IL-15/IL-15Rα Heterodimeric Complex as Cancer Immunotherapy in Murine Breast Cancer Models.

Interleukin 15 (IL-15) has been evaluated as a potential treatment for solid tumors in clinical trials, but the effectiveness of systemic IL-15 administration as a monotherapy has not been realized. IL-15 receptor alpha (IL-15Rα) can stabilize IL-15 and enhance its bioactivity. The goal of this study was to examine the activity of IL-15/IL-15Rα complex (IL-15cx) to CD8+ T cells and evaluate its potential efficacy in murine breast cancer models. The antitumor efficacy was studied in mouse mammary carcinoma models (Her2/neu transgenic and 4T1-luc mammary cancers) treated with systemic recombinant protein with/without the depletion of myeloid-derived suppressor cells or intra-tumoral gene electrotransfer (GET). IL-15cx shows superior in vivo bioactivity to expand CD8 T cells in comparison to an equimolar single chain IL-15. T-bet is partially involved in CD8 T cell expansion ex vivo and in vivo due to IL-15 or IL-15cx. Intraperitoneal administration of IL-15cx results in a moderate inhibition of breast cancer growth that is associated with an increase in the frequency of cytotoxic CD8 T cells and the improvement of their function. The depletion of myeloid-derived suppressor cells (MDSCs) has no impact on mouse breast cancer growth. IL-15cx treatment diminishes MDSCs in murine tumors. However, it also antagonizes the effects of anti-Gr-1 depleting antibodies. Intratumoral GET with plasmid IL-15/IL-15Rα leads to a long-term survival benefit in 4T1 mammary carcinoma model. An early increase of local cytotoxic cells correlates with GET treatment and an increase of long-term memory T cells results from animals with complete tumor regression. Systemic and local administration of IL-15cx shows two distinct therapeutic responses, a moderate tumor growth inhibition or heterogeneous tumor regressions with survival improvement. Further studies are warranted to improve the efficacy of IL-15cx as an immunotherapy for breast cancer.

CpG and interleukin-15 synergize to enhance IFN-γ production by activated CD8+ T cells.

Interleukin-15 (IL-15) regulates the development and maintenance of memory CD8(+) T cells. Paradoxically, we previously reported that IL-15 could enhance CD8(+) T-cell responses to IL-12, a proinflammatory cytokine required for optimal priming of effector CD8(+) T cells. To expand the physiological relevance of these findings, we tested IL-15 for its ability to enhance T-cell responses to bacterial CpG. Expectedly, CpG enhanced the production of IFN-γ by CD8(+) T cells polyclonally activated with anti-CD3. However, addition of IL-15 to CpG-stimulated cultures led to a striking increase in IFN-γ production. The effect of CpG and IL-15 was also evident with CD8(+) T cells recovered from mice infected with the parasite Trypanosoma cruzi (T. cruzi) and restimulated with antigen. The observed synergy between CpG and IL-15 occurred in an IL-12-dependent manner, and this effect could even be demonstrated in cocultures of activated CD8(+) T cells and CD4(+)CD25(+) regulatory T cells. Although IFN-γ was not essential for CpG-induced IL-12, the ability of CpG and IL-15 to act on CD8(+) T cells required expression of the IFN-γ-inducible transcription factor T-bet. These data have important implications for development of vaccines and design of therapies to boost CD8(+) T-cell responses to infectious agents and tumors.

Intranasal vaccination in mice with an attenuated Salmonella enterica Serovar 908htr A expressing Cp15 of Cryptosporidium: impact of malnutrition with preservation of cytokine secretion.

