Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore.

Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH)2D3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. In conclusion: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH)2D3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.     

Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation

Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.     

Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas

We found a unique thymocyte growth-promoting activity in supernatants (SN) from subclones of the B cell lymphoma CH12.LX. We have tentatively named this activity B-TCGF (for B cell-derived T cell growth factor) and characterized the activity produced by the CH12.LX.4866 subclone. This SN did not induce thymocyte proliferation alone, however, it enhanced both adult and fetal (Day 15 of gestation) murine thymocyte proliferation in the presence of IL-2, IL-4, or IL-7. Other known cytokines were screened for a B-TCGF-like activity using both adult and fetal thymocytes. IL-6 was found to be active only on adult thymocytes, while TNF alpha and GM-CSF were found to be active only on fetal thymocytes. However, neutralizing antibodies against these cytokines did not block the B-TCGF activity present in CH12.LX.4866 using either adult or fetal thymocytes. These observations suggest that the B-TCGF activity is mediated by a novel factor(s). The apparent molecular weight of this novel molecule(s) was 27-50 kDa determined by sizing HPLC.     

Transforming growth factor-beta s are equipotent growth inhibitors of interleukin-1-induced thymocyte proliferation

The effects of two forms of transforming growth factor-beta, TGF-beta 1 and TGF-beta 2, upon the proliferative response of murine thymocytes were investigated in this study. TGF-beta 1 and TGF-beta 2 were found to be equipotent growth inhibitors of interleukin-1 (IL-1)- and phytohemagglutinin (PHA)-stimulated thymocytes when added at the initiation of the cultures. These factors suppressed the proliferative response in a dose-dependent fashion between 0.4 and 100 pM. The proliferative response was maximally inhibited (90% inhibition) at 100 pM. The half-maximal inhibitory dose (ID50) was 6 and 4 pM for TGF-beta 1 and TGF-beta 2, respectively. These factors were less effective or ineffective at suppressing the proliferation of thymocytes which had been prestimulated for 24 to 48 hr by IL-1 and PHA. Neither factor inhibited interleukin-2 (IL-2)-dependent thymocyte proliferation or the proliferation of an IL-2-dependent cytotoxic T cell line (CTL-L), suggesting that the anti-proliferative actions of these factors was by inhibition of cellular events triggered by IL-1. Furthermore, anti-TGF-beta 1 antibodies did neutralize the biological actions of TGF-beta 1 and these antibodies did block the binding of 125I-labeled TGF-beta 1 to cell surface receptors showing that the inhibitory action is mediated through specific receptors for TGF-beta 1 on thymocytes. These antibodies, however, did not neutralize the anti-proliferative action of TGF-beta 2. Although TGF-beta 1 and TGF-beta 2 exhibit very similar biological activities, these molecules are antigenically different and, therefore, have different tertiary structures.     

Selective stimulation of human thymocyte subpopulations by recombinant IL-4 and IL-3

Human thymocytes and thymocyte subsets were examined for their proliferative response to recombinant interleukin-4 (IL-4) and interleukin-3 (IL-3) in serum-free cultures. IL-4 induced marked proliferation of thymocytes after PHA and TPA stimulation, in contrast to the marginal response of T cells from adult peripheral blood. However, depletion of thymocytes bearing the CD3 antigen diminished the IL-4-induced proliferation of thymocytes, indicating that the response of thymocytes to IL-4 is mainly mediated by the CD3-positive cells. Phenotypic changes after culture with IL-4 showed an increase in the percentage of total thymocytes expressing mature T cell antigens (CD3, CD5, and TCR-1) and a decrease in CD1-positive cells. In addition there was an increase in the percentage of CD4+8- cells in both nylon wool-separated thymocytes and CD3-depleted cells with the disappearance of most of the CD4+8+ cells. However, an increase in the percentage of CD4-8- cells was also observed. The IL-4-responding cells do, however, express the mature T cell antigen, CD5, in high density. The effect of IL-3 on the proliferation of human thymocytes was very low and detected only when the thymocytes were cultured in serum-free medium. Depletion of CD3-positive cells did not diminish the IL-3-mediated proliferation of thymocytes, indicating that IL-3-responsive thymocytes are more immature than the subset of thymocytes which responds to IL-4. These results suggest that IL-4 and IL-3 play different roles in the development of human T cells.     

pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells.

