activity than did HBJ98 MoAb; HBJ127 MoAb almost completely inhibited the DNA synthesis of the Con A-stimulated lymphocytes at concentrations higher than 13 micrograms/ml. The flow cytometric analysis of the cellular nucleic acid contents with acridine orange-stained cells showed that when B3 MoAb and Con A were simultaneously added to unstimulated rat T cells, progression of the cell cycle was blocked at the G0 to G1 transition. In this culture condition, the appearance of the B3-defined antigen was arrested in a moderate level, as determined with fluorescein-stained cells. On the addition of B3 MoAb to the culture of the T cells after 24-hr Con A stimulation, the MoAb also strongly inhibited the cellular DNA synthesis, but it did not arrest the cell cycle at a certain phase and did not modulate the corresponding antigen. These data suggest that the B3 MoAb-defined antigen on the rat lymphocytes and the HBJ127/HBJ98 MoAb-defined antigen on the human lymphocytes may play some requisite roles not only in lymphocyte activation but also in the subsequent progression through the cell cycle to proliferate.     

HLA-DR antigens and mitogenic factors released by PWM-stimulated T lymphocytes share the framework determinant recognized by the monoclonal antibody Q5/13

Human T lymphocytes release factors which enhance the mitogenic response of B lymphocytes to PWM. These mitogenic factors share with HLA-DR antigens the framework determinant recognized by the monoclonal antibody Q5/13 (MoAb Q5/13) since adsorption of T-cell medium with an excess of insolubilized MoAb Q5/13 significantly reduces the enhancing activity of the T-cell supernatant on the proliferative response of B cells to PWM. On the other hand, incubation of the T-cell supernatant with an excess of insolubilized anti-HLA-A,B MoAb Q1/28 did not effect the activity of the T-cell supernatant in the proliferative assay.     

Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.     

抗人表皮生长因子受体单克隆抗体的制备、鉴定及初步应用

 用人上皮癌细胞系A 431细胞作为抗原免疫BalB/c小鼠,制备七株抗人表皮生长因子受体的单克隆抗体的杂交瘤,这些杂交瘤经三次亚克隆后仍能稳定地分泌单克隆抗体。对其中四株杂交瘤分泌的单克隆抗体进行了鉴定。免疫沉淀放射自显影结果示单克隆抗体3、101和176均可识别A 431细胞膜抗原MW为170000的蛋白质即EGF受体。单克隆抗体59可以识别低分化鼻咽癌细胞膜上EGF受体。单抗3、176和59等可抑制EGF与受体的特异结合,而101和94则不能抑制EGF与受体的结合。 用Protein-A Sepharose CL4B纯化了单抗,纯化的单抗主要为IgG_1亚类。用SDS聚丙烯酰胺凝胶电泳对纯化的单抗进行了纯度测定。     

Alterations in the human immune response to the hepatitis B vaccine among the elderly

The specific binding of hepatitis B (HBs) antigen by lymphocytes from old people immunized with hepatitis B vaccine was explored. For that purpose HBs antigen was combined with fluorescent microspheres, and labeled antigen was allowed to react with lymphocytes from HBs vaccine-responsive or unresponsive people. Lymphocytes from 10 responders and 14 nonresponders were tested for their antigen-binding ability. For controls, lymphocytes were incubated with microspheres bearing human albumin. Lymphocytes from 8 out of 10 responders were able to recognize HBs antigen; for the nonresponders the ratio was 9 out of 14. HBs-binding lymphocytes were B cells but not T lymphocytes. B and T cells from responders and nonresponders were combined and cultivated for 8 days in the presence of HBs antigen, and antibody-producing cells were counted. Neither B cells alone nor B cells plus T cells from nonresponders were able to produce antibody. On the other hand B cells from unresponsive old people produced antibodies when they were cultivated in the presence of HBs antigen and T cells from responsive old people. These data suggest that some elderly individuals who do not produce antibody after in vivo immunization by HBs vaccine do have antibody-producing cells. Instead of a gap in their immune repertoire, these people are suffering from immune dysfunction.     

