Interleukin (IL) 4, in the absence of antigen stimulation, induces an anergy-like state in differentiated CD8+ TC1 cells: loss of IL-2 synthesis and autonomous proliferation but retention of cytotoxicity and synthesis of other cytokines.

Naive T cells in the periphery mainly secrete interleukin (IL) 2 upon activation. After stimulation in the presence of appropriate costimulators, both CD4+ and CD8+ T cells differentiate into effector cells secreting distinct T helper (Th) 1- and Th2-like cytokine patterns. Subsequent to differentiation, both CD4+ (Th1 and Th2) and CD8+ (TC1 and TC2) cells are stable and cannot be induced to differentiate into the opposite pattern or revert to the naive cytokine secretion pattern. We now show that IL-4 caused committed TC1 bulk populations or clones to lose the ability to synthesize IL-2. The cells retained the ability to secrete interferon (IFN) gamma, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor, did not synthesize any Th2 cytokines, and did not alter cell surface marker expression. IL-4 rapidly inhibited IL-2-synthesizing ability in the absence or presence of antigen-presenting cells, thus demonstrating that IL-4 acted directly on TC1 cells. The defect in IL-2 synthesis could not be reversed by subsequent stimulation with potent antigen-presenting cells in the presence of IL-2 and anti-IL-4, or with anti-CD3 plus anti-CD28 antibodies. Both IL-2+ and IL-2- TC1 cells were strongly cytotoxic toward allogeneic but not syngeneic targets. However, IL-2- TC1 cells were unable to proliferate unless exogenous IL-2 was provided. TC1 cells that lose IL-2 synthesis but retain IFN-gamma synthesis and cytotoxicity may be similar to the "anergic" cells induced by stimulation of CD4+ or CD8+ cells in the absence of costimulators. These results suggest that during a mixed type 1/type 2 response in vivo, IL-4 may induce the IL-2+ TC1-->IL-2-TC1 conversion, and thus curtail the expansion of the TC1 response without impairing short-term effector function.

Perforin and Fas killing by CD8+ T cells limits their cytokine synthesis and proliferation.

During an immune response, effector CD8+ T cells can kill infected cells by the perforin-dependent pathway. In comparison to CD4+ T cells, which are major sources of cytokines, normal CD8+ T cells produced less interleukin 2 and interferon gamma, and proliferated less vigorously after antigenic stimulation. Killing of target cells was a major cause of these reduced responses, since perforin-deficient CD8+ T cells showed substantially increased cytokine synthesis and proliferation. Cytotoxicity by the alternate Fas pathway also resulted in self-limitation of CD8+ T cell cytokine synthesis. This relationship between cytotoxicity and cytokine synthesis may regulate CD8+ T function in different phases of an immune response.

Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.

A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.

Chemically modified antigen preferentially elicits induction of Th1-like cytokine synthesis patterns in vivo.

Differential activation of CD4+ T cell subsets in vivo leads to the development of qualitatively different effector responses. We identify an approach that allows selective activation of strongly Th1-dominated immune responses to protein antigens. Whereas in vivo administration of ovalbumin (OVA) induces cytokine synthesis that is neither Th1 nor Th2 dominated, administration of glutaraldehyde polymerized, high relative molecular weight OVA (OA-POL) leads to 20-fold increase in the ratio of interferon gamma (IFN-gamma)/IL-4 and IFN-gamma/IL-10 synthesis observed after short-term, antigen-mediated restimulation directly ex vivo. In contrast, concurrent in vivo administration of anti-IFN-gamma mAb and OVA or OA-POL results in marked increases in IL-4 and IL-10, and decreased IFN-gamma production, reflecting a polarization of the response towards a Th2-like pattern of cytokine synthesis. These observations may be useful in clinical settings including hypersensitivity, autoimmune diseases, and vaccine development where the ability to actively select specific patterns of cytokine gene expression would be advantageous.

Lymphokine-mediated regulation of the proliferative response of clones of T helper 1 and T helper 2 cells.

