Subpopulations of human helper and suppressor T lymphocytes

Mitogen driven differentiation of normal human mononuclear cells is a a well-established model for the study of antibody synthesis in man. In certain rare individuals who are clinically normal, unfractionated mononuclear cells or a mixture of purified B plus T lymphocytes differentiate into immunoglobulin producing cells in response to purified protein derivative of tuberculin (PPD) but not in response to pokeweed mitogen (PWM). To evaluate this observation we have irradiated T cells from such individuals to eliminate naturally occurring suppressor T cell activity and then added the irradiated T cells back to autologous B cells before culture. The B cells then responded to PWM. The original PPD responses of cells from these individuals were now significantly reduced. Although, there was no difference between PWM nonresponders and responders in the number of OKT-8 positive cells, elimination of OKT-8 positive cells in the PWM nonresponders with OKT-8 monoclonal antibody and complement resulted in a significantly increased response to PWM. This study indicates that there are suppressor T cells which specifically inhibit B cell response to PWM without affecting the PPD response. These results also show that the helper T cells involved in the PWM response are radioresistant and those involved in the PPD response are radiosensitive.     

In vitro IgM and IgM rheumatoid factor production in response to Staphylococcus aureus Cowan I and pokeweed mitogen: the contribution of CD5+ (Leu 1) B cells.

The relation between the percentage of circulating CD5+ CD20+ B cells and the ability to synthesize IgM and IgM rheumatoid factor (RF) in vitro in response to pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SAC) was assessed in 21 healthy controls. CD5+ CD20+ cells ranged from 7.3 to 19.9% of total CD20+ B cells. By Spearman's rank correlation, there was an inverse relation between the percentage of CD5+ CD20+ B cells and IgM production in response to PWM (rs = -0.452, p less than 0.05) and a direct correlation with RF production in response to SAC (rs = 0.450, P less than 0.05). The percentage of CD5+ CD20+ B cells was not related to any serologic HLA-A, B, C or D antigen. Healthy individuals may be predisposed to producing IgM or autoantibodies based on the percentage of circulating CD5+ Cd20+ B cells.     

In vitro analysis of B lymphocyte function in uraemia.

We have investigated the immune responses in vitro of uraemic patients undergoing regular haemodialysis or continuous ambulatory peritoneal dialysis. Twenty-five healthy subjects were also studied as controls. In uraemic patients, the number of T and B lymphocytes were within the normal range, but proliferative responses to phytohaemagglutinin (PHA) were impaired. Spontaneous immunoglobulin plaque forming cell (PFC) responses by peripheral blood mononuclear cells (PBMC) from uraemic patients were significantly lower than those of healthy subjects. The PFC response of uraemic PBMC to the T cell independent polyclonal B cell activator (PBA) Epstein-Barr virus (EBV) was comparable to the response of the healthy subjects, indicating that uraemic B cells are still capable of synthesizing immunoglobulin. Pokeweed mitogen (PWM) induced PFC responses of uraemic PBMC were also normal, whereas the response to another T cell dependent B cell activator, Staphylococcus aureus Cowan I (SAC), was very low. Addition of indomethacin to PWM- and SAC-activated cultures of uraemic PBMC enhanced the PFC response to SAC, but had little effect on the PWM response. As full differentiation of B cells in response to SAC depends on helper T cells, we conclude that a defect in T lymphocyte function accounts for the reduced spontaneous and SAC induced production of immunoglobulin by uraemic PBMC. This defect may be mediated by an indomethacin-sensitive mechanism.     

In vitro synthesis of IgM rheumatoid factor by lymphocytes from patients with essential mixed cryoglobulinemia.

Peripheral blood mononuclear cells (PBMC) from patients with Essential Mixed Cryoglobulinemia (EMC) were studied for their ability to synthesize polyclonal IgM and rheumatoid factor (RF) IgM in vitro. Our results indicate: that EMC-PBMC produce smaller amounts of polyclonal IgM but higher quantities of IgM-RF than normal PBMC after pokeweed mitogen (PWM) or Staphylococcus aureus activation, so that the IgM-RF to total IgM ratio is significantly greater in EMC than in normal cultures; that enriched EMC-B lymphocytes display a significantly higher spontaneous synthesis of IgM-RF than normal B lymphocytes and that the IgM-RF B cell clones are receptive to T cell regulation. Taken together these findings suggest an expansion of B cell clones committed to IgM RF production and the presence in peripheral blood of differentiated B lymphocytes capable of secreting IgM-RF in EMC.     

