Heterogeneity of lymphocyte calcium metabolism is caused by T cell-specific calcium-sensitive potassium channel and sensitivity of the calcium ATPase pump to membrane potential.

Calcium management differs in T and B lymphocytes. [Ca2+]i elevation in response to calcium ionophores is up to 10 times greater in T cells than B cells. There is no difference between them in ionophore uptake. T cells, but not B cells, possess a calcium-sensitive potassium channel which produces membrane hyperpolarization at [Ca2+]i above 200 nM. This alters T cell density providing a rapid and easy method of cell separation. In contrast, B cells depolarize when [Ca2+]i is increased. Isolated B cell membrane vesicle ATP-dependent calcium pump activity is higher than T cell vesicles. Membrane depolarization reduces the [Ca2+]i response to ionomycin, most dramatically in T cells because they are hyperpolarized by increased [Ca2+]i. The most likely basis of this behavior is an effect of membrane potential on lymphocyte membrane calcium pump activity. This mechanism provides an explanation of the inhibitory effect of membrane depolarization on T lymphocyte responses.

Early T lymphocytes. Differentiation in vivo of adult intrathymic precursor cells.

A minor subpopulation of adult murine thymocytes (less than 5%) that is Lyt-2-, L3T4-, and expresses low levels of Ly-1 (designated dLy-1 [dull] thymocytes) has been identified, isolated, and characterized. This study assesses the differentiation potential of dLy-1 thymocytes in the thymus in vivo. Using multiparameter flow cytometry, radiation chimeras of C57BL/6 mice congenic at the Ly-1 or Ly-5 locus, and allelic markers to discriminate host and donor, we showed that transferred dLy-1 cells were able to generate thymocytes expressing both cortical and medullary phenotypes in a sequential manner. The proportion of donor-derived thymocytes obtained was directly related to the number of dLy-1 thymocytes transferred. Transfer of purified Lyt-2+ or Lyt-2+ + L3T4+ thymocytes, which constitute greater than 94% of total thymocytes, failed to generate any donor-derived thymocytes in irradiated recipients. Transfer of bone marrow (BM) cells produced the same sequential pattern of differentiation as that produced by dLy-1 cells, but was delayed by 4-5 d. Transferred dLy-1 thymocytes exhibited a limited capacity for self-renewal, and resulted in a single wave of differentiation in irradiated hosts. Thus, thymic repopulation by donor-derived cells after transfer of dLy-1 thymocytes was transient, while repopulation by BM was permanent. These findings suggest that the isolated dLy-1 thymocytes described herein are precursor thymocytes that represent a very early stage in intrathymic development.

T cell receptor-mediated recognition of self-ligand induces signaling in immature thymocytes before negative selection.

Shaping of the T cell repertoire by selection during intrathymic maturation involves T cell receptor (TCR) recognition of major histocompatibility complex/self-antigen complexes. In this communication, we studied the ability of minor lymphocyte stimulating (Mls) determinants to act as self-tolerogens in the selection of the T cell repertoire. We demonstrate that unprimed T cells from normal as well as TCR transgenic mice form Mls-specific conjugates with antigen-presenting cells, and that this TCR-ligand interaction leads to elevation of intercellular Ca2+ ([Ca2+]i). Peripheral T cells from TCR transgenic mice expressing receptors specific for self-Mls antigen show no reactivities to Mlsa. However, a proportion of immature thymocytes from these mice show specific binding and strong [Ca2+]i elevation in response to self-antigen-presenting cells, although these thymocytes do not proliferate. This self-reactivity of thymocytes is inhibited by antibodies specific for TCR, CD4, CD8, class II molecules, lymphocyte function-associated antigen 1, and intercellular adhesion molecule 1. These results demonstrate for the first time that before thymic negative selection, immature T cells can specifically interact with cells bearing self-antigen, and suggest that the resulting TCR-dependent signal transduction events provide a basis for negative selection of self-reactive T cells.

CBA/N X-linked B-cell defect prevents NZB B-cell hyperactivity in F1 mice.

