The impaired Th1 immune response of C3HeB/FeJ mice infected with Leishmania amazonensis: lessons learned from immunotherapy and vaccines

Leishmaniasis is a zoonotic disease caused by intracellular protozoan parasites of the genus Leishmania. Leishmania major and Leishmania amazonensis are both causative agents of cutaneous leishmaniasis in the Old and New World, respectively. While C3H mice are resistant to a L. major infection, they are susceptible to a L. amazonensis challenge and this is characterized by the development of chronic cutaneous lesions. Resistance to L. major is mediated by a T helper 1 (Th1) immune response characterized by CD4+ T cells producing IFN-gamma. In contrast, the susceptibility to L. amazonensis is associated with an impaired Th1 response and CD4 + T cell defects. Dendritic cells (DC) are the most potent antigen-presenting cells in vitro and in vivo and they have been used successfully as vaccine adjuvants to promote Th1 response in mouse studies of Leishmania spp. infection. Our hypothesis was that the induction of a Th1 response in C3H mice would promote resistance to a subsequent L. amazonensis challenge. Using a DC-based immunotherapy, we were able to induce a Th1 response in mice chronically infected with L. amazonensis. However, this did not mediate healing of the infection. When given before infection, a DC-based vaccine also promoted a Th1 response but it was not protective against a subsequent L. amazonensis promastigote challenge. Taken together, this data suggested that a Th1 response may not mediate resistance to L. amazonensis infection. However, we also show that the Th1 response elicited in C3H mice that had healed a previous L. major infection could promote resistance to a subsequent L. amazonensis infection. Therefore, future studies using the immune response of mice that have healed a L. major infection as a model will be needed to determine which immune factors are necessary and/or sufficient to induce protection to a L. amazonensis infection. This will be helpful for the design of future therapeutic and/or vaccine strategies against L. amazonensis

Interplay between CD8α+ Dendritic Cells and Monocytes in Response to Listeria monocytogenes Infection Attenuates T Cell Responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α+ dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8+ T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α+ DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α+ DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b+ DCs primarily secrete low levels of TNFα while CD8α+ DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α+ DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming

The impaired Th1 immune response of C3HeB/FeJ mice infected with Leishmania amazonensis: lessons learned from immunotherapy and vaccines

Leishmaniasis is a zoonotic disease caused by intracellular protozoan parasites of the genus Leishmania. Leishmania major and Leishmania amazonensis are both causative agents of cutaneous leishmaniasis in the Old and New World, respectively. While C3H mice are resistant to a L. major infection, they are susceptible to a L. amazonensis challenge and this is characterized by the development of chronic cutaneous lesions. Resistance to L. major is mediated by a T helper 1 (Th1) immune response characterized by CD4+ T cells producing IFN-gamma. In contrast, the susceptibility to L. amazonensis is associated with an impaired Th1 response and CD4 + T cell defects. Dendritic cells (DC) are the most potent antigen-presenting cells in vitro and in vivo and they have been used successfully as vaccine adjuvants to promote Th1 response in mouse studies of Leishmania spp. infection. Our hypothesis was that the induction of a Th1 response in C3H mice would promote resistance to a subsequent L. amazonensis challenge. Using a DC-based immunotherapy, we were able to induce a Th1 response in mice chronically infected with L. amazonensis. However, this did not mediate healing of the infection. When given before infection, a DC-based vaccine also promoted a Th1 response but it was not protective against a subsequent L. amazonensis promastigote challenge. Taken together, this data suggested that a Th1 response may not mediate resistance to L. amazonensis infection. However, we also show that the Th1 response elicited in C3H mice that had healed a previous L. major infection could promote resistance to a subsequent L. amazonensis infection. Therefore, future studies using the immune response of mice that have healed a L. major infection as a model will be needed to determine which immune factors are necessary and/or sufficient to induce protection to a L. amazonensis infection. This will be helpful for the design of future therapeutic and/or vaccine strategies against L. amazonensis.</p

Antigen-Responsive CD4(+) T Cells from C3H Mice Chronically Infected with Leishmania amazonensis Are Impaired in the Transition to an Effector Phenotype

