Heterogeneity of helper/inducer T lymphocytes. IV. Stimulation of resting and activated B cells by Th1 and Th2 clones

To test the hypothesis that resting and previously activated B lymphocytes differ in their proliferative and differentiative responses to various Th cell-derived stimuli, we have examined the interactions of purified small (resting) and large (activated) murine B cells with rabbit Ig-specific Th1 and Th2 clones in the presence of the Ag analogue, rabbit anti-mouse Ig antibody. Small numbers of Th2 cells induce strong Ag-dependent proliferation of and Ig secretion by both resting and activated B lymphocytes. In contrast, Th1 clones stimulate lower responses of activated B cells and fail to stimulate small resting B cells. An interaction with Th1 clones does make small B cells responsive to the Th2-derived cytokine, IL-4, indicating that Th1 clones are capable of delivering some but not all the stimuli necessary for the induction of humoral immunity. Finally, in order to compare the responses of small and large B cells to cognate interactions and secreted cytokines, we used an autoreactive I-Ak-specific Th2 line. This line induces proliferation of and Ig secretion by I-Ak expressing but not H-2d resting and activated B cells as a result of cognate interactions. However, when the H-2d B cells are bystanders in the presence of cytokine secretion by this Th2 line, or are directly exposed to Th2-derived cytokines, both small and large B cells are induced to proliferate but only the large B cells secrete antibody. These results indicate that the magnitude and nature of antibody responses depend on three principal factors: the cytokines produced by Th cells, the state of activation of the responding B lymphocytes, and whether the B cells are recipients of cognate help or are bystanders at the site of T cell stimulation. Our findings also confirm the view that cognate T-B interactions are most efficient for initiating B cell responses and may allow B cells to subsequently respond to a variety of T cell-derived cytokines.     

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production.

Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Primary response of naive CD4(+) T cells to amino acid-substituted analogs of an antigenic peptide can show distinct activation patterns: Th1- and Th2-type cytokine secretion, and helper activity for antibody production without apparent cytokine secretion

Naive CD4(+) T cells differentiate into two types of helper T cells showing an interferon-gamma-predominant (Th1) or an interleukin-4-predominant (Th2) cytokine secretion profile after repeated antigenic stimulation. Their differentiation can be influenced by slight differences in the interaction between the T cell receptor (TCR) and its ligand at the time of primary activation. However, the primary response of freshly isolated naive CD4(+) T cells to altered TCR ligands is still unclear. Here, we investigated the primary response of splenic naive CD4(+) T cells derived from transgenic mice expressing TCR specific for residues 323-339 of ovalbumin (OVA323-339) bound to I-A(d) molecules. Naive CD4(+) T cells secreted either Th1- or Th2-type cytokines immediately after stimulation with OVA323-339 or its single amino acid-substituted analogs. Helper activity for antibody secretion by co-cultured resting B cells was also found in the primary response, accompanied by either low-level Th2-type cytokine secretion or no apparent cytokine secretion. Our results clearly indicate that dichotomy of the Th1/Th2 cytokine secretion profile can be elicited upon primary activation of naive CD4(+) T cells. We also demonstrate that the helper activity of naive CD4(+) T cells for antibody production does not correspond to the amounts of the relevant cytokines secreted.     

Th2 lymphocyte clone can activate macrophage antileishmanial defense by a lymphokine-independent mechanism in vitro and can augment parasite attrition in vivo.

Antileishmanial defense has been ascribed to the antimicrobial effects induced by soluble macrophage-activating lymphokines (MAFs), such as interferon-gamma and granulocyte-macrophage colony-stimulating factor. Recently, we identified an additional mechanism of T cell-mediated macrophage activation of defense against Leishmania that is apparently lymphokine independent, requires cell-cell contact, and is not cytotoxic to host cells. By employing antigen-specific murine T cell hybridoma lines, we observed that this property was associated with CD4+ subpopulations possessing the characteristics of the Th1 subset. In the present study, we address the question of whether contact-mediated macrophage activation can also be induced by Th2 lymphocytes. We employed as T effector cells in antileishmanial defense assays the Th2 cell line D10.G1.4 (D10) which is specific for conalbumin. We observed that D10 cells were able to induce activation of Leishmania-infected macrophages only when the macrophages were also primed with conalbumin, and that this activation apparently occurred by a mechanism without the secretion of MAF. Moreover, when mice infected with L. major were injected into footpad lesions with conalbumin and D10 cells, in situ parasite replication was partially inhibited. The expression of this antimicrobial mechanism by Th1 as well as Th2 clones suggests that the property of contact-mediated (lymphokine-independent) activation may be shared by certain lymphocytes in both Th1 and Th2 subpopulations. We hypothesize that this activation mechanism may involve the interaction of a lymphocyte membrane-associated MAF (such as tumor necrosis factor) and its receptor on the infected macrophage, resulting in the induction of antimicrobial effects but not cytotoxicity to the host cell.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Development of a monoclonal antibody, anti-6C2, which is involved in the interaction of CD4 T helper cells and activated B cells.