NZB mice and their F1 hybrids produce excessive polyclonal IgM and autoantibodies of both IgM and IgG classes. CBA/N mice and CBA/N-mothered F1 males fail to make antibody to many T-independent antigens and have low levels of serum IgM; further, these mice lack a population of splenic B cells characterized by a low-to-intermediate density of surface IgM. We have studied male CBA/N, NZB, CBA/N X NZB, NZB X CBA/N, and CBA/J mice; female CBA/N X NZB mice; and males of several control crosses of NZB and CBA/N mice. We have found that the CBA/N X-linked defect of T-independent immune response is completely expressed in CBA/N X NZB mice. In marked contrast to NZB mice and to NZB mice and to NZB F1 hybrids bearing at least one normal X chromosome, the CBA/N X NZB males failed to respond to two T-independent antigens, had small numbers of splenic IgM-producing cells, barely detectable splenic IgM production, and splenic B-cell surface-Ig patterns resembling those of CBA/N mice. These data suggest that the NZB B-cell abnormality resulting in excessive IgM production occurs almost exclusively in that population of B cells affected by the CBA/N X chromome-linked defect. Preliminary studies suggest that CBA/N X chromosome retards the spontaneous development of anti-erythrocyte autoantibodies in CBA/N X NZB males. Castration, known to accelerate autoimmune disease in certain NZB F1 males, appears to have no influence on the immune functions examined in this study.

Evidence of suppressor cell activity in spleens of mice bearing primary tumors induced by Moloney sarcoma virus.

Spleens from Moloney sarcoma virus (MSV) tumor-bearing C57BL/6N mice contained four times the normal number of mononuclear cells and displayed a markedly elevated "spontaneous" (mitogen-independent) DNA synthesis on a per cell basis. The number of macrophages were increased three-fold while there was a slight reduction in the percentage of T lymphocytes. The phytohemagglutinin (PHA) response on a per cell basis of spleens from tumor-bearing mice was decreased about 90% when compared with normal control mice. The primary in vitro immune response to sheep red blood cells was also suppressed to levels of less than 10% of normals. The PHA response could be restored by purification of MSV spleen cells by rayon adherence columns and by removal of phagocytic cells by an iron/magnet technique. The activity of suppressor cells in MSV spleens was demonstrated in mixtures with syngeneic normal spleen cells where a marked impairment of the PHA response was observed. Spleen cells from tumor-free nude mice and normal spleen cells treated by anti-theta serum plus guinea pig complement (C'), both totally unreactive to PHA, had no such effect. The inhibitor cell in MSV spleens was shown to be insensitive to inactivation by anti-theta plus C', but could be removed by the adherence columns and the iron/magnet technique. These data suggest that this suppressor cell is a cell of the monocyte/macrophage series. Suggestive evidence was also presented that the suppressor cells belong to a proliferating population in MSV spleens. Similar suppressor cells have been previously demonstrated in spleens of mice during a variety of immune responses. Our data show, that a tumor, although stimulating the immune system, nevertheless may be suppressive on certain immune functions through the activation of suppressor cells.

Differentiation antigens identify subpopulations of rabbit T and B lymphocytes. Definition by flow cytometry.

A panel of six monoclonal antibodies produced against cell surface glycoproteins of a rabbit T lymphocyte line was used with flow cytometry to define rabbit lymphocyte subpopulations. Four thymocyte populations were characterized by size and expression of cell surface antigens and appear to represent stages in thymocyte differentiation. Rabbit spleen contained five subpopulations: two of T lineage, two of B, and a null cell subset. Bimodal distribution of staining of thymocytes and peripheral T cells was observed using an antibody (9AE10) directed against a Thy-1 analogue in the rabbit, suggesting two separate T cell lineages. One of the monoclonal reagents, L11/135, reacted strongly with peripheral rabbit T cells as shown by two-color immunofluorescence. In functional studies, only the L11/135-bearing cells responded to the T cell mitogens concanavalin A and phytohemagglutinin and to allogeneic splenocytes. The thymocyte subpopulations and the peripheral T and B cell subsets differ from those described in mouse and man.

Immune cell cooperation, viruses, and antibodies to nucleic acids in new zealand mice.

The development of autoimmunity in New Zealand mice is related to genetic, immunologic, and viral factors. Evidence is presented to suggest that thymus-dependent immune functions may be depressed and bone marrow-dependent functions augmented in these mice. Antibodies to RNA and DNA appear spontaneously and can also be induced by treatment with rI.rC. Antibodies binding rI.rC-(14)C in human lupus sera, in NZB/NZW F(1) (B/W) mice developing lupus, and in NZB, ALN, and ALN/NZB mice have greatest specificity for reovirus double-stranded RNA. Treatment of B/W mice with RNA and cyclophosphamide induces immunologic tolerance, and suppresses antibodies binding rI.rC-(14)C. During recovery, the specificity of the antibodies is unaltered. Induction of tolerance in this way prevents the accelerated formation of anti-RNA antibodies normally induced by MLV. This finding suggests that virus-accelerated and natural disease occur through a similar mechanism, and supports the hypothesis that viruses may act as antigenic stimuli for a genetically hyper-responsive antibody-producing system.

