Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells.

Coincident production of IL-2 and induction of high-affinity IL-2R upon TCR engagement has precluded a clear distinction for the biological outcome of signaling through TCR/costimulatory molecules vs the IL-2R. Using a novel transgenic mouse on the IL-2Rbeta(-/-) genetic background, this study has separated the relative outcome of signaling through the TCR and IL-2R. We show that stimulation through the TCR and CD28 or CD40 ligand directly leads to T cell activation and several rounds of proliferation in an IL-2-independent fashion. However, this stimulation is insufficient for extended T cell growth to multiple cytokines or differentiation into CTL or IFN-gamma-secreting effector T cells. IL-2 is required for these functions in part by regulation of cyclin D3 and granzyme B. Somewhat less efficiently, IL-4 stimulation of these transgenic T cells redundantly rescued many of these activities. These data demonstrate a fundamental requirement for IL-2 and perhaps other common gamma-chain-dependent cytokines to promote selective gene expression by Ag-activated T cells for their subsequent growth and differentiation into effector T lymphocytes.

Normal lymphoid homeostasis and lack of lethal autoimmunity in mice containing mature T cells with severely impaired IL-2 receptors.

The importance of IL-2Rbeta function for immune regulation is highlighted by the severe impairment in lymphoid cell function in IL-2Rbeta-deficient mice. It has been speculated that failed IL-2/IL-2R signaling in peripheral T cells causes the associated autoimmunity, imbalanced peripheral lymphoid homeostasis, and defective T cell function. This study explored the requirement for IL-2Rbeta function in mature T lymphocytes. We show that transgenic thymic expression of the IL-2R beta-chain in IL-2Rbeta-deficient mice prevents lethal autoimmunity, restores normal production of B lymphocytes, and results in a peripheral T cell compartment that is responsive to triggering through the TCR, but not the IL-2R. The dysfunction of the IL-2R is illustrated by the near complete failure of mature T cells to proliferate to IL-2 in vitro and in vivo, to differentiate into CTL, and to up-regulate IL-2Ralpha expression. These data indicate that lymphoid homeostasis is largely maintained despite a nonfunctional IL-2R in mature T lymphocytes and suggest that IL-2Rbeta provides an essential signal during thymic development to regulate self-reactivity.

Regulation of T lymphocyte function by glycosylphosphatidylinositol (GPI)-anchored proteins.

Monoclonal antibody binding to GPI-anchored proteins, e.g. Thy-1 and Ly-6, on the surface of T lymphocytes usually leads to stimulation of interleukin-2 production. This phenomenon is dependent upon expression of the zeta-chain of the T cell receptor complex and requires the GPI anchor. Recent studies suggest that this activation may proceed through a common pathway resulting in tyrosine phosphorylation of intracellular substrates and association of various GPI-anchored proteins to src-family tyrosine kinases. Several models are discussed to explain the signaling capabilities of GPI-anchored proteins. In contrast, under some experimental conditions antibody binding to selected GPI-anchored proteins, i.e. Ly-6A/E and sgp60 (CD48), leads to inhibition of T cell activation. Furthermore, induction of Ly-6A/E expression on CD4 effector T cells correlates with a decreased capacity to secrete IL-2. These latter results suggest that Ly-6A/E may also function to down-regulate an immune response.

Subsets of activated CD4 T lymphocytes refractory for secretion of IL-2 are distinguished by expression of Ly-6A/E in BALB/c mice.

