Co-stimulation of antigen-specific CD4 T cells by 4-1BB ligand.

4-1BB is a member of the TNF receptor family predominantly expressed on activated T cells, and binds an inducible ligand found on B cells, macrophages and dendritic cells. Whereas ligation of 4-1BB has been shown to enhance response of purified CD8 T cells to mitogens, and to augment NK activity and generation of cytotoxic T lymphocytes in vivo, there are little direct data on 4-1BB action during CD4 responses. Using pigeon cytochrome c-presenting fibroblast antigen-presenting cells transfected with 4-1BB ligand (4-1BBL), we show that engaging 4-1BB on naive CD4 cells promotes proliferation, cell cycle progression and IL-2 secretion, and suppresses cell death, all to a similar extent as B7-1 engagement of CD28. In addition, 4-1BBL synergizes with B7 and ICAM to enhance naive CD4 proliferation when antigen is limiting. 4-1BBL alone, and to a greater extent with B7, also augmented IL-2 secretion resting antigen-experienced CD4 cells, as typified by T helper clones, whereas short-term effector cells showed similar levels of proliferation and cytokine secretion regardless of whether 4-1BB was engaged. A major role in augmenting IFN-gamma, IL-4 or IL-5 was not demonstrated. Blocking studies with activated B cells presenting antigen showed that 4-1BB participates in promoting IL-2 production by resting CD4 cells, confirming that 4-1BBL can play a role in antigen-specific CD4 T cell responses.

Activation of CD4 T cells by somatic transgenesis induces generalized immunity of uncommitted T cells and immunologic memory.

Cellular immune responses were analyzed in vivo after a single intraspleen inoculation of DNA coding for a 12-residue Th cell determinant associated with a 12-residue B cell epitope, a process termed somatic transgene immunization. We show that CD4 T cells are readily activated and produce IL-2, IFN-gamma and IL-4, characteristics of an uncommitted phenotype. Linked recognition of the two epitopes coded in the same transgene promoted IgM-IgG1 switch and enhanced the total Ab response but had no effect on IgG2a Abs. Although originating in the spleen, T cell responsiveness was found to spread immediately and with similar characteristics to all lymph nodes in the body. A single inoculation was also effective in establishing long term immunologic memory as determined by limiting dilution analysis, with memory T cells displaying a cytokine profile different from that of primary effector T cells. These studies provide evidence that by initiating immunity directly in secondary lymphoid organs, an immune response is generated with characteristics that differ from those using vaccines of conventional DNA or protein in adjuvant administered in peripheral sites. Somatic transgene immunization can therefore be used to probe T cell responsiveness in vivo and represents a tool to further understanding of the nature of the adaptive immune response.

Lymphotoxin alphabeta is expressed on recently activated naive and Th1-like CD4 cells but is down-regulated by IL-4 during Th2 differentiation.

Lymphotoxin (LT) is a cytokine that orchestrates lymphoid neogenesis and formation of germinal center reactions. LT exists as a membrane heterotrimer of alpha and beta subunits and is secreted as a homotrimer, LTalpha3. Using LTbetaR.Fc, expression of LTalphabeta on CD4 T cell subsets was investigated in a TCR transgenic model. LTalphabeta was evident 24-72 h after activation of naive T cells with specific Ag, and declined thereafter. Early expression was independent of IFN-gamma and IL-12, however, IL-12 prolonged expression. LTalphabeta was reinduced within 2-4 h after Ag restimulation, but declined by 24 h regardless of IL-12 or IFN-gamma priming. Exposure of naive T cells to IL-4 did not affect early LTalphabeta expression at 24 h, but resulted in subsequent down-regulation. IL-4-differentiated Th2 effectors did not re-express LTalphabeta, and LTalphabeta was transiently found on Th1 clones but not Th2 clones. LTalpha3 and TNF were immunoprecipitated from supernatants and lysates of IL-12 primed cells but not IL-4 primed cells. These studies demonstrate that LTalphabeta is expressed by activated naive CD4 cells, unpolarized IL-2-secreting effectors, and Th1 effectors. In contrast, loss of surface LTalphabeta and a lack of LTalpha3 and TNF secretion is associated with prior exposure to IL-4 and a Th2 phenotype.

Generation, persistence, and modulation of Th0 effector cells: role of autocrine IL-4 and IFN-gamma.

Many studies have classified CD4 responses into either Th1-like or Th2-like, based on cytokine secretion profiles, but little significance has been placed on Th0 cells. This has largely resulted from studies that suggested that Th0 populations primarily comprise individual Th1 and Th2 cells. Here, we show that priming of Ag-specific naive CD4 cells with moderate dose IL-4 generates a Th0 population that is evident after 3 days in vitro and becomes prevalent after successive encounters with Ag over a 9-day period. By intracellular cytokine staining, the majority (>60%) of effector cells generated in this way produce either IL-4, IFN-gamma and IL-2, or IL-4 and IFN-gamma without IL-2. Endogenous IFN-gamma secreted over the initial 3 days of culture was critical for generating Th0 cells, since neutralization allowed IL-4 to induce differentiation into Th2-like cells. Successive encounters with Ag were required for generating Th0 cells, and their stability and persistence were governed by the balance of endogenous IL-4 and IFN-gamma secreted during the later stages of differentiation. Studies blocking Fas-induced cell death showed that this process played no role in Th0 cell generation, and differential death of committed Th1 or Th2 cells was not required for Th0 persistence. These data suggest that Th0 cells can be as prevalent as Th1- or Th2-like cells after naive CD4 activation, that the relative levels of autocrine IL-4 and IFN-gamma are important to the lack of commitment, and that not all cells are predestined to the Th1 or Th2 phenotypes early in the response.

Augmentation of naive, Th1 and Th2 effector CD4 responses by IL-6, IL-1 and TNF.

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-gamma, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-gamma and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.

Partial activation of naive CD4 T cells and tolerance induction in response to peptide presented by resting B cells.

Tolerance is thought to occur when Ag is presented to T cells in the absence of costimulatory interactions from APC accessory molecules. Of the professional APC, the resting B cell may be the main tolerizing cell in vivo. We have analyzed several aspects of activation of naive transgenic CD4 cells stimulated with resting or activated B cells presenting peptide Ag. Similar results were obtained with stimulation from peptide presenting fibroblast APC lacking or expressing B7-1 with intracellular adhesion molecule-1. TCR ligation with little or no accessory molecule coreceptor engagement induced efficient blastogenesis; up-regulation of CD25, CD44, CD69, CD95 and CD71; and down-regulation of CD62L over a 48-h period. Accessory molecule help enhanced the expression of CD25, CD44, CD69, and CD71, but to very modest degrees. Only two molecules, CD40 ligand and IL-2, were found to be extremely dependent on accessory molecule help, with little or no expression evident with peptide presented on resting B cells or class II-positive fibroblasts. T cells induced on resting B cells expanded minimally over 3 days, and this was followed by extensive cell death and hyporesponsiveness of the resulting cells. These studies suggest that under tolerizing conditions, such as Ag presentation by resting B cells, much of the naive CD4 response is induced efficiently. Partial activation, however, may be the overall result due to the lack of CD40 ligand expression, which may regulate costimulatory activity in APC and, in turn, may contribute to limiting the production of IL-2 required for T cell expansion and survival.