Expression of xenotropic murine leukemia viruses as cell-surface gp70 in genetic crosses between strains DBA/2 and C57BL/6.

Flow microfluorometry was used to assess levels of xenotropic murine leukemia virus envelope-related cell-surface antigens (XenCSA) expressed on lymphocytes of mice derived from crosses between C57BL/6 (B6) and DBA/2 (D2); 24 recombinant inbred strains (BXD RIs) and 62 backcross mice were studied. The results suggest that XenCSA expression is affected by more than one gene but that the predominant influence is exerted by a single semidominant gene apparently located on chromosome 4 at or in close proximity to the Fv-1 locus. Studies of spontaneous virus production in B6D2F1 X D2 mice suggest that this locus may also affect production by spleen cells of xenotropic MuLV registering in a fluorescent antibody assay of mink lung cells.

B lymphocyte antigens in sicca syndrome.

All individuals tested in this study with sicca syndrome, a human autoimmune disease, bear two immunologically distinct and genetically unrelated B lymphocyte antigens that appear similar to the immune response associated (Ia) antigens of the mouse. The genes coding for these two antigens are present in only 37 and 24 percent of normal controls. In animal models Ia antigen genes are closely linked to immune response genes. Our findings suggest that two such genes may be required for the development of sicca syndrome.

Human neutrophil heterogeneity identified using flow microfluorometry to monitor membrane potential.

Previous studies of neutrophil nitroblue tetrazolium dye reduction in response to endotoxin and rosetting of IgG-coated erythryocytes have suggested functional heterogeneity of peripheral blood neutrophils. In the following study we utilized flow microfluorometry and the membrane potential-sensitive fluorescent dye 3-3'-dipentyloxacarbocyanine to assess the heterogeneity of neutrophils upon activation by a variety of stimuli. Unstimulated neutrophils from normal subjects exhibited a unimodal distribution of fluorescence, suggesting that all the cells possessed the same resting membrane potential. As neutrophils aged (>5 h), some cells lost fluorescence producing a bimodal distribution. In studies with fresh cells, the secretagogue phorbol myristate acetate (20 ng/ml) stimulated a uniform loss of fluorescence (apparent depolarization). The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) (0.1 muM) caused the neutrophils to assume and maintain (for > 30 min) a bimodal fluorescence distribution in which 65+/-5% of the neutrophils first decreased and then increased fluorescence (apparent depolarization/partial repolarization), and 35+/-5% of the cells exhibited either an increase in fluorescence (apparent hyperpolarization) or no change. Treatment of neutrophils with cytochalasin B before stimulation caused the cells to respond homogeneously to f-Met-Leu-Phe. Additional studies using neutrophils from patients with chronic granulomatous disease, which exhibit abnormal membrane potential responses, indicated that this defect affected all such neutrophils uniformly. These observations demonstrate the need to investigate the physiological significance of the heterogeneity of neutrophil function and indicate that the f-Met-Leu-Phe-induced changes in membrane potential observed in bulk population cell studies are the summation of two different responses.

Responder cells in the human autologous mixed lymphocyte reaction.

Isolated human T4+ cells proliferate in the autologous mixed lymphocyte reaction (AMLR), whereas isolated T8+ cells do not. However, in the presence of Interleukin 2 or T4+ cells, the T8+ cells demonstrated substantial proliferation. These studies suggest that T8+ cells recognize signals from autologous non-T cells, but require an additional factor for the subsequent proliferative response. Since this stimulus can be provided by T4+ cells, the AMLR appears to constitute an inducer circuit. Different defects in this circuit may be responsible for the common abnormality of the AMLR in different diseases.

Human lymphocytes with either the OKT4 or OKT8 phenotype produce interleukin 2 in culture.