Cryptosporidium is a protozoan parasite associated with acute and persistent diarrhea that, even in asymptomatic persons, can impair normal growth and potentially cognitive and physical development in young children. The recent availability of the complete gene sequence for Cryptosporidium hominis antigen Cp15 allows examination of innovative vaccine regimens involving intra-nasal antigen priming with live bacterial vectors applicable to human populations. We used a recently described weaned mouse model of cryptosporidiosis, where nourished and malnourished vaccinated mice receive the Cp15 antigen recombinant with cytolysinA on a Salmonella serovar Typhi CVD 908-htr A vector, followed by parenteral exposure to antigen with adjuvant. After challenge with Cryptosporidium oocysts via gavage, parameters of infection and disease (stool shedding of parasites, growth rates) were quantified, and serum/lymphoid tissue harvested to elucidate the Cp15-specific adaptive immune response. In vaccinated nourished mice, the regimen was highly immunogenic, with strong antigen-specific IL-6 and IFN-γ secretion and robust Cp15-specific immunoglobulin titers. In vaccinated malnourished mice, secretion of cytokines, particularly IFN-γ, and antigen-specific humoral immunity were generally undiminished despite protein deprivation and stunted growth. In contrast, after natural (oral) challenge with an identical inoculum of Cryptosporidium oocysts, cytokine and humoral responses to Cp15 were less than one-fourth those in vaccinated mice. Nevertheless, vaccination resulted in only transient reduction in stool shedding of parasites and was not otherwise protective against disease. Overall, immunogenicity for a C. hominis antigen was documented in mice, even in the setting of prolonged malnutrition, using an innovative vaccine regimen involving intra-nasal antigen priming with a live enteric bacterial vector, that has potential applicability to vulnerable human populations irrespective of nutritional status.

Regulation of proinflammatory Th17 responses during Trypanosoma cruzi infection by IL-12 family cytokines.

Previously, we reported that the transcription factor T-bet (Tbx21) regulates Th17 responses to Trypanosoma cruzi infection in an IFN-γ-independent manner. In an effort to further understand this regulation, we examined the development and plasticity of Th17 cells during T. cruzi infection. Th17 cells recovered from infected Tbx21(-/-) mice were amenable to the inhibitory effects of T-bet, as ectopic expression of T-bet reduced IL-17 expression. We subsequently addressed the role of IL-12 family cytokines IL-12 and IL-27 and report that IL-12p35(-/-) mice infected with T. cruzi exhibited a significant increase in Th17 cells and Th17-associated inflammation. Ex vivo culture of these cells with IL-12 led to a dramatic reduction in IL-17 production and concomitant increase in IFN-γ. Importantly, the ability of IL-12 to suppress IL-17 was independent of IFN-γ. Surprisingly, and contrary to results reported for other pathogens, IL-27 had no inhibitory effect on Th17 development, as Ebi-3(-/-) mice failed to show any increase in their T. cruzi-specific Th17 response. Furthermore, IL-27 could not compensate or synergize with IL-12 to suppress IL-17 production ex vivo. Thus, we have established that IL-12, not IL-27, is critical for regulating Th17 responses to T. cruzi.

Identification and immunological characterization of three potential vaccinogens against Cryptosporidium species.

Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium hominis and Cryptosporidium parvum, leading to acute, persistent, and chronic diarrhea with life-threatening consequences in immunocompromised individuals. In developing countries, cryptosporidiosis in early childhood has been associated with subsequent significant impairment in growth, physical fitness, and intellectual abilities. Currently, vaccines are unavailable and chemotherapeutics are toxic and impractical, and agents for immunoprophylaxis or treatment of cryptosporidiosis are a high priority. Availability of the genome sequences for C. hominis and C. parvum provides new opportunities to procure and examine novel vaccine candidates. Using the novel approach of "reverse vaccinology," we identified several new potential vaccine candidates. Three of these antigens--Cp15, profilin, and a Cryptosporidium apyrase--were delivered in heterologous prime-boost regimens as fusions with cytolysin A (ClyA) in a Salmonella live vaccine vector and as purified recombinant antigens, and they were found to induce specific and potent humoral and cellular immune responses, suggesting their potential as new vaccinogens against Cryptosporidium infection.

T-bet-independent effects of IL-12 family cytokines on regulation of Th17 responses to experimental T. cruzi infection.

