The Journal of Experimental Medicine

Yarchoan, R; Tosato, G; Blaese, R M; Simon, R M; Nelson, D L

doi: 10.1084/jem.157.1.1pmid: 6294212

The Epstein-Barr virus (EBV) is a herpes virus that has the capacity to infect human B cells and to induce them to secrete immunoglobulin (Ig). In the current experiments, Poisson analysis of limiting dilution cultures has been used to study the activation of human peripheral B cells by the B95-8 strain of EBV. Under the culture conditions used, 0.2-1% of peripheral blood B cells were activated by EBV to secrete IgM or IgG. In addition, when multiple replicate cultures containing limited numbers of B cells were tested for IgM and for IgG production, the precursors for IgM and IgG segregated independently; thus, individual B cell precursors matured into cells secreting IgM or IgG but not both classes of Ig. Additional experiments using limiting dilutions of EBV were undertaken to study the viral requirements for B cell activation. These studies indicated that B cell activation by EBV to produce Ig was consistent with a "one-hit" model and inconsistent with a "two-hit" model. Taken together, these results indicate that infection by one EBV virion is sufficient to induce a precursor peripheral blood B cell to secrete Ig and that only one isotype of Ig is then secreted.

Kresina, T F; Nisonoff, A

doi: 10.1084/jem.157.1.15pmid: 6217277

Mice that are suppressed with respect to an idiotype (CRIA) present in A/J anti-p-azophenylarsonate antibodies, hyperimmunized, and allowed to rest were previously found to possess high concentrations of suppressor T cells with anti-idiotypic receptors. We have now observed that the sera of such mice contain soluble factors that can selectively suppress the CRIA component of a humoral response when passively transferred to adult or neonatal recipients. When T cells from suppressed, hyperimmunized mice were transferred into female mice before mating, their offspring, upon immunization, produced anti-Ar antibodies that lacked CRIA. A state of idiotypic suppression was also produced in offspring when the mother was inoculated with serum from suppressed mice a few days before parturition. The results indicate that the suppressor factor is not an immunoglobulin.

Young, W W; Tamura, Y; Johnson, H S; Miller, D A

doi: 10.1084/jem.157.1.24pmid: 6848617

Mice challenged with L5178Y lymphoma cells expression high levels of the glycolipid asialo GM2 (gangliotriosylceramide) were protected from tumor growth by passive administration of a monoclonal antibody specific for the glycolipid; in a few antibody-treated mice, ascites cells eventually proliferated which contained a reduced chemical quantity of the glycolipid antigen (3). We now report that the cells emerging from antibody-treated mice had abnormal marker chromosomes identical to those in the cells used for challenge, indicating that the emergent cells were progeny of the challenge inoculum. Flow cytometric analysis revealed that asialo GM2 was undetectable on the surface of greater than 95% of the tumor cells from antibody-treated mice, whereas surface display of H-2 determinants was unchanged from that of the cells used for challenge. Tumor cells arising in challenged but untreated mice consisted of a mixture of asialo GM2-positive and -negative cells, indicating the presence of selective pressures in these mice as well. None of the cells taken from tumor bearing mice differed significantly from the challenge cells in their susceptibility to natural killer cell attack, suggesting that resistance to natural killer cell lysis was not responsible for the proliferation of these cells in vivo. When cells derived from an antibody-treated mouse were used to challenge mice, serotherapy with anti-asialo GM2 had no effect on mouse survival. These results suggest that serotherapy may complement a host anti-tumor response, from which only asialo GM2 deficient cells can escape.

Jackson, S; Chused, T M; Wilkinson, J M; Leiserson, W M; Kindt, T J

doi: 10.1084/jem.157.1.34pmid: 6600269

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.