We have generated mutant mice that do not express pp59fyn, a nonreceptor protein tyrosine kinase related to pp60src, by homologous recombination in embryonic stem cells. fyn- mice did not display an overt phenotype. Because fyn is associated with the T cell receptor (TCR), thymocyte and T cell signaling was analyzed in the mutant background. Cross-linking of TCR-CD3 in thymocytes led to markedly reduced calcium fluxes and abrogated proliferation, whereas mature splenic T cells retained largely normal proliferation despite depressed calcium movements and IL-2 production. Similarly, proliferation induced by Thy-1 cross-linking was reduced in thymocytes but not in splenic T cells. fyn- thymocytes were impaired at a late stage of maturation and showed limited clonal deletion to the Mls-1a self-super-antigen but not to staphylococcal enterotoxin A. These results implicate fyn as a critical component in TCR signaling in thymocytes and, potentially, in the process that determines T cell repertoire in the adult mouse.     

Human recombinant IL-3 is a growth factor for normal B cells.

IL-3 is a well known hemopoietic cell growth and differentiation factor. However, its functional role in normal B cell differentiation has not been established. We have investigated the effect of IL-3 on the growth and differentiation of human B cells. IL-3 enhanced the proliferation of Staphylococcus aureus Cowan 1 strain-stimulated B cells. The optimal time of IL-3 to stimulate B cell growth was on day 2 to day 3, suggesting that IL-3 was a B cell growth factor acting in the late stage. IL-3 synergized with IL-2 to enhance B cell proliferation and differentiation. Pretreatment of B cells with IL-3 for more than 3 days increased the expression of IL-2R on B cells. However, pretreatment of B cells with IL-2 did not alter the subsequent response to IL-3, suggesting that the synergy between IL-2 and IL-3 may be attributed to the up-regulation of IL-2 response by IL-3. In addition, pretreatment of B cells with IL-4 decreased subsequent response of B cells to IL-3 as well as IL-2, suggesting that IL-3- and IL-2-responding cells passed a similar way during the early stage of B cell activation. It appears that IL-3 and IL-6 mediate normal B cell differentiation via separate mechanisms. IL-3-induced B cell differentiation was mainly mediated by increasing cell growth, whereas IL-6 induced B cell differentiation without affecting proliferation.     

Characterization of a B cell-derived growth-enhancing factor produced by a human B cell line established from a patient with rheumatoid arthritis

A human B cell line, TKS-1, which was established from the peripheral blood of a patient with rheumatoid arthritis, was found to spontaneously produce a factor which enhances the activity of interleukin 1 (IL-1). This factor, designated B cell-derived growth-enhancing factor (BGEF), enhanced IL-1-induced proliferation of peanut agglutinin nonagglutinated thymocytes. BGEF also enhanced IL-1-induced production of interleukin 2 (IL-2) by both thymocytes and a human T cell clone, HSB.2 C5B2. BGEF alone did not induce the production of IL-2. BGEF failed to induce proliferation of the IL-2-dependent T cell clone, and did not enhance its response to IL-2. The activity of BGEF was not blocked by antisera against human IL-1-alpha or human IL-1-beta. Gel filtration analysis revealed that BGEF has a m.w. of 60,000 to 65,000 in its native state. We concluded that BGEF differed from IL-1 and IL-2, but is a novel factor produced by TKS-1 cells. In addition, we found that partially purified B cells from patients with rheumatoid arthritis produced factors which enhanced the activity of IL-1.     

Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin

The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1beta in lipopolysaccharide (LPS)-primed monocytes and macrophages, the latter depending on caspase-1 activation by an unknown mechanism. Here, we investigate the pathway that triggers cell death and activates caspase-1. We show that without LPS priming, nigericin alone triggered caspase-1 activation and IL-18 generation in THP-1 monocytic cells. Simultaneously, nigericin induced caspase-1-independent necrotic cell death, which was blocked by the cathepsin B inhibitor CA-074-Me and other cathepsin inhibitors. Cathepsin B activation after nigericin treatment was determined biochemically and corroborated by rapid lysosomal leakage and translocation of cathepsin B to the cytoplasm. IL-18 maturation was prevented by both caspase-1 and cathepsin B inhibitors in THP-1 cells, primary mouse macrophages and human blood monocytes. Moreover, IL-18 generation was reduced in THP-1 cells stably transformed either with cystatin A (an endogenous cathepsin inhibitor) or antisense cathepsin B cDNA. Collectively, our study establishes a critical role for cathepsin B in nigericin-induced caspase-1-dependent IL-18 maturation and caspase-1-independent necrosis.     