Tbx21 (i.e., T-bet) is an IFN-γ-inducible transcription factor that promotes Th1 differentiation. Previously, we reported that Tbx21(-/-) mice develop a robust Th17 response to the parasite Trypanosoma cruzi, including CD4(+) T cell subsets producing IL-17 and IFN-γ. Because of the known inhibitory effects of IFN-γ on Th17 cells, the purpose of this study was to determine the contribution of IFN-γ to regulation of Th17 differentiation during the course of T. cruzi infection. We observed that infection of IFN-γ(-/-) or Stat-1(-/-) mice generated increased numbers of IL-17-producing cells. In sharp contrast to infected Stat-1(-/-) or Tbx21(-/-) mice, however, IFN-γ(-/-) mice developed a lower overall Th17 response, suggesting that IFN-γ was not required for T-bet-dependent activity, including T-bet-dependent expression of CXCR3. To determine if IFN-γ could influence Th17 responses indirectly by acting on APCs, we neutralized IFN-γ in cultures containing APC and T. cruzi antigens. Although anti-IFN-γ increased IL-17 production modestly, anti-IFN-γ and anti-IL-12 led to a significant enhancement of T. cruzi-specific IL-17 (P<0.01). In contrast to the inhibitory effects of IL-12, IL-23 was able to stimulate Tbx21(-/-) T cells and cause a striking increase in T. cruzi-specific IL-17. These data show that the IL-12 family of cytokines can influence Th17 responses in a T-bet-independent manner and that the effects of IFN-γ are not necessarily related to its ability to induce T-bet expression in T cells.

T-bet-dependent regulation of CD8+ T-cell expansion during experimental Trypanosoma cruzi infection.

The transcription factor T-bet (T-box, expressed in T cells), promotes type I immunity to pathogens through effects involving T cells and dendritic cells. In CD8(+) T cells, many of the functions of T-bet are redundant with those of eomesodermin (Eomes), a paralogue of T-bet. We therefore investigated the role of T-bet in immunity to Trypanosoma cruzi, an intracellular pathogen that causes Chagas disease, and which requires CD8(+) T cells for resistance. T-bet-deficient mice (tbx21(-/-)) were highly susceptible to T. cruzi infection, marked by severe liver pathology. CD8(+) T cells from infected tbx21(-/-) mice expressed typical markers of activation, including CD44 and CD25. In striking contrast, there was a 10-fold reduction in the number of antigen-specific CD8(+) T cells in tbx21(-/-) mice. This reduction was not a consequence of increased apoptosis or altered tissue-specific migration. Further, antigen-presenting cell (APC) functions in tbx21(-/-) mice were normal as we observed comparable levels of B7-1, B7-2 and CD40 expression as well as normal antigen-driven proliferation of wild-type CD8(+) T cells in infected tbx21(-/-) mice. However, adoptive transfer of naïve T cells from tbx21(-/-) donors into infected Rag-2-deficient mice (tbx21(+/+)) demonstrated a similar quantitative defect in CD8(+) T-cell expansion. These data demonstrate that T-bet facilitates immunity to T. cruzi by promoting the expansion of T. cruzi-specific CD8(+) T cells in a T cell-intrinsic manner. They also serve to further illustrate the multifaceted functions of T-box proteins in regulating quantitative aspects of T-cell immunity, in addition to qualitative components such as cytokine production.

T-bet inhibits the in vivo differentiation of parasite-specific CD4+ Th17 cells in a T cell-intrinsic manner.

CD4(+) Th17 cells have emerged as a new T cell subset in the Th1/Th2 paradigm, and efforts have shifted toward understanding the factors that regulate their development in vivo. To analyze the role of the transcription factor T-bet in regulation of Th17 cells, we used a murine model of Trypanosoma cruzi infection, a protozoan parasite that causes Chagas disease in humans. Infection of Tbx21(-/-) mice led to normal, unimpaired development of Ag-specific CD4(+) T cells producing IFN-gamma. However, a robust Th17 response developed concomitant with Th1 responses. Despite significant IFN-gamma production, the physiological effects of Th17 responses prevailed as there was a sharp increase in Gr-1(+)Ly6G(+) neutrophils. Adoptive transfer of T cells from infected Tbx21(-/-) mice into Rag-2(-/-) mice (Tbx21(+/+)) revealed that CD4(+) T cells maintained their IL-17-producing phenotype, including those cells capable of producing both IFN-gamma and IL-17. Furthermore, and in contrast to the effects of IL-2 on Th17 development, IL-2 had no effect on IL-17 production by primed T cells. Importantly, adoptive transfer of T cells from naive Tbx21(-/-) mice into infected Rag-2(-/-) mice recapitulated the differentiation of T. cruzi-specific Th17 cells observed in infected Tbx21(-/-) mice. Conversely, transfer of wild-type T cells into infected Tbx21(-/-) mice did not reveal an increase in Th17 development. These results demonstrate that T-bet regulates the differentiation of T. cruzi-specific Th17 cells in vivo in a T cell-intrinsic manner. These data provide important insight into the role of T-bet in regulation of parasite-specific Th17 responses.

