Jeanne Magram

IL-12-Deficient Mice Are Defective in IFNγ Production and Type 1 Cytokine Responses

Abstract IL-12 is a cytokine that can exert regulatory effects on T and NK cells and promote Th1 responses. To delineate further the physiologic role of IL-12 in immunity, mice deficient for this cytokine were generated. IL-12-deficient mice were impaired but not completely lacking in the ability to produce IFNγ following endotoxin administration and to mount a Th1 response in vivo, as measured by antigen-induced IFNγ secretion by immune lymph node cells in vitro. In contrast, secretion of IL-4 was enhanced, while proliferation and secretion of IL-2 and IL-10 were normal following antigen stimulation. DTH responses were significantly reduced in IL-12-deficient mice, but no defect in allogeneic CTL responses was observed. These results indicate that IL-12 plays an essential role in regulating IFNγ production and in facilitating normal DTH responses. However, other phenomena associated with Th1 responses and cell-mediated immunity, i.e., IL-2 secretion and CTL generation, were not compromised in the absence of IL-12.

Mice Lacking Bioactive IL-12 Can Generate Protective, Antigen-Specific Cellular Responses to Mycobacterial Infection Only if the IL-12 p40 Subunit Is Present

Recent evidence suggests that absence of the IL-12p40 subunit is more detrimental to the generation of protective responses than is the absence of the p35 subunit. To determine whether this is the case in tuberculosis, both p35 and p40 knockout mice were infected with Mycobacterium tuberculosis. Mice lacking the p40 subunit were highly susceptible to increased bacterial growth, exhibited reduced production of IFN-γ, and had increased mortality. In contrast, mice lacking the p35 subunit exhibited a moderate ability to control bacterial growth, were able to generate Ag-specific IFN-γ responses, and survived infection longer. The superior Ag-specific responses of the p35 gene-disrupted mice, when compared with the p40 gene-disrupted mice, suggest that the p40 subunit may act other than as a component of IL-12. A candidate molecule capable of driving the protective responses in the p35 gene-disrupted mice is the novel cytokine IL-23. This cytokine is composed of the IL-12 p40 subunit and a p19 subunit. In support of a role for this cytokine in protective responses to M. tuberculosis, we determined that the p19 subunit is induced in the lungs of infected mice.

IL-12 Receptor β2 (IL-12Rβ2)-Deficient Mice Are Defective in IL-12-Mediated Signaling Despite the Presence of High Affinity IL-12 Binding Sites

Two subunits of the IL-12 receptor (IL-12R), IL-12Rβ1 and IL-12Rβ2, have been identified and cloned. Previous studies demonstrated that the IL-12Rβ1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12Rβ2 in IL-12 signaling, we have generated IL-12Rβ2-deficient (IL-12Rβ2−/−) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12Rβ2−/− mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12Rβ2−/− mice failed to produce IFN-γ or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12Rβ2−/− splenocytes was not induced when incubated with IL-12. IL-12Rβ2−/− splenocytes were deficient in IFN-γ secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12Rβ2−/− mice were deficient in vivo in their ability to produce IFN-γ following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12Rβ1 is the subunit primarily responsible for binding IL-12, IL-12Rβ2 plays an essential role in mediating the biological functions of IL-12 in mice.

IL-12-Deficient Mice Are Defective but Not Devoid of Type 1 Cytokine Responses

Interleukin-12 (IL-12) has been described as a pivotal molecule in the immune response based in part on its ability to influence the differentiation of T helper (Th) cells into a type 1 (Th1) phenotype. This event is crucial in that appropriate differentiation of naive T cells can determine susceptibility or resistance to given pathogens by influencing the balance between cellular and humoral immunity. In order to further delineate the role of IL-12 in the immune response, we generated mice deficient for this cytokine. IL-12 knockout mice were viable, fully fertile, and displayed no obvious developmental abnormalities. Upon immunological analysis, these mice demonstrated an impaired ability to effect a Th1 response as well as an impaired ability to produce interferon-gamma in response to endotoxin in vivo. These data establish an essential role for IL-12 in the generation of optimal Th1 responses in vivo, but weak responses can occur independently of IL-12.

Impaired Th2 Subset Development in the Absence of CD4

Prior studies in CD4-deficient mice established the capacity of T helper (Th) lineage cells to mature into Th1 cells. Unexpectedly, challenge of these mice with Nippostrongylus brasiliensis, a Th2-inducing stimulus, failed to result in the development of Th2 cells. Additional studies were performed using CD4+ or CD4-CD8- (double-negative) T cell receptor (TCR) transgenic T cells reactive to LACK antigen of Leishmania major. Double-negative T cells were unable to develop into Th2 cells in vivo, and, unlike CD4+ T cells, could not be primed for interleukin-4 production in vitro. Similarly, CD4+ TCR transgenic T cells primed on antigen-presenting cells expressing mutant MHC class II molecules unable to bind CD4 did not differentiate into Th2 cells. These data suggest that interactions between the TCR, MHC II-peptide complex and CD4 may be involved in Th2 development.

