Brian Hondowicz

IL-12 Is Required to Maintain a Th1 Response During Leishmania major Infection

IL-12 initiates Th1 cell development and cell-mediated immunity, but whether IL-12 contributes to the maintenance of a Th1 response is unclear. To address this question, we infected IL-12 p40−/− C57BL/6 mice with Leishmania major, an intracellular protozoan parasite controlled by a cell-mediated immune response, and simultaneously administered IL-12. Whereas untreated p40−/− mice developed an uncontrolled infection, p40−/− mice treated with IL-12 for the first 2 or 4 wk of infection developed a Th1 response and resolved their lesions. However, the induction of this protective Th1 cell response by IL-12 treatment was not associated with long term immunity. We observed that on rechallenge in the absence of IL-12, the mice exhibited a susceptible phenotype. In addition, without rechallenge, lesions in the IL-12-treated p40−/− mice developed several weeks after cessation of IL-12 treatment. In both cases, disease was associated with the loss of a Th1 response and the development of a Th2 response. Our observations are not limited to the C57BL/6 strain, because IL-12 treatment was also unable to provide lasting protection to p40−/− BALB/c mice. Finally, we found that although Th1 cells from healed wild-type C57BL/6 mice adoptively transferred protection to L. major-infected RAG−/− mice, they were unable to protect p40−/− mice. In conclusion, these studies provide the first demonstration that IL-12 is required not only to initiate Th1 cell development but also throughout infection to maintain a Th1 cell response and resistance to L. major.

Infection with Leishmania major induces interleukin-12 production in vivo.

Experimental infections of mice with the protozoan parasite Leishmania major provide an excellent model for defining the conditions required for generation of CD4+ Th1 and Th2 cells in vivo. Since interleukin-12 (IL-12) has been implicated in the development of Th1 cells, we investigated whether L. major stimulates IL-12 production in vitro or in vivo. Surprisingly, macrophages cultured in vitro failed to produce IL-12 following L. major infection. In contrast, lymph node cells from C3H mice infected for 2 days with L. major produced elevated levels of IL-12. In order to determine if the inability to stimulate IL-12 production was limited to in vitro infections, we infected macrophages in vivo by inoculating L. major into the peritoneal cavity. Peritoneal cells isolated 24 h later exhibited a significant increase in the number of cells producing IL-12. In addition, supernatants harvested from these cells following culture contained elevated levels of IL-12. These data indicate that L. major infection induces increased IL-12 production in mice.

The Role of IL-12 in Maintaining Resistance to Leishmania major

IL-12p40 is required for the maintenance of resistance during Leishmania major infection. In this study, we addressed how IL-12 mediates this function. First, we demonstrated that both subunits of IL-12, p40 and p35, were required for continued resistance to L. major. Second, using IL-12, IL-4 doubly deficient mice, we investigated the possibility that IL-12 inhibits IL-4-induced outgrowth of Th2 cells that might compete with Th1 cells. We found that even in the absence of a Th2 response, IL-12 was still required to maintain resistance. Next, using adoptive transfer of Thy-1 disparate CD4+ T cells from L. major-healed mice, we were able to show that the loss of a protective response in L. major-infected IL-12-deficient mice is linked with the loss of Th1 cells. In contrast, there was an equal recovery of CD4+ Th1 cells from wild-type and IL-12-deficient mice when transferred into mice that were not challenged with L. major. The ability of Th1 cells to survive regardless of IL-12 levels in the absence of Ag stimulation was confirmed by adoptive transfer studies of CD4+ Th1 cells from DO11.10 TCR transgenic mice. Taken together, these results indicate that, rather than modulating Th2 responses or optimizing IFN-γ production, the critical role for IL-12 in maintaining cell-mediated immunity may be to prevent the loss of Th1 cells during a challenge infection.