A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1

The synthetic peptide CKS-17 has homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, to envelope proteins of HTLV I, II, III, and to that encoded by an endogeneous C-type human retroviral DNA. CKS-17 inhibits the immune response of lymphocytes and the respiratory burst of human monocytes. Because P15E-related antigens are present in human malignant cell lines and cancerous effusions, we sought to determine the effect of CKS-17 on monocyte-mediated tumor cell lysis. Lysis of A375 tumor cells by lymphokine or lipopolysaccharide-activated human monocytes was inhibited by 10 microM CKS-17 (control, 79%; CKS-17-treated, 19%). Another synthesized peptide, CS-2, which has partial homology to CKS-17, failed to block monocyte-mediated killing. Thus, the inhibition by CKS-17 appeared to be specific. Because interleukin 1 (IL-1) is a cytocidal factor produced by activated monocytes, we also investigated the effect of CKS-17 on IL-1 production by monocytes and on direct IL-1-mediated cytotoxicity. CKS-17 did not block production or secretion of IL-1 by lipopolysaccharide- or interferon-gamma-activated monocytes. However, the direct cytocidal activity of both recombinant IL-1 alpha and IL-1 beta against A375 tumor cells was blocked by CKS-17. The cytotoxic activity of IL-1 was inhibited by CKS-17 if (a) IL-1 was preincubated with CKS-17 for 1 hr at 37 degrees C or (b) the A375 cells were incubated with CKS-17 for 1 hr prior to the addition of IL-1. CKS-17 also blocked IL-1-induced proliferation of murine thymocytes, the D10 T cell line, and an IL-1-responsive astrocytoma cell line. These data suggest that CKS-17 may be a potent inhibitor of IL-1.     

Proliferation and production of IL-2 and B cell stimulatory factor 1/IL-4 in early fetal thymocytes by activation through Thy-1 and CD3

To examine which cell surface molecules can operate as transducers of activation signals to early fetal thymocytes, we analyzed the ability of mAb to CD3 and Thy-1 to induce fetal thymocyte activation. Both proliferation and lymphokine secretion were used as measures of activation. We show that anti-CD3 antibodies induce activation of fetal thymocytes as early as day 13 of fetal thymus development, 2 days before CD3 can be detected by flow cytometry. In addition, an alternative activation signal can be delivered to fetal thymocytes through the Thy-1 molecule as early as it is expressed, i.e., day 13. Both CD3- and Thy-1-mediated activation of day 15 fetal thymocytes results in expansion of cells expressing a CD3-gamma delta receptor complex; no CD3-alpha beta receptor complex could be detected. IL-2 production induced by CD3- and Thy-1-induced activation of fetal thymocytes is evident at the 13th day of gestation. Finally, an additional lymphokine B cell stimulatory factor-1 (BSF-1)/IL-4 (so far known only to be produced by mature CD3- cells), is also produced by fetal thymocytes. The results demonstrate that at least two cell surface molecules, Thy-1 and CD3, can function as pathways of activation in fetal thymocytes, and that at least two lymphokines, IL-2 and BSF-1/IL-4, are produced upon activation. These findings may well reflect a role for the early appearance of CD3- cells in thymus ontogeny.     

