Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). I. Analysis of the PITS beta factor

Within populations of mitogenically (PWM) stimulated normal human lymphocytes, the proliferation of B lymphocytes is terminated by T cells. In contrast, T cells limit their own proliferation. T cells thus apparently measure and terminate the proliferation of B cells as well as themselves, suggesting an important role for them in limiting amplification during immune response. Under the culture conditions employed, PWM-induced B- and T-cell proliferation was uncoupled from B-cell differentiation into plasmacytes. Termination of B-cell proliferation in this in vitro model of humoral immune response is independent of B-cell differentiation.     

Functional analysis of monkey lymphocyte subsets defined by OKT4 and OKT8 monoclonal antibodies

The percentages of rhesus monkey blood lymphocytes (PBL) reactive with OKT4 and OKT8 antibodies and the $\text{OKT}\text{4}\text{OKT}\text{8}$ ratio showed significant correlations with the log of the immunoglobulin plaque-forming cell (PFC) response after stimulation with pokeweed mitogen (PWM). These correlations suggested that monkey OKT4⁺ cells function as “helper” cells and OKT8⁺ cells function as “suppressor” cells for the PFC response. This was confirmed by separation and study of enriched T- and B-cell subpopulations. OKT8-depleted (OKT4⁺) and OKT4-depleted (OKT8⁺) cells were obtained by treatment of purified T cells with antibody and complement. OKT4⁺ cells augmented the PWM-induced B-cell differentiation into PFC but OKT8⁺ cells did not. OKT8⁺ cells suppressed the PFC response by mixtures of B cells and OKT4⁺ cells. OKT8 antibodies also detected a suppressive cell subset in African green monkeys since the percentage of OKT8⁺ cells showed a negative correlation with the log PFC response. OKT4 antibodies failed to bind to African green monkey PBL.     

Induction of human immunoglobulin secretion. II. T lymphocyte dependency and radiosensitivity of T-cell help for induction of B-cell differentiation by Staphylococcus aureus strain Cowan I

The induction of peripheral blood B lymphocytes to mature to immunoglobulin-secreting cells (ISC) when stimulated by Staphylococcus aureus strain Cowan I was found to be T helper cell-dependent (J. Immunol.127, 1044, 1981). The nature of T help was studied in B- and T-cell separation and reconstitution experiments. T helper cells for Cowan I were very radiosensitive (D₃₇ < 500 rad) in comparison to helpers for pokeweed mitogen (PWM) (D₃₇ > 2000 rad). PWM synergized with Cowan I in induction of ISC, and helper T cells for dual stimulation were also radioresistant. The ratio of T to B cells was found to be critical in judging reactivity of donors for both PWM and Cowan I. T cells stimulated with PWM, but not Cowan I, produced T cell-replacing factors essential for Cowan I-induced maturation of B cells. Irradiation of T cells prior to PWM stimulation increased the level of such helper factors. Poor responders to Cowan I, as judged by mononuclear cell cultures, had apparently few helpers for the bacterial stimulant, compared to high responders. Cowan I helper T-cell activity did not appear to be due to protein A leaking from the bacteria and stimulating T cells. In all these experiments, induction of ISC by Cowan I was completely dependent on T cells or factor, providing a good model for investigation of B-cell differentiation regulated by a unique subset of radiosensitive T helper cells.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Role of monocytes in pokeweed mitogen-induced differentiation of human peripheral blood lymphocytes

Separate stimulation (“pulsing”) method of different cell populations with pokeweed mitogen (PWM) was used to study the regulatory role of monocytes in the PWM-induced plaque-forming cell response of human peripheral blood lymphocytes. T cells, B cells, and monocytes were separated, pulse-stimulated with PWM, extensively washed, and cocultured with unstimulated cell populations without additional PWM. Pulse-stimulated T cells helped unstimulated B cells to differentiate into immunoglobulin-secreting cells. This generation of helper T cells by PWM-pulsing was enhanced by monocytes in the presence of free PWM, as well as by PWM-pulsed monocytes in the absence of free PWM. A coculture of pulse-stimulated B cells and unstimulated T cells produced more substantial B-cell differentiation than the coculture of stimulated T cells and unstimulated B cells. Further enhancement of the latter response was obtained when B cells were pulse-stimulated in the presence of monocytes. However, pulse-stimulated B cells did not differentiate in the absence of T cells, and monocytes were unable to replace this T-cell function. It appears that there are several pathways by which PWM induces B-cell differentiation and in each, monocytes play an enhancing role.     

Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81.

CD81 (TAPA-1) is a member of the transmembrane 4 superfamily (TM4SF) which is expressed on the cell surface of most cells of the body throughout their cellular differentiation. It has been recognized in several cell surface complexes of lymphocytes, suggesting that it may have diverse roles in lymphocyte development and activation regulation. Mice with a CD81 null mutation revealed normal T- and conventional B-cell development, although CD19 expression on B cells was dull and B-1 cells were reduced in number. However, both T and B cells of mutant mice exhibited strikingly enhanced proliferation in response to various types of stimuli. Interestingly, while proliferative responses of T cells following T-cell antigen receptor (TCR) engagement was enhanced in the absence of CD81, B-cell proliferation in response to B-cell antigen-receptor (BCR) cross-linking was severely impaired. Despite these altered proliferative responses, both tyrosine phosphorylation and intracellular calcium flux in response to cross-linking of cell surface antigen receptors were normal in mutant mice, reflecting apparently normal initial signaling of antigen receptors. In conclusion, though CD81 is not essential for normal T- and conventional B-cell development, it plays key roles in controlling lymphocyte homeostasis by regulating lymphocyte proliferation in distinct manners, dependent on the context of stimulation.     

Redefinition of lymphoid progenitors

Similarities between T and B lymphocytes might have led to the idea that these functionally distinct cells develop from a common lymphoid progenitor. However, investigations with a new clonal assay which allows for T-, B- and myeloid-lineage development indicate that commitment to T-cell and B-cell lineages occurs instead through myeloid/T and myeloid/B bipotential stages, respectively. These findings provide an opportunity to reconsider the ontogeny and phylogeny of T- and B-cell development.     

Bromodeoxyuridine and light treatment enhances responsiveness of pokeweed mitogen-stimulated human lymphocytes to autologous and allogeneic determinants

We have investigated a novel system whereby lymphocytes from normal human subjects can be induced to develop exaggerated reactivity to histocompatibility antigens in vitro. Peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM) showed increased and accelerated subsequent proliferation to both autologous and allogeneic stimulators. Addition of bromodeoxyuridine (BUdR) during the period of maximal PWM-induced DNA synthesis followed by light exposure caused unexpected, but marked enhancement of this secondary proliferation. While untreated cultures contained a preponderance of T8⁺ cells after PWM activation, BUdR plus light-treated cultures were largely T4⁺ cells. Because removal of suppressor cells in nonsuicided cultures with anti-T8 and complement just before restimulation failed to unmask enhanced autoreactivity, events critical in the induction of the enhanced response must have occurred during priming. Cultures of PBMC with medium alone or concanavalin A, as well as purified T cells cultured with PWM, gave no enhanced autoproliferation after BUdR and light; thus T and non-T cells must be acted on by a T- and B-cell mitogenic stimulus to prime T cells for enhanced responsiveness. The interactions between T cells and activated B cells in this in vitro system may be relevant to regulatory mechanisms important in the induction of pathological autoimmune responses.     

Cytotoxic effects of butyrate and other ‘differentiation inducers’ on immature lymphoid cells

A connection between the processes of cell death and differentiation is suggested by observations which show that chemical inducers of differentiation are cytotoxic to CCRF-CEM human leukaemic lymphoblasts, cells which have properties typical of immature lymphoid cells. Sodium $\text{n}$-butyrate, salts of other short-chain fatty acids, 5-azacytidine, hypoxanthine, L-ethionine and dimethyl sulphoxide were all cytotoxic to these cells at concentrations similar to those reported to produce reversible growth inhibition in more mature lymphocytes or growth inhibition and differentiation in other cell types. Only actively cycling cells were susceptible to killing by $\text{n}$-butyrate. Inhibitory effects of these compounds on DNA methylation are postulated to be responsible for their cytotoxic actions.     