Biosynthesis and partial amino acid sequence of the human NDA4 antigen. An activation antigen common to B and T cell lineages

NDA4, a cell surface protein of molecular mass 46 kDa common to activated peripheral blood B and T cells, plays a unique role in the control of B and T cell maturation. NDA4 inhibits B and T cell activation, as mitogen-stimulated B and T cell blastogenic responses are decreased in the presence of mAb NDA4, the antibody recognizing NDA4. After mitogen-activation, however, the regulatory function of NDA4 changes. Addition of mAb NDA4 to cultures of Staphylococcus aureus Cowan strain A-activated B cells or alloreactive T cell clones stimulates their proliferation. NDA4 epitopes are conserved across primate species lines and are present on transformed cells of neuroectodermal origin. NDA4 is synthesized as a molecular mass 50 kDa precursor and is processed to a mature 46 kDa form within 30 min. The NDA4 Ag also exists as soluble forms of 40 and 42 kDa. The membrane and soluble forms of NDA4 have been purified to homogeneity and sequenced by N-terminal Edman degradation.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Distinct and non-cross-reactive epitopes are recognized on B16 melanoma by LAK cells and anti-B16 monoclonal antibodies.

Two rat anti-B16 melanoma monoclonal antibodies (MoAb), designated IB16-6 and IB16-8, recognize an epitope expressed with high density on the surface of B16 parental cells and B16-F1, F10, F10FLR, and BL6 sublines. The purpose of this study was to define by means of cytolytic and clonogenic assays whether these MoAbs reacted with the same or distinct determinants as those recognized on B16 targets by lymphokine-activated killer (LAK) cells. Using 125I-labeled antibody and Scatchard analysis, the affinity constant (KA) of IB16-6 was determined to range from 5.6 to 9.4 x 10(8) liter/M and the number of receptor sites per B16 cell was 4.8 x 10(4) to 2.5 x 10(5). The effects of anti-B16 MoAb on LAK activity were determined by either preincubating 51Cr-labeled B16 target cells with varying concentrations of MoAb, followed by the cytolytic assay, or exposing unlabeled B16 cells to MoAb, and then carrying out a 10-day clonogenic assay. Over a wide range of antibody concentrations, IB16-6 and IB16-8 had minimal effects on LAK activity, and even at MoAb concentrations up to 1 mg there were no changes in target cell sensitivity or colony-forming ability. Enzymatic treatment of B16 melanoma cells with either trypsin or pronase completely removed the epitope recognized by MoAb IB16-6 but did not alter B16 sensitivity to LAK cells. These observations indicate that the LAK recognition unit was distinct from the epitope reactive with MoAb IB16-6 and that the B16 determinant(s) recognized by LAK cells is resistant to proteolytic enzymes. The molecular structure of each of these remains to be determined.     

Malignant human B cells express two populations of p24 surface antigens

Monoclonal antibodies (MoAb) to a leukemia-associated p24 cell surface antigen are currently being used to purge bone marrow of malignant cells before autologous transplantation for acute lymphoblastic leukemia (ALL). Their use as potential diagnostic reagents for hematologic disorders is also under investigation. It has been assumed throughout these investigations that the p24-specific MoAb produced by different laboratories all identify the same antigen. Our present studies indicate that at least two populations of p24 antigens, having different chemical properties and cellular distributions, exist on malignant B cells. For example, eight MoAb raised to ALL cells (ALL-MoAb) identify a p24 antigen on these cells but do not react with the Burkitt's lymphoma cell line Ramos. In contrast, six MoAb raised to Ramos (Ramos-MoAb) identify a p24 antigen on both Ramos and ALL cells. Quantitative binding of both sets of MoAb to ALL cells is comparable. The ALL-MoAb react with platelets, granulocytes, and activated but not resting T lymphocytes, whereas the Ramos-MoAb react with both resting and activated T lymphocytes but not with platelets or granulocytes. The ALL-MoAb react with 11 of 34 human hematopoietic cell lines tested; the Ramos-MoAb react with all 34. Both sets of MoAb react with most of the nonhematopoietic human cell lines tested. Reciprocal exhaustive radioimmune precipitation experiments performed with an ALL cell line indicate that the antigenic determinants recognized by these two sets of MoAb are present on different molecules. Similarly, proteolytic digests of iodinated antigens identified by these two sets of MoAb on ALL cells confirm the unique chemical identities of these molecules and suggest that they reflect the products of different genetic loci. The presence of the antigen identified by the Ramos-MoAb on every cell population tested except granulocytes suggests that it may serve an important cellular function. The existence of two populations of p24 antigens on at least some hematopoietic cells indicates the need for caution when comparing the results of studies of these antigens by groups employing different MoAb.     