The role of interleukin 2 in the regulation of proliferation and IgM synthesis of human newborn mononuclear cells.

The effect of interleukin 2 (IL-2) on cord blood mononuclear cells (CBMC) was studied, with special reference to B cell function. The study shows that CBMCs proliferate in response to recombinant interleukin 2 (rIL-2) without in-vitro preactivation. The response was also detected when whole blood cultures were used. CBMCs not preactivated in vitro proliferated in response to rIL-2 even after T cell and monocyte depletion. Thus, rIL-2 affects both non-T cells and T cells of newborns without preactivation in vitro. IL-2 receptors on unstimulated CBMCs appear to be distinct from Tac antigen, as the mean number of Tac antigen-positive cells among CBMCs was only 0.8%. Furthermore, rIL-2 inhibited PWM-induced proliferation and the response was more prominent in newborns than in adults. Thus, rIL-2 seems to cause a higher increase of suppressor cell function in CBMCs than in adult peripheral blood mononuclear cells. Neither rIL-2 nor Staphylococcus aureus Cowan 1 (SAC) stimulated any IgM synthesis, but a dose-dependent IgM synthesis was obtained by combining SAC and rIL-2. Alone, however, rIL-2 appears to be an insufficient factor to induce differentiation of SAC-activated cord blood B cells, as no IgM production in response to rIL-2 occurred in T cell and monocyte-depleted cell populations. The results of this study suggest both increased suppressor cell function and intrinsic B cell deficiency as causes of a weak humoral immune response in newborns.     

The effects of retinoic acid on immunoglobulin synthesis by human cord blood mononuclear cells

Derivatives of vitamin A have attracted considerable attention as agents which have immune potentiating properties and possibly tumor-suppressive effects. Recent investigations have shown that retinoic acid (RA) can augment immunoglobulin production of B-cell hybridomas from patients with immune deficiency. In this study we examined the ability of RA to modify the mitogen-induced polyclonal immunoglobulin synthesis of cord blood mononuclear cells (CBMC). RA in concentrations ranging from 10(-5) to 10(-7) M augmented IgM synthesis of CBMC in response to formalinized Cowans I strain Staphylococcus aureus (SAC) up to 45.6-fold which was greater at suboptimal responses to SAC. There were no changes in IgG or IgA synthesis and minimal effects on SAC-induced proliferative responses. RA did not produce similar changes in IgM synthesis of SAC-stimulated adult peripheral blood mononuclear cells (PBMC), and RA had no effect on the immunoglobulin synthesis of Epstein-Barr virus (EBV)-stimulated CBMC or adult PBMC. Time course studies showed that peak enhancement occurred when RA was added between 4 and 24 hr after culture initiation and required prior activation by SAC for augmentation of IgM synthesis. Cell separation experiments showed that prior incubation (18 hr) of an enriched T-cell fraction with RA enhanced the IgM synthesis of a T-cell-depleted B-cell fraction. These experiments and the findings that RA-induced augmentation of IgM production in response to SAC, but not to EBV suggest that the immunoregulatory effects of RA may be mediated by either T cells or T-cell products. Further studies will be necessary to understand the mechanism by which RA augments IgM synthesis of CBMC.     

IL-4 and IL-2 upregulate the expression of antigen B7, the B cell counterstructure to T cell CD28: an amplification mechanism for T-B cell interactions.

We recently generated mAb 104 which is specific for the B cell activation antigen Ag B7. With this we studied the regulation of Ag B7 expression on normal tonsillar B lymphocytes as well as the activities of B7+ and B7- activated B cells. SAC and to a lesser extent anti-IgM antibody upregulated Ag B7 and this was further enhanced by IL-2 and most notably IL-4. Ag B7 was expressed on virtually all sIgG+ and sIgA+ B cells and approximately half of the sIgD+ and sIgM+ B cells. SAC-stimulated B7+ B cells proliferated and produced IgM, IgG and IgA in response to IL-2 and IgM and IgG in response to IL-4. SAC-stimulated B7- B cells proliferated and produced only IgM in response to IL-2 and IL-4. Considering that Ag B7 has recently been shown to be the counterstructure of the T cell CD28 and that CD28 triggering strongly enhances cytokine production by T cells, it is likely that the CD28/B7 interaction represents an important amplification phenomenon in T-B cell interaction leading to humoral immune responses. The preferential expression of Ag B7 on IgG and IgA committed cells suggests that CD28/B7 interaction may be more specific to secondary antibody responses provided by memory T and B cells.     

