Infusion of mesenchymal stem cells and rapamycin synergize to attenuate alloimmune responses and promote cardiac allograft tolerance.

The inherent immunosuppressive properties and low immunogenicity of mesenchymal stems cells (MSCs) suggested their therapeutic potential in transplantation. We investigated whether MSCs could prolong allograft survival. Treatment involving infusion of MSCs into BALB/c recipients 24 hours after receiving a heart allograft from a C57BL/6 donor significantly abated rejection and doubled graft mean survival time compared to untreated recipients. Furthermore, combination therapy of MSCs and low-dose Rapamycin (Rapa) achieved long-term heart graft survival (>100 days) with normal histology. The treated recipients readily accepted donor skin grafts but rejected third-party skin grafts, indicating the establishment of tolerance. Tolerant recipients exhibited neither intragraft nor circulating antidonor antibodies, but demonstrated significantly high frequencies of both tolerogenic dendritic cells (Tol-DCs) and CD4(+)CD25(+)Foxp3(+)T cells in the spleens. Infusion of GFP(+)C57BL/6-MSCs in combination with Rapa revealed that the GFP-MSCs accumulated in the lymphoid organs and grafts of tolerant recipients. Thus, engraftment of infused MSCs within the recipient's lymphoid organs and allograft appeared to be instrumental in the induction of allograft-specific tolerance when administered in combination with a subtherapeutic dose of Rapamycin. This study supports the clinical applicability of MSCs in transplantation.

Receptor-interacting protein 2 is a marker for resolution of peritoneal dialysis-associated peritonitis.

There are no predictive factors for peritoneal dialysis-associated peritonitis; however, its resolution correlates with a cell-mediated Th1 immune response. We tested the hypothesis that induction of receptor-interacting protein 2 (RIP2), an assumed kinase linked with Th1 responses, is a useful marker in this clinical setting. Basal RIP2 expression was measured in human immune cells and during dialysis-associated peritonitis. RIP2 increased with bacterial toxin cell activation and the temporal profile for this differed depending on immune cell involvement in the innate or adaptive phases of the response. Importantly, RIP2 expression increased in peritoneal immune cells during dialysis-associated peritonitis and this upregulation correlated with clinical outcome. An early induction in peritoneal CD14(+) cells correlated with rapid resolution, whereas minimal induction correlated with protracted infection and with catheter loss in 36% of patients. These latter patients had higher levels of MCP-1 consistent with a delayed transition from innate to adaptive immunity. Our study shows that upregulation of RIP2 is a useful marker to monitor dialysis-associated peritonitis and in predicting the clinical outcome of these infections.

CTLA-4 (CD152) can inhibit T cell activation by two different mechanisms depending on its level of cell surface expression.

CTLA-4 (CD152) engagement results in down-regulation of T cell activation. Two mechanisms have been postulated to explain CTLA-4 inhibition of T cell activation: negative signaling and competitive antagonism of CD28:B7-mediated costimulation. We assessed the contributions of these two mechanisms using a panel of T cell lines expressing human CTLA-4 with mutations in the cytoplasmic region. Under conditions of B7-independent costimulation, inhibition of IL-2 production following CTLA-4 engagement required the CTLA-4 cytoplasmic region. In contrast, under B7-dependent costimulation, inhibition of IL-2 production by CTLA-4 engagement was directly proportional to CTLA-4 cell surface levels and did not require its cytoplasmic region. Thus, CTLA-4 down-regulates T cell activation by two different mechanisms-delivery of a negative signal or B7 sequestration-that are operational depending on the levels of CTLA-4 surface expression. These two mechanisms may have distinct functional outcomes: rapid inhibition of T cell activation or induction of T cell anergy.

The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation.

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces zeta-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules.