Ly-6A/E, a cell surface glycosylphosphatidylinositol-anchored protein that modulates T cell activation, is expressed on developing and mature T cells and is tightly regulated in a haplotype- and subset-specific manner. We examined whether Ly-6A/E(low)CD4+ and Ly-6A/E(high)CD4+ T cells comprised functional subsets. Peripheral CD4+ T cells were primed in vitro with Con A in the presence or absence of IL-4 or IFN-gamma, sorted for Ly-6A/E expression, and restimulated to induce lymphokine production. Regardless of priming conditions, IL-2 production by Ly-6A/E(high)CD4+ effector T cells was markedly reduced (mean = 83%) compared to the Ly-6A/E(low)CD4+ subset. The Ly-6A/E(high)CD4+ subset also produced less IFN-gamma than Ly-6A/E(low)CD4+ cells and little IL-4 when primed without exogenous IL-4. In contrast, Ly-6A/E(high)CD4+ cells primed with exogenous IL-4 produced ample IL-4 and IFN-gamma. Interestingly, the difference in IL-2 production between Ly-6A/E(low) and Ly-6A/E(high)CD4+ subsets was not due to a failure of the Ly-6A/E(low)CD4+ cells to become primed because substantially greater amounts of IL-2 and IL-4 were produced by the Con A-pretreated Ly-6A/E(low)CD4+ subset in comparison to unprimed Ly-6A/E(low)CD4+ cells. Taken together these data suggest Ly-6A/E marks a subset of CD4+ effector T cells that differs from Ly-6A/E(low)CD4+ cells by a greatly reduced capacity to produce IL-2 but not IL-4.

Role of Ly-6A/E and T cell receptor-zeta for IL-2 production. Phosphatidylinositol-anchored Ly-6A/E antagonizes T cell receptor-mediated IL-2 production by a zeta-independent pathway.

Ly-6A/E molecules were originally implicated in regulation of T cell activation because anti-Ly-6A/E mAb induce IL-2 production. More recently we have shown that anti-Ly-6A/E also inhibits IL-2 production induced by anti-CD3. In the present study we used mutant and transfected cell lines that varied in expression of Ly-6A/E or TCR-zeta to test whether the positive and negative modulations of IL-2 production by anti-Ly-6A/E occur by distinct mechanisms. Anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production for Ly-6E.1-transfected EL4J cells, but did not affect IL-2 production of the parental Ly-6A/E-negative EL4J cells. These results indicate that TCR-mediated IL-2 production can occur in the absence of Ly-6A/E expression and establish that anti-Ly-6A/E-induced inhibition of IL-2 production was the result of antibody binding to Ly-6A/E. As expected, MA5.8 (zeta-negative) or CT108 (zeta-truncated) variants of the 2B4.11 T cell hybridoma did not produce IL-2 when stimulated with anti-Thy-1 or anti-Ly-6A/E mAb. In contrast, anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production by MA5.8 and CT108. Furthermore, anti-Ly-6A/E-induced IL-2 production was restored for zeta-transfected MA5.8. Thus, although induction of IL-2 by anti-Ly-6A/E depends on zeta expression, inhibition of IL-2 by anti-Ly-6A/E occurs by a zeta-independent mechanism. Interestingly, anti-Ly-6A/E, but not anti-Thy-1, inhibited anti-CD3-induced IL-2 production by MA5.8 and Ly-6E.1-transfected EL4J. Therefore, inhibition of IL-2 production by anti-Ly-6A/E was not a general property of a mAb binding to a phosphatidylinositol-linked molecule, as has been suggested for induction of IL-2 production. Taken together these data suggest that the molecular mechanisms of induction and inhibition of IL-2 production by anti-Ly-6A/E are separable and expression of TCR-zeta is one variable that distinguishes these two pathways.

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E.

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.

Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules.

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.

Tumor necrosis factor synergistically acts with IFN-gamma to regulate Ly-6A/E expression in T lymphocytes, thymocytes and bone marrow cells.