A novel monoclonal antibody, 1B3.1, binds to a new epitope of the VLA-1 molecule

A monoclonal antibody, 1B3.1, was raised against a cloned IL-2-dependent T cell line that expresses the T gamma delta T cell receptor. MoAb 1B3.1 reacted with long-term cultured T cell lines of both T gamma delta and T alpha beta lineage, and with in vivo-stimulated T cells, derived from synovial fluid, but not with resting or short-term activated T cells, B cells, or macrophages. Immunoprecipitation of the 1B3.1 target antigens showed that 1B3.1 recognizes a 200/110 kDa molecule that is identical to the VLA-1 heterodimer precipitated by MoAb TS2/7. 1B3.1, however, binds to an epitope of VLA-1 that is distinct from the TS2/7 binding site. This new MoAb could be useful in further studies of the functions of VLA-1, and of the cells that express this molecule.     

Expression of 18.6/CD23 Antigen on Human Lymphoid Progenitor Cell Lines and Phorbol 12-Myristate 13-Acetate (PMA)-Induced Microglia-Shaped Cells

The nature of lymphoid progenitors and factor(s) determining commitment to either the T- or B-lymphocyte pathway are poorly understood in the human system. In this study, we generated a monoclonal antibody (MoAb), 18.6, that recognizes a cell surface antigen on a human lymphoid progenitor cell line (FL4.4). MoAb 18.6 reacted with lymphoid progenitor lines, B lymphoid cell lines, and myelomonocytic cell lines. It did not react with any T cell or erythroid leukemic cell lines. Two color FACS analyses of normal lymphoid tissues showed that MoAb 18.6 reacted with a majority of CD20⁺ mature B cells and a minority of CD64⁺ monocytes. Molecules of 3 different sizes with MW of 34, 45, and 68 Kd were precipitated with MoAb 18.6 from the lymphoid progenitor cell line. The 18.6 antigen was not expressed on a fetal liver-derived lymphoid progenitor-like cell line, FL1.4, which has the capacity to differentiate into microglia-shaped cells upon PMA-stimulation. Stimulation of FL1.4 cells with PMA induced expression of the 18.6 antigen within 24 hr and the microglia-shaped cells stained positively with MoAb 18.6. Finally, cloning of a cDNA that encoded the 18.6 antigen revealed that the 18.6 antigen is identical to the CD23 antigen. Taken together, these data suggest that the 18.6/CD23 antigen is expressed on lymphoid precursors at a very early stage of differentiation.     

Polyclonal activation of murine B lymphocytes by Fc fragments. I. The requirement for two signals in the generation of the polyclonal antibody response induced by Fc fragments

Fc fragments derived from human IgG1 induce murine splenic B lymphocytes to undergo proliferation and differentiation to antibody-secreting cells. The polyclonal antibody response was found to require both the presence of macrophages and T cells. Spleen cell cultures from nude mice or T cell-depleted normal mice proliferate to the level of untreated control mice but do not produce polyclonal antibody unless T cells are added. Regulation of the Fc fragment induced B cell differentiation to antibody synthesis apparently occurs through two distinct signals. One signal is provided by Fc fragments for proliferation and the other by T cells for differentiation. This suggestion is supported by the observation that spleen cell preparations, devoid of T cells, are capable of proliferation to the level of normal spleen cell cultures in response to Fc fragments, but are incapable of making a polyclonal antibody response. The cell population that responds to the differentiation signal also responds to the proliferative signal. "Hot pulse" experiments demonstrated that proliferation precedes polyclonal activation.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.     

Inhibition of lymphocyte proliferation by antibodies to prolactin

Recent in vivo studies have shown that treatments that decrease circulating prolactin (PRL) in rodents result in significant immunosuppression. Our attempts to demonstrate corresponding direct stimulatory effects of PRL on cultured lymphocytes were unsuccessful. However, antibodies against pituitary PRL potently inhibited both murine and human lymphocyte proliferation in response to both T and B cell mitogens. Further studies using IL 2 and IL 4 responsive cell lines (CTLL-2 and HT-2) demonstrated that the same anti-PRL antibodies inhibited the proliferative response to these cytokine growth factors. Thus, antibodies to PRL appear to block an event occurring in the G1 to GS phase transition of these cell lines, which constitutively express growth factor receptors. The inhibitory activity of anti-PRL antibodies could be adsorbed by addition of purified human PRL or by immobilized PRL on an affinity column. Antibodies to other pituitary hormones were without inhibitory effect on CTLL-2 cell proliferation. Proliferation of lymphocytes in serum-free medium was also potently inhibited by anti-PRL antibodies, suggesting that antibody effects were not due to neutralization of PRL or other factors contained in culture serum supplements. We suggest from these data that a protein with homology to PRL and recognized by these anti-PRL antibodies is produced by lymphocytes and plays a critical role in their progression through the cell cycle.     