Specific CD3 epsilon association of a phosphodiesterase 4B isoform determines its selective tyrosine phosphorylation after CD3 ligation.

cAMP-specific phosphodiesterases (PDE) comprise an extensive family of enzymes that control intracellular levels of cAMP and thus regulate T cell responses. It is not known how the function of these enzymes is altered by TCR engagement. We have examined this issue by studying one of the PDE isozymes (PDE4B). PDE4B RNA and protein were detected in resting PBLs, and the levels of PDE4B protein increased with cell cycling. In peripheral blood T cells, two previously reported PDE4B isoforms could be detected: one was 75-80 kDa (PDE4B1) and the other was 65-67 kDa (PDE4B2). These two isoforms differed in their N-terminal sequence, with the presence of four potential myristylation sites in the PDE4B2 that are absent in PDE4B1. Consequently, only PDE4B2 was found in association with the CD3var epsilon chain of the TCR. In addition, although both isoforms were phosphorylated in tyrosines in pervanadate-stimulated T cells, only the TCR-associated PDE4B2 was tyrosine-phosphorylated following CD3 ligation. The kinetics of phosphorylation of TCR-associated PDE4B2 correlated with changes in cAMP levels, suggesting that tyrosine phosphorylation of the TCR-associated PDE4B isoform upon engagement of this receptor may be an important regulatory step in PDE4B function. Our results reveal that selectivity of PDE4B activation can be achieved by differential receptor association and phosphorylation of the alternatively spliced forms of this PDE.

The complete genomic sequence of an HTLV-II isolate from a Guahibo Indian from Venezuela.

A polyclonal CD3(+), CD8(+) T-cell line, G2, was derived from the peripheral blood of a seropositive, PCR-positive, HTLV-IIB infected Guahibo Indian from Venezuela. The cell line is productively infected with HTLV-IIB. The entire HTLV-II G2 proviral DNA was sequenced via PCR using overlapping HTLV-II primer pairs. Phylogenetic analyses indicate that HTLV-II G2 is the most divergent HTLV-IIB strain identified to date. Characterization of its deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

T cell hyperreactivity to IL-6 in chronic nonviremic HBV carriers despite normal IL-6 receptor or gp130 expression.

We previously showed that T cells from chronic nonviremic HBsAg carriers activated with immobilized OKT3 MAb are hyperreactive to monocyte accessory signals, mainly to interleukin-6 (IL-6). We have further characterized this T cell hyperreactivity using phytohemagglutinin (PHA) as the primary activating signal. PHA-stimulated T cells from nonviremic patients had a significantly higher response to addition of monocytes, monocyte supernatants, and IL-6 alone or combined with IL-1 beta when compared to controls. We examined if these effects could be mediated by a differential expression of IL-6 receptor (p80) or gp130 on resting or PHA-stimulated T cells. We found that PHA, IL-6, IL-1 beta, or IL-2 induced only small changes of the dull p80 expression on T cells. In contrast, we found a significant increase of gp130 expression on PHA-activated T cells compared to unstimulated T cells, which was down-regulated by the presence of IL-6. However, no significant differences in p80 or gp130 expression were detected between patients and controls within all the culture conditions tested. Our results confirm that IL-6 is involved in the in vitro T cell hyperreactivity of nonviremic HBV carriers and indicate that this effect is not mediated by disturbances of IL-6 receptor expression.

[Immunoclinical, molecular and immunopathologic approach to chronic viral hepatitis. Therapeutic considerations]. / Abordaje inmunoclinico, molecular e inmunopatológico de la hepatitis crónica viral. Consideraciones terapeuticas.

Through a pilot study which includes a clinical, molecular and immunopathological approach to the chronic Hepatitis induced by HBV or by HCV, we determined that 66% of HBsAg carriers are in the "non viremic" phase. The positive HBeAg "viremic" carriers showed HBV-DNA quantitation which varies between > 50 pg to > 100 pg. Both types of carries are infected with the "wild" type HBV. Each subgroup of positive surface antigemia carriers demonstrated a differential immunopathological response. So far, 96% of the HCV carriers investigated, showed HCV-RNA associated to repeatedly positive anti-HCV antibodies. Those patients with increased ALT values uniformly expressed liver histopathological signs of inflammation caused by HCV; demonstrating also the presence of peripheral blood mononuclear cells infected with HCV. At the present, the genotypes investigation indicates a predominance of HCV genotype II (1b). Autoimmune phenomenons associated to HCV have been detected only in 3 patients. The therapeutic approach with interferon alpha applied to the HCV infection preliminary showed similar results to those reported worldwide. Currently, a comprehensive approach to the chronic HBV and chronic HCV infections requires the application of Immunochemistry, Molecular Biology and Cellular Immunology combined technologies.