Mapping of biologically relevant sites on human IL-1 beta using monoclonal antibodies

mAb have been raised that recognize human IL-1 beta. Using overlapping peptide fragments expressed in yeast and bacteria, we have mapped the regions of the protein to which these antibodies bind. To assess the relevance of the different regions of IL-1 beta for the expression of its biologic activity, the ability of the antibodies to block IL-1 activity was assayed. Antibodies recognizing the regions 133-148 and 251-269 of human IL-1 beta could inhibit the activity of IL-1 beta, but not of IL-1 alpha, in two different biologic assays, the murine thymocyte proliferation and PGE2 release from human fibroblasts. Conversely, antibodies that recognize the region 218-243 have only a moderate inhibitory effect on the IL-1 beta biologic activity in both assays. Finally, an antibody mapping to the region 148-192 did not inhibit IL-1 beta activity either on thymocytes or on fibroblasts. It is suggested that IL-1 beta-induced cell activation involves different regions of the protein and that both N-terminal and C-terminal fragments are involved in the correct functioning of the IL-1 beta molecule.     

LFA-1在胸腺细胞活化信号传导中的功能研究

在ConA和固相抗CD_3单抗刺激系统中,应用抗LFA-1/ICAM-1单抗,研究其在胸腺细胞活化中的功能作用,结果证明,培养初期加入可溶性抗LFA-1可完全阻断ConA活化胸腺细胞增殖,对固相抗CD3单抗诱导的胸腺细胞活化也表现出相同的抑制效应,但对ConA刺激24h后的胸腺细胞应答以及IL-1 IL-2诱导的胸腺细胞增殖无影响。在可溶性抗LFA-1单抗的存在下,ConA诱导胸腺细胞合成IL-2和IL-6的能力显著下降,IL-2R的表达降低。此外,当用固相抗LFA-1和固相抗CD3或用二抗交联LFA-1和CD3刺激胸腺细胞时,抗LFA-1则具有明显地促增殖应答效应,单纯固相抗LFA-1刺激或交联LFA-1均无诱导活化作用,研究结果表明,LFA-1是未成熟胸腺细胞活化的重要辅助分子之一,它可参与TCR/CD3途径介导的早期活化信号的传导,并为胸腺细胞表达IL-2R 和产生IL-2可能提供复合刺激信号。     

Atypical memory phenotype T cells with low homeostatic potential and impaired TCR signaling and regulatory T cell function in Foxn1Delta/Delta mutant mice

Foxn1Delta/Delta mutants have a block in thymic epithelial cell differentiation at an intermediate progenitor stage, resulting in reduced thymocyte cellularity and blocks at the double-negative and double-positive stages. Whereas naive single-positive thymocytes were reduced >500-fold in the adult Foxn1Delta/Delta thymus, peripheral T cell numbers were reduced only 10-fold. The current data shows that Foxn1Delta/Delta peripheral T cells had increased expression of activation markers and the ability to produce IL-2 and IFN-gamma. These cells acquired this profile immediately after leaving the thymus as early as the newborn stage and maintained high steady-state proliferation in vivo but decreased proliferation in response to TCR stimulation in vitro. Single-positive thymocytes and naive T cells also had constitutively low alphabetaTCR and IL7R expression. These cells also displayed reduced ability to undergo homeostatic proliferation and increased rates of apoptosis. Although the frequency of Foxp3+CD4+CD25+ T cells was normal in Foxn1Delta/Delta mutant mice, these cells failed to have suppressor function, resulting in reduced regulatory T cell activity. Recent data from our laboratory suggest that T cells in the Foxn1Delta/Delta thymus develop from atypical progenitor cells via a noncanonical pathway. Our results suggest that the phenotype of peripheral T cells in Foxn1Delta/Delta mutant mice is the result of atypical progenitor cells developing in an abnormal thymic microenvironment with a deficient TCR and IL7 signaling system.     

IL-6 is an intermediate in IL-1-induced thymocyte proliferation

Both IL-1 and IL-6 have been shown to be comitogenic for lectin-stimulated thymocytes. Thymocytes cultured in the presence of IL-1 produce IL-6 themselves. This IL-6 production is caused by a cell population with low buoyant density. After removal of these cells, IL-6 or IL-2 are still co-mitogenic for thymocytes whereas IL-1 is not. Addition of IL-1 to such thymocytes renders them about 100-fold more sensitive to IL-6. At all conditions proliferation is inhibitable with antibodies to IL-2 and to the IL-2R. Our experiments show that IL-1-driven proliferation of thymocytes is dependent on endogenous IL-6 production and that in the classical thymocyte assay IL-1 has a dual role: it induces IL-6 production and it greatly increases the sensitivity for IL-6.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Characterization of a low molecular weight suppressor of lymphocyte proliferation from guinea pig L2C leukemia cells