Profound enhancement of the IL-12/IL-18 pathway of IFN-gamma secretion in human CD8+ memory T cell subsets via IL-15.

Human memory CD8(+) T cell subsets, termed central memory and effector memory T cells, can be identified by expression of CD45RA, CD62 ligand (CD62L), and CCR7. Accordingly, functional differences have been described for each subset, reflecting unique roles in immunological memory. The common gamma-chain cytokines IL-15 and IL-7 have been shown to induce proliferation and differentiation of human CD8(+) T cell subsets, as well as increased effector functions (i.e., cytokines, cytotoxicity). In this study, we observed that addition of IL-15 or IL-7 to cultures of human CD8(+) T cells profoundly enhanced the IL-12-IL-18 pathway of IFN-gamma production. Importantly, IL-15 and IL-7 lowered the threshold concentrations of IL-12 and IL-18 required for induction of IFN-gamma by 100-fold. Comparison of IL-15 and IL-7 demonstrated that IL-15 was superior in its ability to enhance IL-12-IL-18-induced IFN-gamma, without evidence of a synergistic effect between IL-15 and IL-7. We also observed that IL-15- and IL-7-mediated enhancement of IL-12-IL-18-induced IFN-gamma production was a functional property of effector memory CD8(+) T cells. Despite a lack of association between cell division and acquisition of IL-12-IL-18-induced IFN-gamma, down-regulation of CD62L expression correlated well with increased IL-12-IL-18-induced IFN-gamma. Purified central memory T cells stimulated with IL-15 and IL-7 down-regulated CD62L and acquired potent IL-12-IL-18-induced IFN-gamma similar to effector memory T cells. Thus, in addition to its known role in development of T cell memory, IL-15 may amplify memory CD8(+) T cell effector functions by increasing sensitivity to proinflammatory cytokine stimulation.

Transforming growth factor-beta1 enhances the interferon-gamma-dependent, interleukin-12-independent pathway of T helper 1 cell differentiation.

Transforming growth factor (TGF)-beta, a pleiotropic cytokine that has multiple effects on immune responses, has been shown to inhibit interleukin (IL)-4/GATA-3 expression as well as T helper 2 (Th2) differentiation. Consistent with these reports, we found that priming T cells from DO11.10 transgenic mice with antigen in the presence of TGF-beta inhibited GATA-3 expression and the development of IL-4-producing T cells. Unexpectedly, the inhibition of Th2 development was accompanied by a substantial increase in the number of interferon-gamma (IFN-gamma)-producing cells. T cells primed with TGF-beta secreted IFN-gamma in response to both T-cell receptor ligation and IL-12/IL-18 stimulation, and expressed high levels of T-bet and low levels of GATA-3. The TGF-beta-mediated enhancement of T helper 1 (Th1) priming was independent of IL-12 and signal transducer and activator of transcription (STAT)-4, but required endogenous IFN-gamma. TGF-beta-mediated enhancement of the IFN-gamma-dependent, IL-12-independent pathway of Th1 priming was mediated primarily by the inhibition of IL-4 produced by memory/activated T cells in the unfractionated CD4+ responder population. Nevertheless, TGF-beta did not inhibit this pathway of Th1 differentiation when purified naive CD4+ T cells were used as responders. These data have important implications for strategies being considered for the use of TGF-beta-producing T cells for the treatment of autoimmune disorders.

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression.

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.