In this study, we demonstrate that both highly purified T4+ and T8+ lymphocytes can produce substantial amounts of Interleukin 2(IL 2) when stimulated with the combination of concanavalin A (Con A) and phorbol myristate acetate. Furthermore, addition of IL 1 to macrophage-depleted T lymphocytes significantly increased IL 2 production by lymphocytes of either the T4+ or T8+ phenotype. These findings provide a basis for further studies of the molecular mechanisms involved in human immune cell interactions.

Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

We used monoclonal antibodies and flow cytometry to study the expression of the receptors for the complement fragments C3bi (CR3) and C3b (CR1) on human polymorphonuclear neutrophil leukocytes (PMN). Expression of both receptors was minimal on cells stained in anticoagulated whole blood incubated at 0 degree or 37 degrees C. PMN isolated with Percoll density gradients and held at 0 degree C also had only minimal expression of both receptors. With the isolated cells, however, a spontaneous increase in expression of both receptors occurred upon warming to 37 degrees C. This did not represent complete expression of either receptor since additional increments in surface expression could be induced upon stimulation with N-formyl-methionyl-leucyl-phenylalanine or Raji cell supernatant. The increases in complement receptor (CR) expression appeared to be specific since there were no changes in expression of the Fc gamma receptor or beta-2-microglobulin under any of these conditions. The increased CR expression seems to involve translocation from an intracellular pool since it is complete within minutes and is not blocked by puromycin or cycloheximide. These results demonstrate that both CR3 and CR1 expression increase rapidly upon activation of PMN and that isolated cells can be used to study this phenomenon, which may be a critical part of neutrophil function in vivo.

Proliferation of infected lymphoid precursors before Moloney murine leukemia virus-induced T-cell lymphoma.

NFS/N mice inoculated with Moloney murine leukemia virus (M-MuLV) developed T-cell lymphoma after a 10-week latent period. Expression of lymphoid differentiation antigens, appearance of M-MuLV-encoded cell surface antigens, and rates of cellular proliferation were measured in splenic and bone marrow subpopulations during this latent period. At 2 weeks of age, Thy-1-and surface immunoglobulin-negative null cells of spleen and bone marrow expressed M-MuLV antigens whereas T- and B-lymphocytes did not. During the 3d and 4th weeks, the number of splenic null cells increased to six times the number found in uninfected controls. These null cells included the precursors of lymphocytes and hematopoietic cells. For the remainder of the latent period, the percentage of null cells undergoing proliferation was three times greater in the infected mice, while the total number of null cells remained constant. This proliferation was not accompanied by terminal differentiation or emigration of mature cell types from the spleen. Proliferation was substantially delayed in CBA mice, which are resistant to lymphoma induction.

Diversity of immunological phenotypes of lymphoblastic lymphoma.

Eleven cases of lymphoblastic malignancy, presenting as lymphoma, were investigated for immunological and differentiation markers prior to the onset of therapy. Biopsy specimens exhibited the typical morphological features of lymphoblastic lymphoma (convoluted T-cell lymphoma). Intranuclear terminal deoxynucleotidyl transferase was detected in the neoplastic cells from each case by indirect antibody staining of cytocentrifuge preparations. Eight cases were T-cell type as evidenced by unsensitized sheep erythrocyte rosette formation and staining with the monoclonal antibody OKT11. Three T-cell cases were OKT4 positive, two were OKT8 positive, and none were positive with both OKT4 and OKT8. Three cases failed to react with any monoclonal antibodies specific for T-cells and did not form unsensitized sheep erythrocyte rosettes or stain for surface immunoglobulin. However, these three cases were Ia positive and J5 (common acute lymphoblastic leukemia antigen) positive. Cells from two of these erythrocyte rosette-negative, Ia-positive, common acute lymphoblastic leukemia-positive cases contained intracytoplasmic mu heavy chains and were therefore of pre-B-cell phenotype. These cases were histologically indistinguishable from the T-cell cases. However, clinically, they were distinguished by the absence of mediastinal masses and by a clinical presentation as isolated lytic lesions of bone in two of the three. OKT9 and OKT10 stained neoplastic cells from T-cell, as well as pre-B-lymphoblastic, lymphoma. Although morphologically homogeneous, lymphoblastic lymphomas are comprised of an immunologically diverse group of neoplasms which include cells of "common" and "mature" thymocyte, non-T, non-B, and pre-B phenotypes and are closely related to the cells of acute lymphoblastic leukemia. In addition, intratumor heterogeneity was observed in most instances and may reflect growth or differentiation differences between subpopulations of individual neoplastic clones.