The Ly-6 alloantigens represent a family of phosphatidylinositol anchored proteins that function in the process of T lymphocyte activation and whose expression are often induced on T and B lymphocytes after activation by mitogens or Ag. Previous studies have shown that the induction of Ly-6 alloantigens in T cells is at least in part due to the action of IFN-alpha/beta or IFN-gamma. In the present report, we have demonstrated that IFN-gamma also induced Ly-6 molecules on B lymphocytes, several B cell tumors, and bone marrow cells. Furthermore, we now show that TNF also participates in the induction of at least one of the Ly-6 proteins, Ly-6A/E. TNF was found to synergize with IFN-gamma to induce Ly-6A/E expression in thymocytes, T lymphocytes, bone marrow cells, but not B lymphocytes. For T lymphocytes, the synergistic induction of Ly-6A/E by TNF was restricted to cells from the Ly-6.1 haplotype, whereas IFN-gamma was sufficient to fully induce Ly-6A/E expression in cells from the Ly-6.2 haplotype. This result is consistent with the notion that there is more complex regulation of the Ly-6A/E molecules in T cells obtained from the Ly-6.1 haplotype. For T lymphocytes from BALB/c (Ly-6.1) mice, Ly-6A/E, but not Ly-6C, molecules were synergistically induced by IFN-gamma and TNF. The induction of Ly-6A/E molecules on BALB/c T cells resulted in an enhanced capacity to activate these cells through the Ly-6 T cell activation pathway. One transformed T cell line, 5.1.2, was also identified whose Ly-6A/E molecules were synergistically induced by IFN-gamma and TNF. Optimal expression of Ly-6A/E molecules on 5.1.2 cells required continuous culture of this cell line with these two cytokines and resulted in the detection of optimal levels of cytoplasmic Ly-6A/E mRNA by Northern blot analysis. This latter result suggests that IFN-gamma and TNF regulate Ly-6A/E at the level of transcription and/or mRNA stabilization.

Expression of Ly-6A/E alloantigens in thymocyte and T-lymphocyte subsets: variability related to the Ly-6a and Ly-6b haplotypes.

We have studied the cellular basis for differential expression of the Ly-6A/E alloantigen on T cells obtained from mice of the Ly-6a (10-20% Ly-6A/E+) and Ly-6b (50-60% Ly-6A/E+) haplotypes. During T-cell ontogeny only a small fraction (less than 12%) of thymocytes expressed Ly-6A/E. By 4 weeks of age adult levels of Ly-6A/E bearing lymphocytes were seen in peripheral lymphoid tissue. Immunohistochemical studies of the thymus revealed that Ly-6A/E+ cells were located predominantly in the medulla with small clusters of Ly-6A/E+ cells throughout the cortex. Consistent with this result, phenotypic studies showed that in the adult thymus the majority of Ly-6A/E expression was on mature CD4+CD8- and CD4-CD8+ cortisone-resistant and precursor CD4-CD8- thymocytes. However, a much higher percentage of CD4+CD8- and CD4-CD8- thymocytes as well as CD4+CD8- peripheral T cells expressed Ly-6A/E from Ly-6b mice. Furthermore, although gamma interferon induced increased Ly-6A/E expression in certain thymocyte and T-cell subsets, this induction functioned preferentially for cells obtained from Ly-6b mice. Studies using F1 hybrid mice (Ly-6a x Ly-6b) indicated that the "basal" level of Ly-6A/E expression on these subsets appeared to be under codominant genetic control, whereas gamma interferon-induced regulation of Ly-6A/E expression appeared to be under dominant genetic control. Collectively, these results suggest that the expression of Ly-6A/E on a particular T-cell subset is established in the thymus and is a stable characteristic of each haplotype. In addition, the low levels of Ly-6A/E expression for the Ly-6a haplotype appear to be partially due to the inability of the majority of resting CD4+ T cells to express Ly-6A/E and to the relatively poor induction of this protein by gamma interferon.

Distribution of serotypes of the Mycobacterium avium-intracellulare-scrofulaceum complex in Georgia.

Cultures of the Mycobacterium avium-intracellulare-scrofulaceum (MAIS) complex, isolated from patients who lived throughout the state of Georgia, were studied using the serotyping scheme of Schaefer. One hundred four (77 per cent) of the 135 isolates tested could be classified into 16 serotypes. The rest were not typable. Fifty-eight per cent of all the typable strains were serotypes 1, 14, 16, and 42. Results of the serotyping were recorded, mapped, and analyzed by county of residence. Most frequent isolations of these mycobacteria were from patients who resided in the coastal plain of Georgia. This finding correlates with the mycobacterial skin test studies of Edwards and associates, who found that the largest percentage of reactors to PPD-Battey resided in the costal plain. The disparate distributions of certain serotypes suggest the possible importance of regional factors that may affect the serotypes of mycobacteria indigenous to an area. The data do not suggest that chicken or swine reservoirs are highly significant sources of human mycobacteria.