INTERLEUKIN-12 AN INTEGRAL CYTOKINE IN THE IMMUNE RESPONSE

Interleukin 12 (IL-12) is a heterodimeric cytokine that is produced primarily by antigen-presenting cells and plays a primary role in the induction of cell-mediated immunity. This function is promoted by the IL-12 induced production of interferon-gamma (IFN-gamma) from both resting and activated NK and T cells, by the proliferative activity of IL-12 on activated NK and T cells, by enhancing the cytotoxic activity of NK cells, and by supporting cytotoxic T lymphocyte generation. IL-12 and IL-12-induced IFN-gamma promote the development of naive T cells into Th1 cells and the proliferation and IFN-gamma secretion by differentiated Th1 cells in response to antigen. IL-12 has been found to exhibit many of these activities in vivo, as well as in vitro, and thus IL-12 plays an important role in both innate resistance and antigen-specific adaptive immunity to intracellular bacterial, fungal, and protozoan pathogens. Due to its effects on T cells, recombinant IL-12 has been shown to have therapeutic activity in a variety of mouse tumor and infectious disease models and is being evaluated in clinical trials in human cancer patients. IL-12 also appears to play a role in the genesis of some forms of immunopathology, including endotoxin-induced shock and some autoimmune diseases associated with aberrant Th1 activity. Therefore, IL-12 antagonists may also have therapeutic potential in the treatment of auto immune disorders.

Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits

Interleukin‐12 (IL‐12) is a heterodimeric cytokine composed of two disulfide‐bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL‐12, a homodimer of the p40 subunit, designated (p40)2, has been shown to be a potent IL‐12 antagonist. To study the interaction between (p40)2 and the known IL‐12 receptor (IL‐12R) subunits, IL‐12Rβ1 and IL‐12Rβ2, we directly measured the binding activity of mouse (p40)2 to ConA‐activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (Kd about 5 pM) and low affinity (Kd about 15 nM) binding sites for mouse 125I‐labeled (p40)2. To elucidate which of the IL‐12R subunits binds mouse (p40)2, binding studies of mouse 125I‐labeled (p40)2 to Ba/F3 cells expressing recombinant mouse IL‐12Rβ1 and/or mouse IL‐12Rβ2 were carried out. Mouse IL‐12Rβ1 bound mouse 125I‐labeled (p40)2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL‐12Rβ2 bound mouse 125I‐labeled (p40)2 very poorly. These data demonstrate that similar to IL‐12, mouse (p40)2 binds with both high and low affinity to Con A blasts and B cells, and that IL‐12Rβ1 is responsible for mediating the specific binding of mouse (p40)2.

12. – Interleukin-12

Publisher Summary This chapter focuses on interleukin-12 (IL-12), which is a potent modulator of natural killer (NK) and T cell functions. Through the activities on these two cell types, the cytokine provides an important link between natural resistance (NK cells) and adaptive immune responses (T cells). IL-12 is unusual among the known cytokines as it is a heterodimer composed of two subunits (p35 and p40) that represent unrelated gene products. The cDNAs for both IL-12 subunits were originally cloned from EBV-transformed induced human B cell lines NC-37 and RPMI 8866. The p40 subunit, having a molecular mass of 40 kDa, is composed of 306 amino acids (aa) and contains 10 cysteine residues, while the p35 subunit, having a molecular mass of 35 kDa, consists of 197 aa and has 7 cysteine residues. The primary cellular source of IL-12 production is the monocyte/macrophage. In addition, B cells produce IL-12 but at significantly lower levels. When cultures of human peripheral blood mononuclear cells (PBMC) are incubated with Staphylococcus aureus (SAC), lipopolysaccharides (LPS), or killed Mycobacterium tuberculosis, a dramatic increase in IL-12 production is observed. IL-12 upregulates the cell-surface expression of adhesion/activation molecules and cytokine receptors on cytolytic cells, including CD2, CD11a, CD54, CD56, CD69, CD71, HLA-DR, the 75 kDa tumor necrosis factor (TNF) receptor, and receptors for IL-2 (α and β subunits), IL-4, and IL-12. In addition to its effects on NK cells, IL-12 can also facilitate the induction of specific human CTL responses to weakly immunogenic allogeneic melanoma tumor cells.