Response of human B cells to Staphylococcus aureus Cowan I: T-independent proliferation and T-dependent differentiation to immunoglobulin secretion involve subsets separable by rosetting with mouse erythrocytes

We separated T-depleted mononuclear cells into subsets by rosetting with mouse erythrocytes and studied proliferation and differentiation responses to Staphylococcus aureus Cowan I (SAC), pokeweed mitogen (PWM), and a combination of the two polyclonal activators. All of the T cell-independent proliferation of unfractionated B cells in response to SAC was attributable to mouse erythrocyte rosette-forming cells (BMR+). BMR- cells were not stimulated to proliferate by SAC in the presence or absence of T cells, but did proliferate to PWM plus irradiated T cells. Co-stimulation of BMR+ cells with SAC and PWM in the presence of autologous T cells did not lead to immunoglobulin secretion. The B cells stimulated to divide by SAC apparently do not become responsive to B cell differentiation factors and are distinct from those that undergo T cell-dependent differentiation.     

T-cell receptor and immunoglobulin genes are rearranged together in Abelson virus-transformed pre-B and pre-T cells.

We have assessed the state of rearrangement and expression of B- and T-cell antigen receptor genes in cells of Abelson murine leukemia virus-transformed thymomas and other tumors. We found that unrearranged TcR gamma genes are expressed, as are unrearranged C mu genes, in pre-T, pre-B, and myeloid cells. We also found TcR gamma genes rearranged and expressed in putative pre-T cells and in cells apparently committed to the B-cell lineage. This is in contrast to the data from more mature T- and B-cell tumors. We conclude that in immature lymphoid cells both immunoglobulin and TcR gamma genes are accessible for rearrangement. We discuss the implications of these observations for an understanding of the B-T lymphoid differentiation event.     

Fluorescence-activated cell sorting of human T and B lymphocytes: I. Direct evidence that lymphocytes with a high density of membrane-bound immunoglobulin are precursors of plasmacytes

Human peripheral lymphocytes bearing either a high or a low amount of membrane-bound immunoglobulin were studied. Cells were “tagged” with fluorescein-labeled antiimmunoglobulin reagents and separated by means of a new electronic instrument, a fluorescence-activated cell sorter (FACS), into populations with either > 10⁵ or < 5 × 10³ immunoglobulin molecules per cell. Fractions of high purities were obtained. (>80% and >99.9%, respectively). In vitro, different functional properties were observed: lymphocytes with high densities of membrane-Ig gave a late proliferative response after stimulation with Pokeweed mitogen (PWM). A considerable proportion of stimulated cells developed into mature plasmacytes as detected by cytoplasmic staining. Those lymphocytes with a low density or complete absence of membrane-Ig could be stimulated by both Phytohemagglutinin (PHA) and Pokeweed mitogen, but no differentiation into plasmacytes occurred. The functions are similar to those of bone marrow-derived (B) and thymus-derived (T) lymphocytes in mice. Thus, the designation as B lymphocytes for human lymphocytes with a large quantity of membrane-bound immunoglobulin seems justified.     

Regulation of the natural killer activity of lymphocytes from healthy donors and patients with chronic lympholeukemia through the interaction of T- and non-T cells

The role of T- and B-cell cooperative interaction in the regulation of natural killer (NK) activity in human peripheral blood lymphocytes was studied. It has been shown that preincubation of normal donor mononuclear cells (MNC) for 48 h is followed by the loss of NK activity, while the incubation of the isolated T- and non-T-cell subsets does not lead to an analogous fall in the killer lymphocyte function. NK activity of MNC and isolated lymphocyte subsets in normal donors is shown to exceed that of CLL patients. The absence of preincubation effect on NK activity level of MNC, T- and non-T-cells in CLL patients has been also found. The findings obtained suggest that as a result of T- and B-cell interaction during preincubation differentiation of young T lymphocytes with NK cellular properties takes place. It is followed by the loss in NK activity. B-cell defect in CLL patients might cause the absence of preincubation effect on NK activity of T cells.     