Biotinylation of human C3.

Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.

Characterization of T lymphocyte and monocyte populations in HLA B8/DRw3 normal individuals and in patients with dermatitis herpetiformis.

Normal individuals who possess the HLA B8/DRw3 haplotype as well as patients with dermatitis herpetiformis have been found to have a number of immunologic abnormalities including decreased numbers of E rosette-positive, Fc IgG receptor-bearing lymphocytes (referred to as TG cells), and increased numbers of cells which spontaneously secrete immunoglobulin. HLA B8/DRw3-positive normal individuals also have an increased risk for the development of a number of immunologically mediated diseases. Since many of these findings are suggestive of B-cell hyperreactivity and since TG cells were initially thought to represent a portion of the T suppressor cell network, we have examined the peripheral blood mononuclear (PBM) cell populations of 14 normal HLA B8/DRw3-positive individuals, 14 patients with dermatitis herpetiformis (all of whom were HLA B8/DRw3-positive), and 9 non-HLA B8/DRw3 individuals using flow cytometry and monoclonal antibodies of the OK and Leu series directed against cell surface antigens. Normal HLA B8/DRw3 individuals were found to have a significantly lower percentage of PBM cells that expressed both OKT8 and Leu-2a when compared to normal non-HLA B8/DRw3 individuals (p less than .05 Student's t-test). When the ratio of T helper cells (OKT4 and Leu-3a) to T suppressor cells (OKT8 and Leu-2a) was calculated for each individual studied, normal HLA B8/DRw3 individuals were found to have a significantly elevated ratio (Leu-3a/Leu-2a = 2.41 +/- .16, mean +/- SEM) when compared to non-HLA selected individuals (Leu-3a/Leu-2a = 1.73 +/- .05) (p less than 0.025). In addition, normal HLA B8/DRw3 individuals had decreased numbers of TG cells when compared to normal non-HLA B8/DRw3 individuals (B8/DRw3 = 6.4 +/- .74%, non-B8/DRw3 = 13.2 +/- 1.0%, mean +/- SEM, p less than .01). In order to determine the cell surface marker characteristics of TG cells, purified TG cells from both normal HLA B8/DRw3 individuals and non-HLA B8/DRw3 individuals were studied using the Leu series monoclonal antibodies and OKM1. Good agreement was found in the percentages of cells expressing each cell surface marker between the two groups. In addition, the TG cells were found to be predominately T cells (78% Leu-1-positive), with both T helper cells (40% Leu-3a-positive) and T suppressor cells (30% Leu-2a-positive) present. These results suggest that the suppressor cell activity associated with the TG subset is not due to a depletion of the T helper cell subset, and that the decreased numbers of TG cells in HLA B8/DRw3 individuals is not due to a preferential loss of cells bearing Leu-1, Leu-2a, Leu-3a, or OKM1.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of human basophils by flow microfluorometry.

Human mononuclear cells labeled with fluorescein-conjugated anti-IgE antibody were subjected to flow microfluorometric analysis. Basophils were enriched to 97-99% purity with a 2-step cell sorting procedure. Trypan blue exclusion of sorted cells exceeded 90% and the net yield of the procedure was 15%.

Selective elimination of lymphocyte subpopulations by monoclonal antibody-enzyme conjugates.

A novel method for the selective depletion of lymphocyte subpopulations has been developed. Conjugates of glucose oxidase (GOx) and phospholipase-C (PL-C) coupled to a monoclonal mouse anti-rat IgG (MAR) were shown to be selectively cytotoxic for targeted lymphocyte subsets in the presence of various rat monoclonal antibodies directed toward murine cell surface antigens. The ability of both conjugates to bind specifically to rat monoclonal antibodies was demonstrated by flow cytometry. The PL-C-MAR conjugate was more stable than the GOx-MAR conjugate. The PL-C conjugate, in conjunction with primary rat anti-mouse monoclonal antibodies, produced selective killing of T or B cells as detected by a loss of proliferative capacity in response to mitogens and by specific cell depletion demonstrated by flow cytometry. Normal mouse serum protected against the cytotoxicity of free enzymes but had no protective effect on enzyme conjugates. Because the substrates of these enzymes are abundant in vivo and serum did not interfere with their cytotoxicity, these enzyme-antibody conjugates may be valuable for selective lymphocyte depletion in vivo.