Murine Th1 and Th2 subsets differ not only in the lymphokines they produce, but also functionally. It is not clear what factors influence the preferential activation of one subset versus the other and what regulatory interactions exist between them. The purpose of this study was to examine the effect of lymphokines produced by clones of Th1 cells (IL-2 and IFN-gamma), Th2 cells (IL-4), and APC (IL-1) on the proliferative response of Th1 and Th2 cells after antigenic stimulation. Activation of both types of clones in the presence of antigen and APC resulted in the acquisition of responsiveness to the proliferative effects of both IL-2 and IL-4, although Th2 cells were more responsive to IL-4 than Th1 cells. Responsiveness of Th1 and Th2 cells to both lymphokines decreased with time after initial antigenic activation; Th1 cells lost their responsiveness to IL-4 more rapidly and to IL-2 more slowly than Th2 cells. IFN-gamma partially inhibited the IL-2 and IL-4-mediated proliferation of Th2, but not Th1 cells. Although the presence of IL-1 was not required for the response of Th1 or Th2 cells to IL-4, its presence resulted in a synergistic effect with IL-2 or IL-4 in Th2 but not in Th1 cells. Both subsets responded to a mixture of IL-2 and IL-4 in synergistic fashion. Delayed addition and wash-out experiments indicated that both IL-2 and IL-4 had to be present simultaneously in order for synergy to occur. These results suggest that Th cell subsets might regulate each other via the lymphokines that they secrete and that the pathways of IL-2 and IL-4 mediated proliferation are interrelated.

Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies.

Lymphokine synthesis patterns of a panel of 19 T cell clones have been evaluated, using mRNA hybridization methods to examine 11 different mRNAs induced by Con A. The two types of CD4+ Th cell clone described previously were clearly distinguished by this procedure, and the differences between the two types have now been extended to six induced products. With minor exceptions, only Th1 clones synthesized mRNA for IL-2, IFN-gamma, and lymphotoxin, and only Th2 clones synthesized mRNA for IL-4, IL-5, and another induced gene, P600. Four more induced products were expressed preferentially but not uniquely by one or another type of clone: mRNAs for GM-CSF, TNF, and another induced, secreted product (TY5) were produced in larger amounts by Th1 clones, whereas preproenkephalin was preferentially expressed by Th2 clones. IL-3 was produced in similar amounts by both types of clone. mAbs were used to establish three bioassays that were functionally monospecific for IL-2, IL-3, and IL-4, and a new anti-IFN gamma mAb, XMG1.2, was used to establish an ELISA for IFN-gamma. These four assays were used to show that secreted protein and mRNA levels correlated well for all cell lines. The implications of these findings for normal T cells are discussed.

Interleukin 10: a novel stimulatory factor for mast cells and their progenitors.

We have characterized the mast cell stimulating activity of murine cytokine synthesis inhibitory factor, referred to as interleukin 10 (IL-10). It was found that IL-10 alone failed to support the growth of mast cell lines and mast cell progenitors. Nevertheless, it dramatically enhanced their growth when combined with IL-3 or IL-4. Moreover, IL-4 plus IL-10 supported the proliferation of mast cells as well as IL-3, suggesting that these two factors may provide a pathway for their development independent of IL-3. However, optimal mast cell growth was stimulated by the combination of IL-10, IL-4, and IL-3. This particular set of cytokines are coordinately produced by activated T cells and may constitute an effective network regulating early and late stages of mast cell development during certain immune responses.

Interleukin 10, a novel B cell stimulatory factor: unresponsiveness of X chromosome-linked immunodeficiency B cells.

Highly purified, small dense splenic B cells from unstimulated mice showed increased expression of class II major histocompatibility complex (MHC) antigens and enhanced viability when cultured with affinity-purified recombinant interleukin 10 (rIL-10), compared with B cells cultured in medium alone. These responses were blocked by a monoclonal antibody (mAb) specific for IL-10, but not by an isotype-matched control antibody. IL-10 did not upregulate the expression of Fc epsilon receptors (CD23) or class I MHC antigens on small dense B cells or induce their replication as monitored by [3H]thymidine incorporation. While these B cell-stimulatory properties of IL-10 are also mediated by IL-4, the two cytokines appear to act independently in these assays; anti-IL-10 antibodies blocked IL-10 but not IL-4-mediated B cell viability enhancement, and vice versa. Similarly, since IL-4 upregulates CD23 on small dense B cells, the inability of IL-10 to do so argues against its acting via endogenously generated IL-4. Finally, IL-10 did not upregulate class II MHC antigens on B cells from X chromosome-linked immunodeficiency (XID) mice, while the same cells showed normal upregulation of class II antigens in response to IL-4. This report also extends our understanding of the relationship between IL-10 and the highly homologous Epstein-Barr virus (EBV)-encoded Bam HI fragment C rightward reading frame no. 1 (BCRFI) protein. It has previously been shown that BCRFI protein exhibits the cytokine synthesis inhibitory activity of IL-10. This report indicates that BCRFI protein also enhances in vitro B cell viability, but does not upregulate class II MHC antigens on B cells. One explanation for these data is that IL-10 contains at least two functional epitopes, only one of which has been conserved by EBV.

Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI.

Complementary DNA clones encoding mouse cytokine synthesis inhibitory factor (CSIF; interleukin-10), which inhibits cytokine synthesis by TH1 helper T cells, were isolated and expressed. The predicted protein sequence shows extensive homology with an uncharacterized open reading frame, BCRFI, in the Epstein-Barr virus genome, suggesting the possibility that this herpes virus exploits the biological activity of a captured cytokine gene to enhance its survival in the host.

Expression of interleukin-10 activity by Epstein-Barr virus protein BCRF1.

Cytokine synthesis inhibitory factor (CSIF; interleukin-10), a product of mouse TH2 T cell clones that inhibits synthesis of cytokines by mouse TH1 T cell clones, exhibits extensive sequence similarity to an uncharacterized open reading frame in the Epstein-Barr virus BCRF1. Recombinant BCRF1 protein mimics the activity of interleukin-10, suggesting that BCRF1 may have a role in the interaction of the virus with the host's immune system.

Cytokines: is there biological meaning?

In the immune system, amidst the bewildering array of cytokine functions, it is sometimes difficult to discern the relative physiological importance of functions that have been determined in tissue culture. Cytokine functions are indeed complex, but the analysis of relatively simple regulatory networks suggests that activities determined in vitro are highly relevant to genuine physiological functions.

Both CD4+ and CD8+ T-cells in syngeneic islet grafts in NOD mice produce interferon-gamma during beta-cell destruction.

Syngeneic pancreatic islet grafts in diabetic NOD mice are infiltrated by mononuclear leukocytes, beta-cells are selectively destroyed, and autoimmune diabetes recurs. This model was used to identify islet graft-infiltrating mononuclear leukocytes associated with beta-cell destruction and diabetes recurrence. We compared cell surface antigen and cytokine-producing phenotypes of mononuclear leukocytes in islet grafts from NOD mice that were protected from diabetes recurrence by complete Freund's adjuvant (CFA) administration (beta-cell nondestructive insulitis) and in islet grafts from control phosphate-buffered saline (PBS)-injected NOD mice (beta-cell destructive insulitis). Islet grafts from CFA-injected mice contained fewer CD4+ and CD8+ cells and more B cells; also fewer interferon gamma (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-alpha)-positive cells and more IL-4 and IL-10 positive cells. By performing two-color immunostaining of cell surface antigens and intracellular IFN-gamma, we found that IFN-gamma positive cells in islet grafts from CFA- and PBS-injected mice were approximately equally divided between CD4+ and CD8+ T-cell subsets. Also, the frequencies of both CD4+ IFN-gamma + and CD8+ IFN-gamma + cells were decreased in islet grafts from CFA-injected mice. These findings suggest that destruction of beta-cells in syngeneic islets transplanted into NOD mice is promoted by cells producing Th1-type cytokines (IFN-gamma, IL-2, and TNF-alpha) and prevented by cells producing TH2-type cytokines (IL-4 and IL-10). Furthermore, both CD4+ and CD8+ IFN-gamma-producing T-cells in the islet grafts appear to be involved in beta-cell destruction and diabetes recurrence.

Specific assays for cytokine production by T cells.

Recent advances in understanding of cytokine function have indicated the need for more stringent cytokine assays, and have at the same time provided the information and tools necessary for establishing monospecific assays for a number of T cell-derived cytokines. Monoclonal antibodies are invaluable for improving the specificity of bioassays, both by removing unwanted activities, and in the most stringent assays, for confirming the identity of the cytokine detected. Several cytokines can also be directly measured by ELISA techniques using monoclonal antibodies.