The role of IL‐12 in the maintenance of an established Th1 immune response in experimental leishmaniasis

IL‐12 initiates the development of cell‐mediated immunity by promoting the differentiation of naive T cells into the Th1 phenotype, and is essential in the development of a Th1 immune response to the intracellular protozoan parasite, Leishmania major. The present study investigated whether IL‐12 is also required for the maintenance and effector function of an established Th1 immune response in L. major ‐infected mice. While neutralization of IL‐12 com promised the ability of a leishmanial antigen‐reactive Th1 cell clone to produce IFN‐γ in vitro, lymphnode cells taken from 2‐week L. major ‐infected mice were able to secrete IFN‐γ in an IL‐12‐independent manner. However, when a short‐term T cell line was established in vitro from lymph node cells, the production of IFN‐γ again became IL‐12 dependent. These results suggest that other factors may compensate for IL‐12 in vivo in promoting IFN‐γ production during L. major infection. To directly assess if IL‐12 was required in vivo for resistance to L. major, we studied the effect of IL‐12 neutralization on both a primary and secondary L. major infection in C3H mice. L. major infection in C3H mice is characterized by the development of a small lesion that heals by 8 weeks, and these animals are resistant to reinfection. As previously reported, administration of anti‐IL‐12 monoclonal antibody (mAb) during a primary infection led to severe disease. However, mice that had healed from a primary infection with L. major and were treated with anti‐IL‐12 mAb were as resistant as control animals. These findings suggest that once Th1 cells have developed, their effector function in vivo is independent of IL‐12, and that this independence is not due to an intrinsic property of the T cell, but to the microenvironment created by the infection.

Modulation of Kv Channel Expression and Function by TCR and Costimulatory Signals during Peripheral CD4+ Lymphocyte Differentiation

Ionic signaling pathways, including voltage-dependent potassium (Kv) channels, are instrumental in antigen-mediated responses of peripheral T cells. However, how Kv channels cooperate with other signaling pathways involved in T cell activation and differentiation is unknown. We report that multiple Kv channels are expressed by naive CD4+ lymphocytes, and that the current amplitude and kinetics are modulated by antigen receptor–mediated stimulation and costimulatory signals. Currents expressed in naive CD4+ lymphocytes are consistent with Kv1.1, Kv1.2, Kv1.3, and Kv1.6. Effector CD4+ cells generated by optimal TCR and costimulation exhibit only Kv1.3 current, but at approximately sixfold higher levels than naive cells. CD4+ lymphocytes anergized through partial stimulation exhibit similar Kv1.1, Kv1.2, and/or Kv1.6 currents, but approximately threefold more Kv1.3 current than naive cells. To determine if Kv channels contribute to the distinct functions of naive, effector, and anergized T cells, we tested their role in immunoregulatory cytokine production. Each Kv channel is required for maximal IL-2 production by naive CD4+ lymphocytes, whereas none appears to play a role in IL-2, IL-4, or IFN-γ production by effector cells. Interestingly, Kv channels in anergized lymphocytes actively suppress IL-4 production, and these functions are consistent with a role in regulating the membrane potential and calcium signaling.

Maintenance of IL‐12‐responsive CD4+ T cells during a Th2 response in Leishmania major‐infected mice

BALB / c and anti‐IL‐12‐treated C3H mice infected with Leishmania major develop a Th2 cell response. However, in contrast to BALB / c mice, C3H mice treated transiently with an anti‐IL‐12 monoclonal antibody switch from a Th2 to a Th1 response and resolve their lesions once treatment is terminated. We report here that the critical difference in the Th2 response between BALB / c and C3H mice is in their ability to respond to IL‐12. Thus, C3H mice with a Th2 response maintain a CD4+ T cell population that expresses IL‐12 receptor β1 and β2 mRNA and produces IFN‐γ after exposure to IL‐12. These results indicate that Th2 cell populations from different genetic backgrounds differ in their stability, and that this difference can be related to differential regulation of the IL‐12 receptor.

Regulation of T Cell Activation and Tolerance by Phospholipase Cγ-1-Dependent Integrin Avidity Modulation

Ag receptor engagement without costimulation induces a tolerant state in CD4+ T cells termed anergy. Anergic CD4+ T cells are primarily characterized by the inability to produce IL-2, but the biochemical basis for this functional defect is not completely understood. We demonstrate that primary CD4+ T cells anergized by costimulatory blockade exhibit impaired TCR-coupled phospholipase C (PLC)γ-1 activation. This defect is associated with the marked reduction of multiple downstream signaling events required for IL-2 transcription, including mobilization of intracellular Ca2+ and activation of the mitogen-activated protein kinase cascade. We also found that primary anergic CD4+ T cells fail entirely to modulate their integrin binding avidity in response to TCR stimulation. Integrin avidity modulation is required for full T cell activation and effector function, and as we show in this study, is completely dependent upon PLCγ-1 activity. Finally, analogs that mimic the actions of diacylglycerol and inositol 1,4,5-triphosphate, the immediate products of PLCγ-1 activity, restored integrin avidity modulation and IL-2 production by anergic T cells. Thus, deficient coupling of PLCγ-1 to the TCR appears to be a central biochemical defect that could potentially account for the failure of multiple functional responses in primary anergic CD4+ T cells.

