IL-4 enhances long-term survival of CD28-deficient T cells.

CD28 signaling is critical for IL-2 production by established Th1 clones, but CD28 does not appear to play a role in the activation of established Th2 clones. To determine the role of CD28 in the generation of polarized T cells, clones were derived using cells from CD28-deficient (CD28-/- mice, which had been bred with mice that express the DO11.10 transgene, a CD4+ TCR-alphabeta receptor that recognizes OVA peptide 323-339 bound to I-Ad. Most T cell clones derived from CD28+/+ mice survived multiple stimulations, while T cell clones derived from CD28-/- mice survived only if they were derived initially in the presence of IL-4 or both IL-2 and IL-4. Signaling through the CD28 molecule did not appear to be important in the initial activation of T cell clones, as the precursor frequency of clones derived from normal (CD28+/+) and CD28-/- mice was similar. Primary stimulation in the presence of IL-4 increased cell number and viability of both CD28+/+ and CD28-/- T cells in primary culture. However, the survival of CD28-/- cells is more dependent on IL-4 than is the survival of CD28+/+ cells. The continued presence of anti-IL-4 mAb dramatically decreased the number of viable cells in the CD28-/- cultures but had little effect on the viability of the CD28+/+ clones. Thus, initial culture with IL-4 allows the isolation of CD28-/- T cell clones that produce IL-4. In these clones, IL-4 acts as both an autocrine growth and survival factor.

Outcome of Kupffer cell antigen presentation to a cloned murine Th1 lymphocyte depends on the inducibility of nitric oxide synthase by IFN-gamma.

The interaction between lymphocytes and the resident hepatic macrophage, the Kupffer cell (KC), is relevant to the phenomenon of immune tolerance to Ags entering the liver. Tolerance to Ag administered via the portal vein can be prevented by the rare earth lanthanide metal, gadolinium (Gd). Therefore, we studied the ability of OVA-responsive, H-2d-restricted Th1 clones to proliferate in response to KCs from DBA/2J (H-2d) mice that had been injected with either saline (control) or a Gd solution. Whereas control KCs functioned as effective APCs, KCs from Gd-injected mice (GdKC) were incapable of sustaining the proliferative response of the Th1 clone to the 16 mer of OVA (323-339). This lack of proliferation was determined not to be caused by impaired Ag processing, but rather was the result of IFN-gamma-stimulated nitric oxide (NO) release by the APC: 1) In vitro addition of the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (NMMA) restored the ability of the Gd-treated KC to stimulate clone proliferation. 2) Additional of anti-IFN-gamma, but not anti-IL-2 or anti-IL-4, prevented the induction of NOS in the Gd-exposed KC and was associated with clone proliferation. 3) IFN-gamma levels from clone-GdKC-OVA cocultures closely paralleled the nitrite released by GdKCs. 4) Only the addition of rIFN-gamma, and not IL-2 or IL-4, to cultures of purified GdKCs resulted in the release of nitrite. The results of the study suggest an autocrine loop initiated by the interaction of the clone's TCR with the class II MHC molecule presenting processed OVA on the surface of KC. This interaction stimulates the Th1 lymphocyte to release IFN-gamma, which in turn induces NO release by KCs isolated from Gd-injected mice. This release of NO blocks Th1 proliferation. Such a feedback loop may have particular relevance to Ag-specific tolerance, which is not only induced by the administration of Ag into the portal vein, but is also prevented by Gd pretreatment of the recipient animal.

IL-4 treatment of small splenic B cells induces costimulatory molecules B7-1 and B7-2.

IL-4 has been shown to be involved in the early stages of B cell maturation. Changes induced by IL-4 include cell enlargement, increased viability, and increased MHC class II expression. However IL-4 alone does not induce B cell activation as defined by proliferation, lymphokine production, or Ig class switching. In this study, we demonstrate that incubation with IL-4 enhances the ability of small splenic murine B cells, normally poor stimulators of murine Th1 clones, to stimulate lymphokine production and proliferation by Th1 clones. Moreover, small resting B cells induce anergy, whereas IL-4-treated B cells do not. IL-4-treated B cells were found to express both B7 (B7-1) and a second ligand for CTLA4Ig (B7-2). Although IL-4 induces both B7-1 and B7-2, the kinetics of expression of these molecules are different: B7-2 is detected by 6 h, whereas B7-1 is not detectable until 48 h. In addition, only CTLA4Ig fully blocks IL-4 induced costimulatory activity; a mAb to B7-1 does not. Thus, these results suggest that IL-4 may function indirectly as a costimulatory factor by inducing costimulatory molecules on resting B cells. Additionally, these findings support our previous findings that an alternative ligand for CD28 and CTLA4 is important in providing costimulation.