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

Mechanisms of T cell organotropism

F.M.M.-B. is supported by the British Heart Foundation, the Medical Research Council of the UK and the Gates Foundation

Cracking the BAFF code.

The tumour necrosis factor (TNF) family members B cell activating factor (BAFF) and APRIL (a proliferation-inducing ligand) are crucial survival factors for peripheral B cells. An excess of BAFF leads to the development of autoimmune disorders in animal models, and high levels of BAFF have been detected in the serum of patients with various autoimmune conditions. In this Review, we consider the possibility that in mice autoimmunity induced by BAFF is linked to T cell-independent B cell activation rather than to a severe breakdown of B cell tolerance. We also outline the mechanisms of BAFF signalling, the impact of ligand oligomerization on receptor activation and the progress of BAFF-depleting agents in the clinical setting

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11ccreIL-4Rα−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11ccreIL-4Rα−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms

Selective killing of Burkitt's lymphoma cells by mBAFF-targeted delivery of PinX1

Increased expression of BAFF (B cell-activating factor belonging to the TNF family) and its receptors has been identified in numerous B-cell malignancies. A soluble human BAFF mutant (mBAFF), binding to BAFF receptors but failing to activate B-lymphocyte proliferation, may function as a competitive inhibitor of BAFF and may serve as a novel ligand for targeted therapy of BAFF receptor-positive malignancies. Pin2/TRF1-interacting protein X1 (PinX1), a nucleolar protein, potently inhibits telomerase activity and affects tumorigenicity. In this study, we generated novel recombinant proteins containing mBAFF, a polyarginine tract 9R and PinX1 (or its C/N terminal), to target lymphoma cells. The fusion proteins PinX1/C–G4S–9R–G4S–mBAFF and PinX1/C–9R–mBAFF specifically bind and internalize into BAFF receptor-positive cells, and subsequently induce growth inhibition and apoptosis. The selective cytotoxicity of the fusion proteins is a BAFF receptor-mediated process and depends on mBAFF, PinX1/C and 9R. Moreover, the fusion proteins specifically kill BAFF receptor-expressing Burkitt's lymphoma (BL) cells by inhibiting telomerase activity and the consequent shortening of telomeres. Therapeutic experiments using PinX1C–G4S–9R–G4S–mBAFF in severe combined immunodeficient (SCID) mice implanted with Raji cells showed significantly prolonged survival times, indicating the in vivo antitumor activity of the fusion protein. These results suggest the potential of PinX1/C–G4S–9R–G4S–mBAFF in targeted therapy of BL

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

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations

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

Accelerated healing of cutaneous leishmaniasis in non-healing BALB/c mice using water soluble amphotericin B-polymethacrylic acid.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that causes prominent skin scaring. No water soluble, non-toxic, short course and low cost treatment exists. We developed a new water soluble amphotericin B-polymethacrylic acid (AmB-PMA) using established and scalable chemistries. AmB-PMA was stable for 9 months during storage. In vitro, it was effective against Leishmania spp. promastigotes and amastigote infected macrophages. It was also less toxic and more effective than deoxycholate-AmB, and similar to liposomal AmB. Its in vivo activity was determined in both early and established CL lesion models of Leishmania major infection in genetically susceptible non-healing BALB/c mice. Intradermal AmB-PMA at a total dose of 18 mg of AmB/kg body weight led to rapid parasite killing and lesion healing. No toxicity was seen. No parasite relapse occurred after 80 days follow-up. Histological studies confirmed rapid parasite clearance from macrophages followed by accelerated fibroblast mediated tissue repair, regeneration and cure of the infection. Quantitative mRNA studies of the CL lesions showed that accelerated healing was associated with increased Tumour Necrosis Factor-α and Interferon-γ, and reduced Interleukin-10. These results suggest that a cost-effective AmB-PMA could be used to pharmacologically treat and immuno-therapeutically accelerate the healing of CL lesions

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