Distinct calcium channels regulate responses of primary B lymphocytes to B cell receptor engagement and mechanical stimuli

Intracellular Ca2+ plays a central role in controlling lymphocyte function. Nonetheless, critical gaps remain in our understanding of the mechanisms that regulate its concentration. Although Ca2+-release-activated calcium (CRAC) channels are the primary Ca2+ entry pathways in T cells, additional pathways appear to be operative in B cells. Our efforts to delineate these pathways in primary murine B cells reveal that Ca2+-permeant nonselective cation channels (NSCCs) operate in a cooperative fashion with CRAC. Interestingly, these non-CRAC channels are selectively activated by mechanical stress, although the mechanism overlaps with BCR-activated pathways, suggesting that they may operate in concert to produce functionally diverse Ca2+ signals. NSCCs also regulate the membrane potential, which activates integrin-dependent binding of B cells to extracellular matrix elements involved in their trafficking and localization within secondary lymphoid organs. Thus, CRAC and distinct Ca2+ permeant NSCCs are differentially activated by the BCR and mechanical stimuli and regulate distinct aspects of B cell physiology.

Influence of Parasite Load on the Ability of Type 1 T Cells To Control Leishmania major Infection

ABSTRACT BALB/c mice infected with Leishmania major developed a type 2 immune response which failed to control parasite replication. We found that scid mice that received splenocytes from BALB/c mice that had been infected for 3 weeks with L. major (a type 2 cell population) and that were subsequently infected with L. major were protected when they were treated with interleukin 12 (IL-12). In contrast, IL-12 was ineffective at protecting BALB/c mice infected for 3 weeks, suggesting that a high parasite load regulates the development of protective immunity. To determine how this regulation operates, we performed a series of adoptive transfers of naïve, type 1 or type 2 splenocytes into scid mice. The recipient scid mice were infected either for 5 weeks prior to cell transfer (and thus had a high parasite load) or at the time of cell transfer. scid mice that were infected for 5 weeks and received a type 1 cell population were able to cure their lesions. However, when 5-week-infected scid mice received both type 1 and 2 cell populations, they were unable to control their infections. In contrast, the same type 1 and 2 cells transferred to naïve scid mice, which were subsequently infected, provided protection. In addition, we found that naïve cells mediated protection in scid mice with established lesions. These results show that high parasite numbers do not block type 1 protective responses or the development of type 1 responses. Instead, the influence of a high parasite load is dependent on the presence of a type 2 cell population.

Regulation of the production of soluble IL-4 receptors in murine cutaneous leishmaniasis. The roles of IL-12 and IL-4.

These studies were undertaken with the purpose of elucidating the key signals involved in the regulation of the production of soluble interleukin‐4 receptors (sIL‐4R) in mice during Th1 and Th2 responses to infection with the parasite Leishmania major. Our results showed that the production of sIL‐4R was consistently higher in lymph node cell cultures from animals mounting a predominant Th2 response (BALB/c mice), and that sIL‐4R production paralleled that of IL‐4 in both mouse strains, even in the presence of a dominant Th1 response (C3H/FeJ mice). Consistently, administration of anti‐IL‐12 antibodies to infected C3H/FeJ mice induced a switch from a Th1‐ to a Th2‐type response and resulted in enhanced production of sIL‐4R. Addition of rIL‐12 to splenic cell cultures, however, was found not to have a direct effect on sIL‐4R production induced by IL‐4 or T cell mitogens. Moreover, the production of sIL‐4R appears to be little influenced by Th1‐produced cytokines, inasmuch as recombinant interferon‐γ or supernatants derived from antigen‐stimulated Th1 clones did not affect the production of sIL‐4R by activated splenic cultures. Despite its correlation with Th2 responses, the presence of IL‐4 was not an absolute requirement for the up‐regulation of the expression of sIL‐4R because increased levels could be induced on cells obtained from IL‐4−/− mice. These results indicate that, although enhanced sIL‐4R production is a feature related to the activation and/or generation of Th2 responses, it is not absolutely dependent on IL‐4 or directly inhibited by IL‐12 or Th1 cytokines. J. Leukoc. Biol. 66: 481–488; 1999.