Hibbs, M S; Postlethwaite, A E; Mainardi, C L; Seyer, J M; Kang, A H

doi: 10.1084/jem.157.1.47pmid: 6549655

The cell-cell interactions between fibroblasts and mononuclear leukocytes (MNL) which promote alterations in collagen accumulation were examined using a system of co-culture of human fibroblasts and peripheral blood MNL. The stimulation of collagen production was optimal after 48 h of co-culture and the increase in collagen correlated directly with the number of MNL added. The enhancement of collagen production was seen in both autologous and allogeneic co-cultures. Stimulation of non-collagenous protein was also noted. Co-culture supernatants contained soluble substances that were capable of stimulating collagen production, although they stimulated collagen production to a lesser degree than direct co-culture. Fractionation of these supernatants on Sephadex G-200 revealed a predominant area of stimulatory activity at 160,000 mol wt. Lesser areas of activity were noted at molecular weights of 80,000 and 25,000. Determination of the types of collagen produced by fibroblasts during co-culture with MNL showed that the ratio of type I:III collagen was decreased. These alterations in both the quantitative and qualitative accumulation of collagen mimic the changes often seen in wound healing and early inflammation suggesting that cellular interactions between fibroblasts and MNL may be important in the modulation of collagen production in normal and pathologic states.

Butler, J L; Muraguchi, A; Lane, H C; Fauci, A S

doi: 10.1084/jem.157.1.60pmid: 6600272

The success of long-term culture of normal human and murine B cells has been hampered by the limited availability of soluble factors capable of maintaining proliferation of activated B lymphocytes. Previous experiments using various culture-derived supernatants in a human system were unable to separate the activities of B cell growth factor (BCGF) and interleukin 2 (IL-2) by immunochemical means. Thus, purified factors with BCGF activity in the absence of IL-2 activity have not been available for study. In the present study, normal human peripheral blood T cells were fused with the hypoxanthine/aminopterin/thymidine-sensitive human T-leukemic cell line, CEM-6. Supernatants from the resulting hybrid cells were tested for the ability to maintain proliferation of normal human B cells in a recently described assay system for human BCGF. Hybrids demonstrating BCGF activity were cloned by limiting dilution. One hybrid clone, 2B11, continued to support proliferation of B cells in both long-term cultures and 6-d assays at a level significantly above that seen with conventionally produced growth factors. No IL-2 activity was found in the supernatant from hybrid 2B11. The hybridoma supernatant was fractionated by gel filtration, and maximum proliferation of B cells was supported by the 18-20,000 mol wt protein fraction. Thus, a human T-T cell hybridoma that has BCGF activity in the absence of any demonstrable IL-2 activity has been developed. Human T-T cell hybridomas secreting discrete immunoregulatory factors should prove to be powerful tools in dissecting the mechanisms of immunoregulation of human lymphocyte function.

Mongini, P K; Paul, W E; Metcalf, E S

doi: 10.1084/jem.157.1.69pmid: 6600273

The IgM, IgG subclass, IgE, and IgA anti-trinitrophenyl (TNP) antibody (Ab) response of B cells to the type 2 antigen TNP-Ficoll was studied in athymic nude mice and in the in vitro splenic focus assay. Results from the splenic focus assay in which purified B lymphocyte preparations had been transferred to irradiated nu/nu recipients indicate that many TNP-Ficoll stimulated B cell clones secrete multiple isotypes and hence appear to be undergoing intraclonal isotype switching. Although the frequency of clones secreting each of the IgG subclasses was found to correlate with 5' to 3' Igh-gamma gene order, the frequency of IgE and IgA-secreting clones did not appear to be influenced by the respective position of Igh-epsilon and Igh-alpha on the chromosome. Unlike clones that secreted anti-TNP Ab of the IgG subclasses, IgE and IgA anti-TNP Ab-secreting clones did not have a high propensity for coexpression of isotypes encoded by 5' Igh-C genes. These data suggest that three distinct switching pathways may be employed by B cells responding to TNP-Ficoll: a common IgG pathway, an IgE pathway, and an IgA pathway. The presence of T cells resulted in a preferential enhancement of the production of anti-TNP Ab of those IgG subclasses which were least represented in the absence of T cells, i.e., IgG2b and IgG2a. No significant enhancement of IgE anti-TNP clonal frequency was found in the presence of T lymphocytes, but T cells were found to significantly enhance the clonal expression of IgA anti-TNP Ab. Although a relatively large number of B cell clones were found to synthesize IgE and IgA anti-TNP Ab in the splenic focus assay, relatively little or no secretion of these isotypes was detected in immune mice. Possible explanations for this apparent discrepancy are discussed.