The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire

In this Opinion article, I address the role of the pre-B-cell receptor (pre-BCR) in the development of antigen-specific B cells in terms of immunoglobulin heavy chain (IgH) variable-region repertoire selection, precursor B-cell differentiation and proliferation, and IgH allelic exclusion. Comparisons with the role of the pre-T-cell receptor (pre-TCR) in T-cell development raise provocative questions. Why do B- and T-cell lineages both use a surrogate chain - the surrogate light chain and the pre-TCR alpha-chain, respectively - as a step to develop their repertoires of antigen-recognizing cells? What are the functions of the pre-BCR and pre-TCR in lymphocyte differentiation and antigen-receptor allelic exclusion? This article, together with the accompanying article by Harald von Boehmer, hopes to answer some of these questions.     

Salmonella typhimurium mitogen induces proliferation of human B lymphocytes

The maturation of human B lymphocytes can be described as a sequence of activation, proliferation, and differentiation into immunoglobulin-secreting cells. A variety of mitogens which are T cell dependent or independent have been employed to study this process. These moieties generally induce B-cell activation and proliferation followed by differentiation, making the study of initial events difficult. This study characterizes the mitogenic activity of Salmonella typhimurium mitogen (STM), a protein fraction of S. typhimurium. Glass-nonadherent peripheral blood lymphocytes were rosetted with affinity-purified rabbit anti-human F(ab')2-coupled ox erythrocytes and separated on a Ficoll-Hypaque gradient. This population of B lymphocytes, when cultured in dilutions of STM showed dose-dependent proliferation by [3H]thymidine incorporation. Maximal proliferation occurred on Day 7 using STM at 100 micrograms/ml (control, 5692 +/- 1704 cpm; STM, 58,541 +/- 5655 cpm). On Day 7 the percentage of blast cells by Giemsa stain was 14 +/- 4% in control cultures and 52.5 +/- 8.7% with STM. ELISA quantitation of IgG and IgM in culture supernatants revealed no secretion above unstimulated controls. When B lymphocytes were enriched by a negative selection technique, significant proliferation was not observed. STM is a novel B lymphocyte mitogen which induces proliferation but not activation or differentiation of human B lymphocytes into immunoglobulin-secreting cells.     

Carbon monoxide and hydroxymercuribenzoate sensitivity of a fatty acid (omega-2) hydroxylase from Bacillus megaterium.

DNA-free minicells of $\text{Escherichia coli}$ will not allow growth of phage T-7, nor is RNA synthesis stimulated by phage infection. Thus, these miniature cells seem not to contain $\text{in vivo}$ RNA polymerase activity. However, DNA-dependent RNA polymerase activity can be unmasked in extracts with poly(dA-T) and Mn²⁺. This activity may represent a cytoplasmic pool of inactive RNA polymerase in normal cells.     

Cytotoxic T-lymphocyte and spontaneous killer cell activity against human T- and B-lymphoid cell lines.

We have investigated cell-mediated immune responses to cultured human T- and B-cell lines. Two effector mechanisms were explored and found to have different capabilities for mediating cytotoxic reactions. Cytotoxic T lymphocytes were generated by stimulation with irradiated B-cell lines and demonstrated cross-reactive cytotoxicity against these lines but not against T-cell lines. Unseparated mononuclear cells showed spontaneous cytotoxicity for both T- and B-cell lines; however, T-cell lines appeared more susceptible. Cell separation procedures were employed to determine functional differences in effector cells. In contrast to cytotoxic T lymphocytes induced in vitro, spontaneous killer cells (SKC) were shown to be nylon wool adherent, non-T lymphocytes with receptors for IgG-coated sheep erythrocytes.     