We have developed a mAb anti-6C2, by immunizing mice with T cell line derived from the Callithrix jacchus (common marmoset). Anti-6C2 is reactive with approximately 50% of unfractionated T cells, 50% of CD4+ cells, and 40% of CD8+ cells. Regarding CD4+ cells, anti-6C2-reactive cells substantially overlap with the CD29+CD45RO+ Th cell population. Moreover, anti-6C2 can divide these T cells into 6C2+ and 6C2- subpopulations. The CD4+CD45RO+6C2+ cells maximally respond to soluble Ag such as tetanus toxoid and provide strong helper function for PWM-driven B cell IgG synthesis. Most interestingly, anti-6C2 was also reactive against activated B cells but not resting B cells; furthermore, this epitope was inducible through activation of resting B cells or B cell line. Biochemical characterization showed that anti-6C2 precipitated two glycoproteins with the relative molecular weights of 180,000 and 95,000 from 125I-surface labeled cell lysate. Sequential immunoprecipitation studies demonstrated that these two glycoproteins were the lymphocyte function-associated antigen (LFA-1) Ag complex (CD11a/18). Significantly, although this antibody did not inhibit cytotoxic killer T cell responses and Ag-induced T cell proliferation as did conventional anti-LFA-1, it did inhibit PWM-driven B cell IgG synthesis. Because 6C2 expression was induced after B cell activation, the above results strongly suggest that the 6C2 molecule may play a role in the interaction of CD4 helper cells and activated B lymphocytes.     

Regulation of lymphocyte clustering by CD30-mediated ICAM-1 up-regulation

CD30 is expressed transiently on activated B and T lymphocytes and constitutively on several B- and T cell lymphomas. CD30 functions include participation in negative selection of thymocytes, costimulation of activated T cells, isotype switching of B cells, and regulation of the effector activity of cytotoxic lymphocytes. Although CD30 is not a marker for T helper 2 (TH2) cells, it may participate in the polarization of TH1 and TH2 cells. The pleiotropic functions of CD30 are initiated by interaction of CD30-expressing cells with other immune competent cells expressing CD30-L and providing the signals for modulation of effector cell activity. Here, we report that CD30 signals generated by anti-CD30 on activated, normal murine T cells strongly up-regulate the expression of intercellular adhesion molecule 1 (ICAM-1, CD54), and to a lesser extent, ICAM-2 (CD102). CD30 signals moreover delay the subsequent decline of ICAM expression. CD30 cross-linking did not alter the expression of CD11a/CD18 (LFA-1), the counter receptor for ICAM abundant on T cells. CD30-mediated ICAM-1 up-regulation is independent of cytokine secretion and appears to be transmitted directly through NF-kappaB activation. CD30-mediated up-regulation of ICAM-1 expression led to a significant increase in cluster formation of lymph node cells. Increased lymphocyte self-aggregation mediated by CD30 may set the stage for fraternal signaling to modulate lymphocyte function.     

Cutting edge: molecular mechanisms of synergy between CD40 and the B cell antigen receptor: role for TNF receptor-associated factor 2 in receptor interaction

Optimal Ag-specific B lymphocyte activation requires both recognition of Ag by the B cell Ag receptor (BCR) and contact-mediated interactions with Ag-specific Th lymphocytes. One of these interactions involves ligation of B cell CD40 by T cell-expressed CD154. CD40 signaling is crucial for Ab production, isotype switching, up-regulation of surface molecules, development of germinal centers, and the humoral memory response. The signaling pathways emanating from the BCR and CD40 are able to cooperate, but the molecular mechanisms responsible for this interaction are incompletely understood. The present study explored the roles of signaling motifs in the CD40 cytoplasmic tail in this synergy. We find that threonine in the PXQXT motif in the TNFR-associated factor-2 binding site is critical for synergistic effects of CD40 and BCR signals, independent of its phosphorylation. Furthermore, data suggest an indirect role for TNFR-associated factor-2 in the cooperative signaling.     

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

Expression and function of the B and T lymphocyte attenuator (BTLA/CD272) on human T cells

Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.     

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.     

NTB-A, a new activating receptor in T cells that regulates autoimmune disease

The CD28 co-stimulatory pathway is well established for T cell activation; however, results from CD28 -/- mice suggest the existence of additional co-stimulatory pathways. Here we report the further characterization of a new member of the CD2 superfamily, NTB-A, important in T cell co-stimulation. NTB-A is expressed on T cells, and its expression is up-regulated on activated cells. Triggering of NTB-A with monoclonal antibodies in the absence of CD28 signals leads to T cell proliferation and interferon-gamma secretion but not interleukin-4. Cross-linking of NTB-A also induces phosphorylation of NTB-A and the association of SAP (SLAM-associated protein), the protein absent in X-linked lymphoproliferative disease. T helper cells differentiated by cross-linking NTB-A and CD3 developed predominantly into Th1 cells not Th2 cells. In vivo blocking of NTB-A interactions with its ligands by using soluble NTB-A-Fc fusion protein inhibits B cell isotype switching to IgG2a and IgG3, commonly induced by Th1-type cytokines. Most important, treatment of mice with NTB-A-Fc delays the onset of antigen-induced experimental allergic encephalomyelitis in myelin basic protein-T cell receptor transgenic mice, suggesting a role in T cell-mediated autoimmune disease. Regulation of interferon-gamma secretion, and not interleukin-4 in vitro, as well as inhibition of Th1 cell-induced isotype switching and attenuation of experimental allergic encephalomyelitis indicate that NTB-A is important for Th1 responses. The observation that cross-linking of NTB-A induces T cell activation, expansion, and Th1-type cytokine production suggests NTB-A is a novel co-stimulatory receptor. The identification of NTB-A as a regulator of T cell response paves the way to provide novel therapeutic approaches for modulation of the immune response.     