Keesee, S K; Owen, F L

doi: 10.1084/jem.157.1.86pmid: 6129278

The T cell alloantigen Tthyd appears on a subpopulation of thymocytes in mice bearing the Igh-1d or e heavy chain haplotype. Ontogenetic studies have suggested that this antigen precedes the appearance of two other T cell alloantigens in the same linkage group, Tindd and Tsud. The purpose of this study was to determine if the Tthy-bearing cell is a precursor for cells in the periphery expressing Tind and Tsu. CAL-20 mice were treated at 48-h intervals beginning on the day of birth with a monoclonal antibody recognizing Tthy. Tthy alloantigen expression, monitored by cytotoxicity assays, was found to be significantly depressed in the thymuses of treated animals; Tind and Tsu also failed to appear in the periphery. Treatment with anti-Tthy caused no significant changes in frequency or surface intensity in the expression of surface Ig. Thy-1.2, Thy-1.2, and Lyt-2.2, as studied by cytofluorograph analysis. We conclude that the T-thyd-bearing cells in the thymus represent a subpopulation that may be a precursor for Tindd- and Tsud-bearing cells. However, Tthyd-bearing cells are more mature than the Thy-1.2 common T cell precursor, pre-T.

Clarke, S H; Claflin, J L; Potter, M; Rudikoff, S

doi: 10.1084/jem.157.1.98pmid: 6401319

Complete variable (V) region amino acid sequences were determined for four heavy (H) and one light (L) chain from C57BL phosphocholine (PC)-binding monoclonal antibodies. Additional NH2-terminal sequences were obtained from H and L chains of C57BL and CBA/J origin. When these V regions were compared with previously reported anti-PC sequences, a number of observations could be made regarding the function and evolution of L and H chain segments used in these antibodies. (a) L and H chain V segments are remarkably conserved in these inbred strains, although there has been an accumulation of point mutations identifying apparently allelic forms of VK and VH. (b) Mice of each genotype use the same three VK segments in combination with a single VH segment to produce most anti-PC antibodies. An exception has been noted that indicates the occasional use of a second VH gene segment. (c) Multiple, different DH regions are used by mice of each strain, which suggests that the DH segment sequence plays no critical role in either antigen binding or VH-VL pairing. Furthermore, the DH segments and their corresponding gene families appear to be highly conserved in the inbred strains studied. (d) Most PC-binding antibodies use the JH1 joining segment. All JH1 sequences from C57BL mice differ from the BALB/c JH1 at position 105, which identifies allelic forms of the JH1 region. These studies are a first assessment of the nature of mutational events associated with the evolution of specific multigene immunoglobulin families and indicate that homologous VH, DH, JH, VK, and JK genes are similarly assembled and expressed in PC antibodies from three diverse genotypes.

Hokland, P; Rosenthal, P; Griffin, J D; Nadler, L M; Daley, J; Hokland, M; Schlossman, S F; Ritz, J

doi: 10.1084/jem.157.1.114pmid: 6571733

Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual fluorescence techniques disclosed that these cells were heterogenous with respect to the expression of a series of differentiation and activation antigens defined by monoclonal antibodies. Thus, whereas all CALLA+ cells were Ia+ and expressed two activation antigens, J2 and T10, only 30-50% expressed B1 antigen. Furthermore, using methanol-fixed cells, it could be shown that approximately 20% contained intracytoplasmic mu chains (cyto-mu) and that approximately 15% were positive for the terminal transferase enzyme (TdT) marker. The CALLA+ fetal cells thus closely resemble the childhood acute lymphoblastic leukemia cell with respect to surface marker phenotype. A population of CALLA- cells devoid of mature erythroid and myeloid surface markers was found to contain higher numbers of TdT+ cells but lower numbers of cyto-mu, B1, and Ia+ cells than the CALLA+ subset. In vitro analysis of normal, purified CALLA+ cells demonstrated that incubation at 37 degrees C with J5 monoclonal antibody specific for CALLA resulted in the specific modulation of surface antigen. Similar results have previously been obtained with CALLA+ tumor cells. Although phenotypic analysis of CALLA+ cells suggests that these cells are relatively immature lymphoid cells, CALLA+ cells do not appear to contain either myeloid precursor cells (CFU-G/M) or the earliest lymphoid stem cells.