Concanavalin A promotes bromodeoxyuridine induction of enodgenous C-Type virus in B cells

Spleen lymphocytes stimulated in vitro by concanavalin A, aT-cell mitogen, in the presence of bromodeoxyuridine, release C-type viruses. Since spleen cultures are noninducible by other T-cell mitogens in contrast to several inducing B-cell mitogens, we have analyzed the subpopulation of cells releasing virus following concanavalin A stimulation. In one approach lymphocyte suspensions were enriched for B or T cells by treatment with anti-Thy 1.2 plus complement or by nylon wool filtration, respectively. Mitogen-triggered virus release in these two enriched, as well as in reconstituted populations, was then measured. In a second line of investigation, increasing doses of X irradiation were used to preferentially block B- but not T-cell proliferation. Mitogen-stimulated virus release was also determined in these irradiated cultures. These methods independently demonstrated that the presence of both B and T cells was necessary for virus induction by concanavalin A in the presence of bromodeoxyuridine. Results of these experiments suggested that concanavalin A/bromodeoxyuridine-induced virus release occurred in a B-cell- through T-cell-dependent stimulation.     

The direct effects of interleukin 1, interleukin 2, interferon-alpha, interferon-gamma, B-cell growth factor, and a B-cell differentiation factor on resting and activated human B cells

A wide variety of cytokines have been demonstrated to affect B-cell function. However, it is unclear which of these mediators actually exert direct effects on the B cells themselves. In the present study, the direct role of interleukin (IL) 1, IL-2, Interferon-gamma, or Interferon-alpha in human B-cell activation, proliferation, or differentiation was examined and compared with the effects of a B-cell growth factor (BCGF) or a B-cell differentiation factor (BCDF). Highly purified human B lymphocytes were separated according to size into two nonoverlapping populations. The fraction of small B cells was incubated with IL-1, IL-2, Interferon-gamma, Interferon-alpha, BCGF, or BCDF, and cell size changes, RNA synthesis, DNA synthesis, or supernatant immunoglobulin (Ig) production were measured. Neither IL-1, IL-2, Interferon-alpha, Interferon-gamma, nor the BCGF induced substantial cell size changes, RNA synthesis, DNA synthesis, or Ig production by the small fraction of B lymphocytes; however, the BCDF could directly activate a proportion of resting B lymphocytes to secrete Ig. The fraction of large B cells was also incubated with these cytokines. While neither IL-1, Interferon-alpha, nor Interferon-gamma enhanced DNA synthesis or Ig production by the fraction of large B lymphocytes, DNA synthesis was augmented 23-fold by BCGF and IgG production was increased 7-fold by BCDF. Additionally, IL-2 slightly enhanced both proliferation and differentiation of large B cells but substantially less so than BCGF and BCDF; DNA synthesis was increased 4-fold, while Ig production in the presence of IL-2 was increased by approximately 50%. Thus, the most important lymphokines modulating the function of these two fractions of tonsillar lymphocytes were a BCGF and a BCDF.     

Jacalin, an IgA-binding lectin, inhibits differentiation of human B cells by both a direct effect and by activating T-suppressor cells

Jacalin, a lectin extracted from the seeds of Artocarpus intergifolia (jackfruit), has been reported to bind specifically to IgA while inducing B-cell polyclonal immunoglobulin secretion. We confirmed that jacalin only binds to IgA and not to IgG or IgM and extended these findings by showing that it does not bind to IgE. Addition of jacalin to either unfractioned peripheral blood lymphocytes or purified B cells failed to induce immunoglobulin synthesis; indeed immunoglobulin production was diminished in the presence of jacalin. We found that jacalin directly inhibited the induction of immunoglobulin synthesis from B cells in the presence of T-cell replacing factor. Cell lines making IgG, IgM, and IgA were inhibited by jacalin. Furthermore, T cells incubated with jacalin also inhibited immunoglobulin production by stimulated B cells. Under these conditions jacalin was found to be a potent mitogen for T cells but to induce little or no activation of B cells. Jacalin appears to be a potent T-cell mitogen which can induce suppressor T cells for Ig production. It also has a direct inhibitory effect on B-cell Ig production.