Parasite-induced Th2 polarization is associated with down-regulated dendritic cell responsiveness to Th1 stimuli and a transient delay in T lymphocyte cycling

The nature of the signals that bias Th effector choice is still not completely understood. Using parasite extracts from pathogens known to induce polarized Th1 or Th2 responses and an in vitro experimental model for priming murine CD4(+) cells, we demonstrated that splenic dendritic cells (DC), but not B cells, promote Th1/Th2 differentiation of naive CD4(+) lymphocytes. Th polarization in this system was found not to depend on DC secretion of the polarizing cytokines IL-12/IL-4, but instead correlated with distinct states of DC activation induced by the different parasite preparations. As expected, conditioning of DC for Th1 development was associated with up-regulation of costimulatory molecules and enhanced chemokine production and required intact MyD88 signaling. In contrast, conditioning of DC for Th2 differentiation correlated with down-regulation of many of the same functions and was MyD88 independent. This dampened DC activation was accompanied in the cocultures by a reduction in the frequency of CD4(+) lymphocytes exiting the first division of the cell cycle. When the latter was mimicked by drug-induced arrest of peptide-primed CD4(+) cells after the S phase of the first cycle, a marked Th2 polarization was also observed. Together, these findings suggest that the emergence of IL-4-producing CD4(+) lymphocytes results from a suppression in DC function leading to a temporary delay in initial T cell cycling.     

Induction of a Th1 response from Th2-polarized T cells by activated dendritic cells: dependence on TCR:peptide-MHC interaction, ICAM-1, IL-12, and IFN-gamma

Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of costimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this study, we demonstrate that the addition of activated DC to spleen cultures containing established Th2-polarized CD4(+) T cells was sufficient to suppress Th2 and induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite Ag specificity. The DC activator B7-DC cross-linking Ab (XAb) was >10,000-fold more efficient at inducing phenotype reversal than the TLR agonists CpG-oligodeoxynucleotide and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR:peptide-MHC complex, the expression of the costimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFN-gamma by the activated DC. The requirement for the costimulation/adhesion molecule SLAM (signaling lymphocytic activation molecule) was found to be quantitative. Thus, activation of DC, particularly by crosslinking B7-DC, can modulate well-established Th2 T cell responses in an Ag-specific manner. Because the regulation of mouse and human DC by B7-DC XAb overlaps in several significant ways, immune modulation with B7-DC XAb is a potential strategy for treating Th2-mediated diseases.     

Up-regulation of gene related to anergy in lymphocytes is associated with Notch-mediated human T cell suppression

A growing body of literature indicates that the Notch pathway can influence the activation and differentiation of peripheral murine T cells, though comparatively little is known about the effects of Notch signaling in human T cells. In the present report we demonstrate that Jagged-1-induced Notch signaling (using immobilized Jagged-1 fusion protein) during stimulation of purified human CD4+ and CD8+ T cells potently inhibits T cell proliferation and effector function, including both Th1- and Th2-associated cytokines. Inhibition of T cell activation is not due to apoptosis or disruption of proximal TCR signaling, but is associated with up-regulation of GRAIL (gene related to anergy in lymphocytes) in CD4+ T cells, with modest effects on other E3 ubiquitin ligases such as c-Cbl and Itch. When evaluated for its effects on CD4+ T cell differentiation, Jagged-1-mediated signaling inhibits T cell cytokine secretion with no significant effect on proliferative responses. Collectively, these data demonstrate that Notch signaling in human T cells induced by Jagged-1 promotes a novel form of T cell hyporesponsiveness that differs from anergy, whereby primary T cell proliferation and cytokine secretion are potently inhibited, and effector function but not proliferative capacity are ameliorated upon secondary stimulation.     

CD4 T-cell memory can persist in the absence of class II

To understand how memory CD4 T cells are generated we have re-examined the requirements for continuing antigen stimulation in the generation and persistence of this population. We find that specific antigen is only required for a short period during the activation of naive CD4 T cells and is not required for memory generation from activated CD4 T cells or for persistence of resting memory cells generated by transfer of activated CD4 to adoptive hosts. Moreover, transfer of activated CD4 T cells to class-II-deficient hosts, indicates that TcR-class II major histocompatibility interaction is also unnecessary for either the transition from activated CD4 T cell to resting memory cells or for persistence over an eight-week period. Thus the signals regulating generation and maintenance of memory are fundamentally different from those which regulate the expansion of effector CD4 T-cell populations which include antigen itself and the CD4 T-cell autocrine cytokines induced by antigen.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.