Effect of dexamethasone on mechanisms responsible for regulation of polyclonal B-cell response

The effect of dexamethasone (Dex) on the differentiation of pokeweed mitogen (PWM), or Staphylococcus aureus Cowan I (SAC)-stimulated human peripheral blood mononuclear cells (PBMC), into immunoglobulin secreting cells (ISC) was studied with special emphasis on the regulatory role of IL-2 in these systems. Dex, known to reduce endogenous IL-2 production and expression of IL-2 receptors, reduced the proliferation of pokeweed mitogen-activated T-cells, and the proliferation was restored by exogenous recombinant interleukin 2 (rIL-2). Furthermore, Dex enhanced in PWM and in SAC-stimulated cultures, the number of ISC. Addition of rIL-2 resulted in a further increase of ISC in SAC-stimulated cultures, whereas in PWM-stimulated cultures the enhancing effect of Dex was reversed. When IL-2 receptors were blocked by a monoclonal anti-IL-2 receptor antibody rIL-2 was no longer suppressive. Addition of monocytes to PWM-stimulated cultures resulted in suppression or the number of ISC, which was even more pronounced when monocytes were pretreated with rIL-2. In contrast to ISC, neither a suppressive effect of rIL-2 nor an enhancing effect of Dex was observed when PWM-stimulated cultures were evaluated for cells with intracytoplasmic immunoglobulin (plasma cells). From these results we conclude that Dex, by blocking IL-2 production and receptor expression, interferes with IL-2 mediated induction and/or activation of suppressor mechanisms.     

Cord blood B cell differentiation. Synergistic effect of pokeweed mitogen and Staphylococcus aureus on in vitro differentiation of B cells from human neonates

B lymphocyte differentiation into immunoglobulin secreting cells is a process depending on the presence of functionally mature B lymphocytes, monocytes and regulatory T lymphocytes. Cord blood B lymphocytes present in isolated cord blood mononuclear cell (MNC) preparations are normally unable to differentiate into immunoglobulin secreting plaque forming cells (PFC) when cultured in the presence of pokeweed mitogen (PWM) alone or killed Staphylococcus aureus Cowan 1 (SAC1) alone. However, each one of these activators induces PFC formation by B lymphocytes in adult MNC cultures. In the present study we show that these two activators can act synergistically to induce a significant in vitro PFC response in cord blood MNC's. The synergism of PWM and SAC1 exhibits a requirement for a specific sequence of addition in order to induce a positive response in neonatal cells. If both activators are not added simultaneously at the initiation of culture, only the initial addition of SAC1 followed by PWM will result in increased PFC production. The action of PWM and SAC1 on cord blood MNC can each be replaced by conditioned media. Supernatants from monocyte cultures containing soluble factors such as interleukin-1 (IL-1) can substitute for the activity of SAC1 while supernatants containing soluble T-cell factors (interleukin-2[IL-2], T cell replacing factor (TRF), B cell differentiation factor (BCDF), etc) can replace PWM in the cord blood MNC cultures. The results suggest that the synergistic effect of these two activators overcomes a partial immaturity or an excessive suppressor activity of human cord blood MNC.     

The activation of T cells with Staphylococcus aureus Cowan 1 (SAC)

We reported our findings on the activation and functional capacity of human T cells stimulated by Staphylococcus aureus Cowan 1 (SAC). Serial determinations of surface markers on T cells stimulated by SAC showed that OKT4+ T cells remained relatively constant with the decrease of OKT8+ T cells and that Tac antigen was predominantly expressed on OKT4+ T cells. When the mixture of OKT4+-depleted T cells and OKT8+-depleted PBL was stimulated with SAC or PWM, PWM-stimulated OKT4+-depleted T cells suppressed immunoglobulin production by OKT8+-depleted PBL in a dose-dependent fashion, but SAC-stimulated OKT4+-depleted T cells did not show suppressive activity.     

Immunoglobulin production of human lymphocytes stimulated by Staphylococcus aureus Cowan I and pokeweed mitogen: differential effects of recombinant interleukin-2.