CD3- and CD28-activating pathways in HCV infection.

We investigated the proliferative response and IL-2 receptor (IL-2R) expression in peripheral blood mononuclear cells (PBMC) activated with anti-CD3 mAb alone or in combination with anti-CD28 mAb in a group of hepatitis C virus (HCV)-infected patients with detectable viremia demonstrated by "nested" PCR. PBMC from HCV patients and controls showed similar proliferative responses either to anti-CD3 mAb, 64.1, and/or to anti-CD28 mAb, 9.3. No differences were found in anti-CD3 or anti-CD3 plus anti-CD28-induced proliferative responses between patients who demonstrated circulating PBMC bearing HCV-RNA when compared to those with negative HCV-RNA PBMC. Moreover, flow cytometry studies confirmed that anti-CD3 alone or in combination with anti-CD28 were able to induce a significant increase of IL-2R expression in patients or controls PBMC. Both groups showed similar basal CD28 expression. These results indicate that both CD3- and CD28-activating pathways are preserved in HCV-infected patients with chronic active liver disease.

Anti-CD3-activated T cells from chronic nonviremic HBV carriers are hyperreactive to monocytic accessory signals.

We analyzed T cell responses through the CD3 activation pathway in a group of chronic HBV carriers. PBMC stimulated with the mAb OKT3 showed higher proliferative response in HBV-DNA(-) carriers compared to HBV-DNA(+) carriers and to controls. In contrast, no differences in proliferative responses were observed between HBV-DNA(-) carriers and controls in cell cultures stimulated with immobilized 64.1 mAb (SPB-64.1) which induces proliferation in the absence of monocytes. We further examined T cell responses in the presence of monocytes and their soluble factors to immobilized OKT3 mAb (SPB-OKT3). Purified T cells did not proliferate to SPB-OKT3. When autologous monocytes were added, higher proliferative response, IL-2 production, and IL-2 receptor expression were observed in HBV-DNA(-) carriers than in controls. An enhanced cell proliferation was also obtained when monocyte supernatants were added to T cells cultured with SPB-OKT3. Moreover, when IL-6 alone or combined with IL-1 was added to SPB-OKT3-stimulated T cell cultures, a significantly higher increase in T cell proliferation was detected in HBV-DNA(-) carriers. Our results thus show a T cell hyperreactivity to accessory signals from monocytes (mainly IL-6) in HBV-DNA(-) carriers, that is probably related to an ongoing viral clearance.

Enhanced CD3-mediated T lymphocyte proliferation in patients with systemic lupus erythematosus.

Nonfractionated peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE) showed enhanced proliferative responses when stimulated via the CD3 pathway. In contrast, proliferative responses induced by phytohemagglutinin were diminished in SLE patients. Levels of CD3-induced interleukin-2 production and interleukin-2 receptor expression were comparable with normal levels. Highly purified T cells also showed augmented CD3 responses, but only in the presence of phorbol myristate acetate or a combination of phorbol myristate acetate plus calcium ionophore A23187, and not with calcium ionophore alone. The data suggest integrity of the T cell receptor/CD3 pathway for T cell activation in patients with SLE, as examined in cultures stimulated with specific anti-CD3 monoclonal antibodies rather than with multivalent lectins. An increased response via the CD3 complex could contribute to the autoimmune activity in human SLE.

Differences in the stimulating capacity of immobilized anti-CD3 monoclonal antibodies: variable dependence on interleukin-1 as a helper signal for T-cell activation.

