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

Discovery of T Cell Antigens by High-Throughput Screening of Synthetic Minigene Libraries

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery

Switching an established Th2 cell response to a Th1 phenotype in Leishmania major infected mice

Infections in mice with Leishmania major are used to study Th cell subset development and regulation, since some strains of mice, such as C3H, develop a Th1 cell response and heal, while other strains, such as BALB/c, develop a Th2 phenotype and exhibit chronic disease. It is widely believed that once the Th2 cell response is established in BALB/c mice, it is difficult to alter. However, a combination of IL-12 treatment and chemotherapy can induce healing in BALB/c mice with an established Th2 cell response. Moreover, we found that while C3H mice develop a Th2 phenotype when given anti-IL-12 mAb, they can switch to a Th1 cell response once mAb treatment is terminated. Therefore, the goal of this thesis was to characterize the factors controlling the switch from a Th2 to a Th1 cell phenotype in these two models. We examined the effects of a low and high parasite burden on the regulation of BALB/c T cell subsets by adoptively transferring cells with defined phenotypes into BALB/c scid mice. In these studies, we found that a high parasite burden does not directly inhibit type 1 cell development or effector function, but rather exerts its inhibitory function only in the presence of a type 2 cell population. Even though anti-IL-12-treated C3H mice contained high numbers of parasites during the first 4 weeks, similar to BALB/c mice, once the anti-IL-12-treatment was terminated these animals developed a Th1 cell response and healed. We show that the critical factors for developing Th1 cells in the presence of an ongoing Th2 cell response in anti-IL-12-treated C3H mice are the continued production of, and responsiveness to, IL-12. Thus, we found that, in contrast to a population of Th2 cells from BALB/c mice, a Th2 cell population from anti-IL-12-treated C3H mice contained a T cell population which responded to IL-12 by increasing the IL-12 receptor and IFN-γ production. Overall, our results are the first to demonstrate that the maintenance of a Th2 cell response during an infection, and the ability to alter that response, may differ depending upon the genetic background of the host

Switching an established Th2 cell response to a Th1 phenotype in Leishmania major infected mice

Infections in mice with Leishmania major are used to study Th cell subset development and regulation, since some strains of mice, such as C3H, develop a Th1 cell response and heal, while other strains, such as BALB/c, develop a Th2 phenotype and exhibit chronic disease. It is widely believed that once the Th2 cell response is established in BALB/c mice, it is difficult to alter. However, a combination of IL-12 treatment and chemotherapy can induce healing in BALB/c mice with an established Th2 cell response. Moreover, we found that while C3H mice develop a Th2 phenotype when given anti-IL-12 mAb, they can switch to a Th1 cell response once mAb treatment is terminated. Therefore, the goal of this thesis was to characterize the factors controlling the switch from a Th2 to a Th1 cell phenotype in these two models. We examined the effects of a low and high parasite burden on the regulation of BALB/c T cell subsets by adoptively transferring cells with defined phenotypes into BALB/c scid mice. In these studies, we found that a high parasite burden does not directly inhibit type 1 cell development or effector function, but rather exerts its inhibitory function only in the presence of a type 2 cell population. Even though anti-IL-12-treated C3H mice contained high numbers of parasites during the first 4 weeks, similar to BALB/c mice, once the anti-IL-12-treatment was terminated these animals developed a Th1 cell response and healed. We show that the critical factors for developing Th1 cells in the presence of an ongoing Th2 cell response in anti-IL-12-treated C3H mice are the continued production of, and responsiveness to, IL-12. Thus, we found that, in contrast to a population of Th2 cells from BALB/c mice, a Th2 cell population from anti-IL-12-treated C3H mice contained a T cell population which responded to IL-12 by increasing the IL-12 receptor and IFN-γ production. Overall, our results are the first to demonstrate that the maintenance of a Th2 cell response during an infection, and the ability to alter that response, may differ depending upon the genetic background of the host

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 ÷ Thl and Th2 cells in vivo. Since interleukin-12 (IL-12) has been implicated in the development of Thl 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