The number of immunoglobulin-secreting cells (ISC) upon stimulation with pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I (SAC) in recombinant interleukin-2 (rIL-2)-supplemented cultures of human peripheral blood mononuclear cells (PBMC) and co-cultures of B and T cells was studied. It has been shown that the addition of rIL-2 to culture can enhance or depress the number of ISC depending on the polyclonal B-cell activator used for culture stimulation. The SAC-induced response was enhanced in the presence of rIL-2, while the number of ISC in PWM-stimulated cultures was depressed. Moreover, in cultures stimulated simultaneously by both activators, the suppressive effect of rIL-2 prevailed, indicating that the reported direct effect of the lymphokine on SAC-activated B cells cannot overcome the suppressive activity of PWM-induced suppressor T cells. rIL-2 could not activate suppressor T cells in the absence of PWM, and has no effect on the number of helper or suppressor cells in the culture.     

Staphylococcus aureus Cowan I-induced human immunoglobulin responses: Preferential IgM rheumatoid factor production and VH3 mRNA expression by protein A-binding B cells

Protein A (PA), a cell wall component of SAC, activates human B cells by cross-linking the Fabs of membrane immunoglobulins. Recent data indicate that PA binds only to Fabs that use VH3 heavy chains, and thus it has been designated as a B-cell superantigen. We previously reported thatStaphylococcus aureus Cowan I (SAC) -induced IgM rheumatoid factor (RF) by human PBMC was mediated by PA. Therefore, we sought to determine if SAC-induced IgMRF production was confined to PA-binding B cells and if these B cells were enriched for the expression of VH3 heavy chains. We observed that the elicitation of IgMRF in response to SAC was limited to a subset of B cells that bind PA and that this subset was enriched for VH3 mRNA expression. Taken together, these results suggest that IgMRFs produced in response to SAC are enriched for usage of VH3 heavy chains. Thus, this SAC-induced autoantibody response appears to represent a new B-cell superantigenic property of PA.     

Characterization of the suppressor activity in lymphocytes from patients with common variable hypogammaglobulinemia: evidence for an associated primary B-cell defect

The pathogenetic mechanisms responsible for the impaired immunoglobulin production in common variable hypogammaglobulinemia (CVH) are diverse with abnormalities in both B cells and immunoregulatory T cells. Production of IgG, IgM, and IgM-rheumatoid factor (IgM-RF) was measured in pokeweed mitogen (PWM) or Epstein-Barr virus (EBV)-stimulated cultures using various combinations of CVH, cord blood mononuclear cells (CBMC), and normal adult control B and T cells. The following results were obtained. First, the proportion of OKT3+ and OKT8+ cells were increased in CVH patients. Second, the T cells from four CVH patients and CBMC suppressed PWM-induced IgG, IgM, and IgM-RF production by normal B cells. Furthermore, major suppressor activity was found in the OKT8+ T-cell subpopulations in CBMC and three out of four CVH patients. There was no significant difference in relative suppression by OKT8+ cells from normal adults, CVH patients, or CBMC. However, in one CVH patient suppressor T cells were found in both OKT4+ as well as OKT8+ fractions. In the CVH patient with OKT4+ suppressor cells, X irradiation (1250 rads) abrogated suppressor activity and restored helper activity in the OKT4+ T-cell fraction. Irradiation of normal OKT4+ cells did not increase helper activity. When non-E-rosetting cells from normal subjects, CVH, and CBMC were stimulated with EBV it was observed that normal adult B cells could be induced to secrete IgG, IgM, and Ig-RF whereas CVH and CBMC could only produce IgM and IgM-RF but not IgG. The present study demonstrates for the first time that a radiosensitive OKT4+ suppressor cell is present in some CVH patients.     

Induction of IgM production by pokeweed mitogen and interleukin-2: different responses by human peripheral blood cells and tonsil cells

Stimulation of tonsils or peripheral blood lymphocytes with either pokeweed mitogen (PWM) or interleukin-2 (IL-2) results in significant IgM production. However, the combination of PWM and IL-2 shows a synergistic IgM synthesis in tonsil cells, whereas the secretion in peripheral blood cells is diminished compared to activation with either stimulus alone. The heavy-chain isotype distribution does not significantly change in the two cell types when stimulated with either PWM, IL-2, or the combination. The Ig synthesis by tonsil cells to PWM or IL-2 drastically increases in the presence of monocytes, but the response to PWM + IL-2 together is not affected. The reason for the synergistic IgM production to PWM + IL-2 in tonsil cells is the relative lack of monocytes and the low number of T8+-suppressor cells. The decreased IgM production in peripheral blood cells after stimulation with both PWM and IL-2 is due to the presence of T8+ cells. These data show that the monocyte-dependent IL-2 production is an essential step in PWM-induced Ig secretion; the subsequent induction of the T-cell-helper signal for B-cell differentiation is determined by the balance between the strength of the stimulus for T cells and the T4/T8 ratio. Hence, these parameters should be included when the B-cell function is studied in vitro with the use of PWM and IL-2.     