Monoclonal antibodies to the CD3 antigen on human T lymphocytes have been shown to induce accessory cell-dependent T-cell activation. One function of the accessory cells is cross-linking of CD3 by Fc receptor-binding of the anti-CD3 antibodies. Whether additional accessory signals are still required when anti-CD3 is presented in immobilized form is controversial. In the present study we stimulated purified human T cells with several anti-CD3 monoclonal antibodies, which were immobilized by coating the culture wells with goat anti-mouse IgG. A first group of immobilized anti-CD3 antibodies (anti-Leu-4, UCHT1, anti-T3, WT32 and 64.1) induced vigorous T-cell proliferation in the complete absence of monocytes, even when anti-interleukin-1 beta antiserum was added to the cultures. Other immobilized anti-CD3 antibodies (OKT3, WT31) required interleukin-1 beta in order to induce T-cell proliferation. However, when OKT3 was immobilized by direct coating of the culture wells with OKT3, it was also able to induce accessory cell-independent production of interleukin-2 and T-cell proliferation. Interleukin-1 beta further enhanced the interleukin-2-dependent proliferative response and it could provide help to induce proliferation at doses of immobilized OKT3 which, by themselves, were insufficient for full T-cell activation. We conclude that the requirement for interleukin-1 beta to induce interleukin-2-dependent proliferation of T cells when stimulated with anti-CD3 antibodies is not absolute, but depends on the CD3 epitope recognized, on the way of antibody presentation, on the antibody concentration and on other, still undefined, characteristics of the monoclonal antibodies used.

IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation.

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.

Accessory cell-derived helper signals in human T-cell activation with phytohemagglutinin: induction of interleukin 2-responsiveness by interleukin 6, and production of interleukin 2 by interleukin 1 [corrected].

Interleukins (IL-) 1 and 6 have been shown to represent accessory signals for T-cell activation. In the present study, we further examined the effects of both cytokines on accessory cell-depleted human T cells stimulated with phytohemagglutinin (PHA). The addition of IL-6 to the cultures resulted in T-cell proliferation; however, IL-1 was unable to support PHA-induced T-cell growth. The addition of IL-1 consistently induced a low level of IL-2 production and strongly enhanced T-cell proliferation in the presence of IL-6. Thus, the effect of IL-1 on T-cell growth becomes apparent only in the presence of IL-6. Blocking the IL-2-receptor (IL-2R) with the monoclonal antibodies anti-Tac and MikBêta 1 (directed to the alpha and bêta chains of the IL-2R, respectively) had no effect on PHA/IL-6-supported proliferation, but completely eliminated the growth-enhancing effect of IL-1. On the other hand, a neutralizing anti-IL-4-antiserum did not affect PHA/IL-6- or PHA/IL-6/IL-1-induced proliferation. Further experiments showed that IL-6 enhances T-cell responsiveness to IL-2, as evidenced by enhanced IL-2-induced proliferation. However, we could not find an effect of IL-6 on the expression of IL-2R as measured by staining with anti-Tac and with MikBêta 1 or by binding of (125I)-IL-2 to T cells. It can be concluded from these studies that IL-1 and IL-6 have different helper effects on PHA-induced T-cell activation. In the presence of PHA, IL-6 induces limited IL-2/IL-4-independent growth, and more importantly it renders T cells responsive to IL-2. IL-1 provides a signal leading to IL-2 production. The combination of IL-1 and IL-6 represents a synergistic helper signal, leading to an IL-2-dependent pathway of proliferation.

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens.

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.

Human T cell activation with phytohemagglutinin. The function of IL-6 as an accessory signal.