Conditioned medium (CM) from 24-hr culture of guinea pig L2C B lymphoblastic leukemia cells contained an inhibitor(s) of mitogen- and antigen-stimulated proliferation of syngeneic (strain 2 guinea pigs), allogeneic (Hartley guinea pigs), and xenogeneic (Balb/c mouse, NZW rabbit) lymphocytes. The proliferation of several lymphoid and nonlymphoid cell lines also was inhibited in the presence of CM. The inhibitor(s) in CM was not toxic to any of the cultures studied. CM inhibited the mitogen-stimulated proliferation of lymphocytes when added to cultures up to 52 hr after addition of mitogen. Normal responsiveness to mitogens could be restored by washing the CM-treated lymphocytes with medium during the first 6 hr of culture. The addition of exogenous IL-2 to lymphocyte cultures did not overcome the CM-mediated suppression of mitogen- or antigen-stimulated proliferation. CM also inhibited the IL-2-dependent proliferation of murine CTLL-2 cells. Preincubation of guinea pig lymphocytes in CM did not inhibit the capacity of these cells to release IL-2 after exposure to mitogen. The antiproliferative activity of CM was stable to heating at low pH (100 degrees C, 10 min, pH 4.0), was resistant to treatment with papain, pronase, DNase, and RNase and did not bind to Con A-Sepharose. Incubation of the L2C cells in indomethacin did not inhibit the release of the inhibitor(s). The inhibitor(s) in CM had an apparent molecular weight of 500-3500 Da as determined by dialysis and ultrafiltration analysis. The inhibitory activity was recovered in the organic phase after extraction with chloroform:methanol and eluted distinct from the thymidine standard after gel filtration on Sephadex-G 25. These data suggest that the inhibitor(s) in CM is a nonspecific, low molecular weight, lipid-like component (not prostaglandin) that exerts its antiproliferative effects subsequent to cell activation. The inhibitor(s) did not appear to suppress other biologic functions associated with activation, such as IL-2 secretion. The inhibitor in CM may be important in promoting tumor survival in vivo by suppressing potential anti-tumor cellular immune responsiveness.     

Control of helper-T-cell proliferation by recognition of Ia and Mac-1 antigens on phagocytic cells of the thymic reticulum

Phagocytic cells of the thymic reticulum (P-TR) have been previously described as being Ia-positive, Mac-1-positive accessory cells which pursue a close relationship with thymocytes. They form rosettes with thymocytes, and these rosettes are inhibited by antibody directed against the complement receptor type 3 CR3 (anti-Mac-1). P-TR induce the proliferation of syngeneic thymocytes. In the present paper, we show that thymocytes enriched in mature medullary type are induced to proliferate in coculture with syngeneic P-TR, while the cortical type does not. After 5 days of culture, 85% of the thymocytes are of helper L3T4+Lyt-2- phenotype. As previously shown by others for syngeneic reactions, antibodies directed against related class II antigens (anti-I-A and anti-I-E) block this helper-T-cell syngeneic proliferation. A new finding is the blockage of helper-T-cell proliferation by anti-Mac-1 as well as with anti-LFA-1 antibodies, showing that accessory molecules may be as important as specific recognition of class II antigen molecules in the control of thymocyte proliferation and hence in thymocyte selection. Mac-1, like LFA-1, belongs to a novel family of differentiation antigens involved in cell interactions. The blockage of cell recognition and interaction between P-TR and thymocytes by either anti-Ia or anti-Mac-1 during the early induction phase of the syngeneic response leads to its inhibition. We demonstrate that P-TR/thymocyte interaction stimulates the enhanced expression of IL-2 receptors on thymocytes, a step which is necessary for helper-T-cell proliferation. The mechanism of syngeneic proliferation inhibition by anti-Ia, anti-Mac-1, and LFA-1 antibodies may be the prevention of IL-2 receptor expression on thymocytes, and/or the inhibition of IL-2 secretion. Although this is an in vitro model, which may not totally reflect in situ situation, our results indicate that thymic accessory cells may participate in a positive selection process which leads to helper-T-cell proliferation.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.