Relationship between T cell subpopulations and the mitogen responsiveness and suppressor cell function of peripheral blood mononuclear cells in normal individuals

A simultaneous analysis was made of numbers and proportions of T cell subsets (T mu and T gamma cells), lymphocyte responsiveness to non-specific mitogens in vitro and 'short-lived suppressor cell activity' in peripheral blood mononuclear cells (PBMC) of normal individuals. No correlation was found between either T gamma or T mu cells and the 'short-lived suppressor cell activity', suggesting that suppression in this system is not a reflection of quantitative alteration in these subsets. However, a highly significant positive correlation was found between numbers of T mu cells and PBMC responses to the mitogens phytohaemagglutinin, concanavalin A and pokeweek mitogen. This may reflect either a helper effect of T mu cells on lymphocyte proliferation in response to mitogens or the presence of the majority of mitogen-responsive cells within this subpopulation. As in normal individuals lymphocyte responsiveness correlates with the number of circulating T mu cells, it is possible that a reduction in these cells in disease states may contribute to defects in cell-mediated immunity.     

The CD8+ Leu-7+ subset of T cells in Crohn''s disease: distinction between cytotoxic and covert suppressor functions.

An expanded T cell subpopulation (CD8+ Leu-7+) has previously been reported in the peripheral blood of patients with Crohn's disease. This subpopulation of T cells was associated with a 'covert suppressor' function, particularly in patients with mild/early Crohn's disease, suppressing immunoglobulin production in vitro when cultured in the presence of pokeweed mitogen. T cells with the same CD8+ Leu-7+ phenotype have also been shown to exhibit non-major histocompatability complex-restricted cytotoxicity when triggered by anti-CD3 antibodies, and this cytotoxic activity has also been shown to be elevated in patients with Crohn's disease. Because cytotoxic cells can have immunoregulatory properties, we investigated the possible relationship between the cytotoxic and 'covert suppressor' functions of the CD8+ Leu-7+ subset of T lymphocytes in patients with mildly active Crohn's disease. Although the correlation between T cell cytotoxic activity and the CD8+ Leu-7+ cells was confirmed, no evidence for covert suppressor activity was found; there were no significant differences between the amount of IgM secreted by B cells from normal subjects and patients with Crohn's disease when cultured with T cells at increasing T:B ratios. In addition, IgM production by peripheral blood B cells did not correlate with either the number of CD8+ Leu-7+ cells or with the level of cytotoxic T cell activity. Furthermore, when B cells and CD4+ T cells were co-cultured with increasing numbers of CD8+ T cells, there was no evidence for excessive suppressor T cell activity in Crohn's disease. Although some patients exhibited low levels of IgM production, this was due to diminished B cell function, rather than excessive T suppressor activity or defective T helper activity. We conclude that the CD8+ Leu-7+ T cell subset is associated with cytotoxic but not with enhanced or covert suppressor activity in Crohn's disease. The previously described covert suppressor function attributed to cells with this phenotype in Crohn's disease was not found to account for diminished B cell responsiveness in vitro and is unlikely to be of major pathophysiologic significance in the majority of patients.     

Bacterial peptidoglycan induces in vitro rheumatoid factor production by lymphocytes of healthy subjects.

The present studies were carried out to further characterize the polyclonal B cell activating properties of bacterial peptidoglycan (PG) and to determine if this ubiquitous agent induces in vitro IgM rheumatoid factor (RF) production by lymphocytes from healthy volunteers. Peripheral blood mononuclear cells (PBMC) were cultured in the presence of peptidoglycan, pokeweed mitogen (PWM), a standard polyclonal B cell activator, or additional culture medium. Supernatants were harvested on days 7-8 for determination of total IgM, total IgG, and IgM RF by an enzyme-linked immunosorbent assay (ELISA). PG and PWM induced comparable amounts of total IgM production but PG was a less potent stimulant of total IgG production. PG induced in vitro IgM-RF production in 9/33 experiments, a frequency of response of less than that observed in corresponding PWM stimulated cultures (22/33 experiments). PG-induced IgM-RF production depended upon active protein synthesis and did not correlate with the magnitude of PG-induced total IgM production. The latter finding suggests that PG-induced IgM-RF may not merely reflect polyclonal B cell activation. These results add to a growing list of PG's functional properties and provide further impetus for considering this ubiquitous agent as a potential stimulant for in vivo RF production.     