Activation of human B lymphocytes. IV. Regulatory effects of corticosteroids on the triggering signal in the plaque-forming cell response of human peripheral blood B lymphocytes to polyclonal activation.

In vitro hydrocortisone in physiologic and pharmacologically attainable concentrations caused a marked enhancement of the PWM-induced PFC response of normal human peripheral blood B lymphocytes. This effect was seen only when hydrocortisone was added within the first 24 hr of culture and only when hydrocortisone and PWM were present together in cultures. Only suprapharmacologic concentrations of hydrocortisone (10(-3) M) were capable of suppressing early B cell activation. Late stages of antibody production and secretion were resistant to suppression by even these extraordinarily high concentrations. Hydrocortisone did not replace the T cell requirement of PWM-induced PFC responses. A single dose of in vivo hydrocortisone (400 mg) to normal adult volunteers did not produce this enhancing effect when PFC responses were measured in vitro in the absence of hydrocortisone. The data strongly suggest that the enhancing effect of hydrocortisone was due not to elimination of naturally occurring suppressor cells, but to a modulation of the triggering signal either directly on the B cell itself or via the balance of positive and negative T cell regulation of B cell activation.     

LAK1: a novel leucocyte differentiation antigen shared by lymphoid and endothelial cells

Lymphokine activated killer (LAK) cells have been utilized as a useful tool in cancer adoptive immunotherapy. The lineage origin of this population has always been controversial since it shares phenotypic markers with both myelomonocytic cells and T lymphocytes. Recently we described a new monoclonal antibody (MoAb), termed LAK1, which recognizes a 120 Kd surface molecule expressed on human large granular lymphocytes (LGL) and LAK precursors and effectors. LAK1 MoAb defines two different populations of positive cells amongst peripheral lymphocytes: the first subset (20%), represented by brightly stained cells, belongs to the non T-LGL population, whereas the second subset (30%) displays low fluorescence intensity and was partially composed of T lymphocytes. More interestingly, LAK1 is shared by some other cell types, such as monocytes and vascular endothelial cells. Immunohistochemical staining performed on muscle, endometrium and lymphoid or other non lymphoid tissues shows that LAK1 antigen is selectively expressed by the reticuloendothelial system.     

Monoclonal antibodies to the human C3b/C4b receptor (CR1) enhance specific B cell differentiation

The addition of monoclonal antibodies against the human C3b/C4b receptor (CR1) to cultures of peripheral blood lymphocytes in the presence of suboptimal amounts of TNP bound to polyacrylamide beads enhanced by 150 to 400% the specific anti-TNP response, as measured by a plaque-forming cell assay on day 7. Anti-CR1 antibodies similarly enhanced the anti-fluorescein antibody response. Enhancement only occurred in cultures performed in the presence of the relevant antigen. No enhancing effect on the anti-TNP response was observed on addition to cultures of monoclonal antibodies directed against other surface antigens of B cells or an anti-T cell antibody of the same subclass as that of anti-CR1 antibodies. Anti-CR1 antibodies alone did not induce nonspecific B cell proliferation and did not provide B cells with a first signal for proliferation in the presence of a source of B cell growth factors. Anti-CR1 antibodies did not enhance the nonspecific proliferative response of B cells to growth factors derived from PHA-stimulated T cells, semi-purified BCGF 20 KD, BCGF 50 KD, or recombinant IL 2 in the presence of anti-mu. In this respect, the effect of anti-CR1 antibodies differs from that of anti-CR2 antibodies which interact with early stages of B cell activation. In contrast, anti-CR1 antibodies enhanced specific differentiation of antigen-activated B cells in the absence of T cells when soluble T cell factors were provided. Similar results were obtained by using either of two sources of differentiation factors, the MLA-144 supernatant or a 30 to 15 KD fraction from PHA-stimulated T cells. These results indicate that triggering of CR1 on B cells positively regulates the specific antibody response to low doses of antigen by enhancing B cell differentiation whether T cell help is provided by intact T cells or by T cell-derived differentiation factors.