T cell proliferation induced by the lectin PHA requires the presence of monocytes. Here, we report that IL-6 represents part of the monocyte-derived helper activity for human T cell stimulation with PHA. Resting T cells isolated from peripheral blood or tonsils and completely depleted of accessory cells (monocytes and NK cells) did not proliferate in response to PHA alone. Addition of a cell-free monocyte culture supernatant (not containing IL-2) to the PHA-stimulated cultures resulted in T cell proliferation. We identified IL-6 as an essential helper factor in these monocyte supernatants, because antisera to IL-6 (but not anti-IL-1-beta) completely neutralized the helper effect of the monocyte supernatant. Moreover, addition of purified human IL-6 (but not IL-1-beta) to T cell cultures resulted in proliferation of PHA-stimulated T cells. Although IL-1 could not restore the PHA-induced proliferative response of isolated T cells by itself, it strongly enhanced the helper effect of IL-6. In contrast to intact monocytes, IL-6 did not induce detectable IL-2 production in cultures of PHA-stimulated T cells. Furthermore, an anti-IL-2R mAb (anti-Tac) did not block the proliferative response induced by PHA and IL-6, suggesting that an IL-2-independent pathway of T cell proliferation was involved. In conclusion our results show that human IL-6, previously identified as a B cell hybridoma growth and a B cell differentiation factor, represents part of the monocyte helper function for PHA-induced human T cell activation and proliferation.

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2.

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.

Defect in the membrane expression of high affinity 72-kD Fc gamma receptors on phagocytic cells in four healthy subjects.

Three different receptors for the Fc portion of IgG (FcR) have been characterized on human leukocytes. We have identified four healthy members of one family, whose blood phagocytic cells lack functional 72 kD high-affinity FcRI. Their monocytes were unable to bind the Fc portion of mouse (m)-IgG2a and of monomeric human IgG, and they were unreactive with two anti-FcRI monoclonal antibodies. Thus, FcRI is either absent, expressed at very low density, or is so structurally altered as to be unable to bind both its ligand and the anti-FcRI antibodies. The failure to bind the Fc portion of mIgG2a underlies the previously reported inability of these monocytes to support T cell mitogenesis on OKT3 stimulation. FcRI was not inducible upon incubation of their monocytes or neutrophils in gamma interferon. However, their monocytes were able to bind aggregated human IgG, and to phagocytose IgG-coated particles in vitro. Both functions could be blocked with a monoclonal antibody to the 40-kD low-affinity FcRII and therefore apparently were mediated exclusively through FcRII. This also demonstrates that FcRII can mediate phagocytosis independently. Despite the FcRI defect, these subjects had no circulating immune complexes, no evidence of autoimmune pathology and no increased susceptibility to infections.

Down-regulation of immunoglobulin and IgM-rheumatoid factor synthesis by oral treatment of rheumatoid arthritis patients with a nonsteroidal antiinflammatory drug.

We examined the effect of treatment with piroxicam, a nonsteroidal antiinflammatory drug (NSAID), on immunoglobulin (Ig) and IgM-rheumatoid factor (IgM-RF) synthesis in vitro by lymphocytes of patients with rheumatoid arthritis (RA). Oral treatment with piroxicam induced a progressive decrease of spontaneous IgM-RF production by unstimulated lymphocyte cultures during 12 weeks of observation. Also, pokeweed mitogen (PWM)-driven Ig synthesis was significantly diminished and the effect on total IgM production was sustained until the end of the study. This modulation of humoral responses is consistent with the drop in RF sera level. In addition, we also showed that treatment with NSAIDs can decrease RF levels in the synovial space. NSAIDs may have a long-term beneficial effect in patients with RA due to their modulatory role of lymphocyte responses.

Helper activity by human large granular lymphocytes in in vitro immunoglobulin synthesis.

In the present study we have examined the effect of human large granular lymphocytes (LGL) from healthy donors on Ig synthesis by autologous B lymphocytes. The results showed that this cell population has a consistent helper activity in pokeweed mitogen-activated cultures even when added at very low numbers. LGL can mediate their effect by secreting soluble helper factors capable of modulating B-cell responses as evidenced by the enhancement of IgG and IgM production by supernatants obtained from LGL cultures. Preincubation with interferon gamma further potentiated the helper activity by LGL.