IL-2 enhances polyclonal IgM but not IgM-rheumatoid factor synthesis by activated human peripheral blood B cells.

IgM-rheumatoid factor (RF) is thought to be involved in the pathogenesis of rheumatoid arthritis (RA). Several cytokines are known to regulate immunoglobulin synthesis. In this study the effects of IL-2 on polyclonal IgM and IgM RF synthesis were compared. Cytokines were added to peripheral blood B cells from normal subjects and patients with RA after activation by Staphylococcus aureus Cowan 1 (SAC). The addition of IL-2, but not IL-4 or IL-6, resulted in significant enhancement of IgM synthesis in cultures from both healthy subjects and patients with RA. Similar degrees of enhancement were seen in both peripheral blood mononuclear cell and highly purified B cell cultures. IgM-RF was synthesized after activation in cultures from healthy subjects and spontaneously in cultures from RA patients. In contrast to polyclonal IgM synthesis, IL-2 failed to augment IgM-RF synthesis in cell cultures from either healthy subjects or RA patients. This study demonstrates different effects of IL-2 on IgM and IgM-RF synthesis.     

Specific antibody responses by high- and low-density human peripheral blood B cells: T-helper cells and T-cell replacing factor (TRF) act on different B-cell subpopulations.

Antibody production to influenza A strain virus X31 (H3N2) was measured in cultures of peripheral blood mononuclear cells (PBMC) stimulated with either antigen (X31) or pokeweed mitogen (PWM). With some donors, X31 antibody was produced in response to antigenic stimulation, but not as part of the polyclonal response to PWM, suggesting that antigen and PWM may be acting on different B-cell subpopulations. To test this hypothesis, T-cell depleted PBMC (E-) cells were fractionated on discontinuous Percoll gradients and assayed for antibody production in response to antigen or PWM. Fraction I (FrI = SG less than 1.070) cultured in the presence of T cells responded well to PWM, but not at all to X31. FrII (1.070 less than SG less than 1.075) and FrIII (SG greater than 1.075) cultured in the presence of T cells both responded well to X31, but only the medium-density B cells (FrII) were able to make specific antibody when T cells were replaced with T-cell replacing factor (TRF). Specific X31 antibody responses by medium- and high-density B cells (FrII and FrIII) were suppressed equally by the addition of allogeneic T-suppressor (Ts) cells. When allo-activated Ts cells were inactivated by irradiation, allogeneic T-helper (Th) cells were able to collaborate with both FrII and FrIII B cells in specific antibody responses to X31. Since TRF was not able to substitute for T cells in specific antibody responses by FrIII B cells, this result shows that allogeneic T-cell help was not mediated by non-specific 'allogeneic effect' factors and apparently requires cognate T cell-B cell interactions.     

Relationship between immune system and gram-negative bacteria. III. Functional analysis of human peripheral blood lymphocyte subpopulations separated by cytoadherence with Salmonella minnesota Rb.

Spontaneous adherence of bacteria to human peripheral blood mononuclear cells (PBMC) represents a useful tool for analysis of lymphocyte subsets with different functions. We have recently shown that PBMC can be divided into 2 populations based on their ability to bind Salmonella minnesota R345 (Rb) bacteria. By using these procedures, here, we provide evidence that Rb-bound and Rb-unbound PBMC populations give similar proliferative responses to phytohemagglutinin (PHA) and concanavalin A (Con A), while the pokeweed mitogen (PWM)-induced proliferative and differentiative responses are higher in the Rb-unbound than in the Rb-bound PBMC fraction. Moreover, enhanced PWM-induced responses are obtained in Rb-unbound cell cultures enriched for T4+ cells. When B (non-E rosetting) cells are cultured with purified T lymphocytes from the Rb-bound (T-Rb+) and Rb-unbound (T-Rb-) fractions, comparable PWM-induced mitogenic responses are observed. The T-Rb- population contains a higher percentage of cells expressing T4+ phenotype, and when added to B cell cultures a more elevated PWM-induced IgA, IgG and IgM synthesis is observed than in B cell cultures containing T-Rb+ cells. These results suggest that the T-Rb- fraction is enriched for T cells which help IgA, IgG and IgM responses.