C3HeB/FeJ mice challenged with Leishmania major develop a polarized Th1 response and subsequently heal, whereas Leishmania amazonensis challenge leads to chronic lesions with high parasite loads at 10 weeks postinfection. In this study, a comparison of draining lymph node cells from L. amazonensis- and L. major-infected mice at 10 weeks postinfection showed equivalent percentages of effector/memory phenotype CD44(hi) CD4(+) T cells producing interleukin-2 (IL-2) and proliferating after antigen stimulation. However, these cells isolated from L. amazonensis-infected mice were not skewed toward either a Th1 or Th2 phenotype in vivo, as evidenced by their unbiased Th1/Th2 transcription factor mRNA profile. In vivo antigen stimulation with added IL-12 failed to enhance gamma interferon (IFN-γ) production of CD4(+) T cells from L. amazonensis-infected mice. Antigen stimulation of CD4(+) T cells from L. amazonensis-infected mice in vitro in the presence of IL-12 resulted in production of only 10 to 15% of the IFN-γ produced by T cells from L. major-infected mice under identical conditions. These results suggest that the CD4(+) T-cell response during chronic L. amazonensis infection is limited during the transition from an early activated CD4(+) T-cell population to an effector cell population and demonstrate that these T cells have an intrinsic defect beyond the presence or absence of IL-12 during antigen stimulation

CD4(+) Th1 Cells Induced by Dendritic Cell-Based Immunotherapy in Mice Chronically Infected with Leishmania amazonensis Do Not Promote Healing

The susceptibility of mice to Leishmania amazonensis infection is thought to result from an inability to develop a Th1 response. Our data show that the low levels of gamma interferon (IFN-γ) produced by the draining lymph node (DLN) cells of chronically infected mice could be enhanced in vitro and in vivo with L. amazonensis antigen-pulsed bone marrow-derived dendritic cells (BM-DC) and the Th1-promoting cytokine interleukin-12 (IL-12). Given intralesionally to chronically infected mice, this treatment induced the upregulation of mRNA levels for IFN-γ, the transcription factor T-box expressed in T cells, and IL-12 receptor β2 in CD4(+) T cells from the DLN and an increase in parasite-specific immunoglobulin G2a in the serum. However, this Th1 response was not associated with healing, and the antigen-specific enhancement of IFN-γ production remained impaired in the DLN. However, addition of IL-12 to the in vitro recall response was able to recover this defect, suggesting that antigen-presenting cell-derived IL-12 production may be limited in infected mice. This was supported by the fact that L. amazonensis amastigotes limited the production of IL-12p40 from BM-DC in vitro. Altogether, our data indicate that the immune response of mice chronically infected with L. amazonensis can be enhanced towards a Th1 phenotype but that the presence of Th1 CD4(+) T cells does not promote healing. This suggests that the phenotype of the CD4(+) T cells may not always be indicative of protection to L. amazonensis infection. Furthermore, our data support growing evidence that antigen-presenting cell function, such as IL-12 production, may limit the immune response in L. amazonensis-infected mice

Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

<div><p>HIV-1-specific CD4⁺ and CD8⁺ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4⁺ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8⁺ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4⁺ and CD8⁺ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4⁺ T cells. Approximately 50% of AdC7-GRN-induced memory CD8⁺ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4⁺ and CD8⁺ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4⁺ and CD8⁺ T-cell responses and antibodies in macaques. Besides, adenoviral vector priming modulated the cytokine-expression profile of the protein-induced CD4⁺ T cells. Each regimen induced HIV-1-specific T-cell responses in systemic/local tissues in mice. This suggests that prime-boost regimens combining adjuvanted protein and low-seroprevalent chimpanzee adenoviral vectors represent an attractive vaccination strategy for clinical evaluation.</p></div

HIV-1-specific CD8+ T-cell responses in NHP.

<p>Rhesus macaques were immunized intramuscularly with F4/AS01 (‘P’) and/or AdC7-GRN (‘A’), according to the following regimens: weeks 0 and 4 (PP group), weeks 0 and 12 (AA group), weeks 0, 4, 16 and 28 (PPAA group) and weeks 0, 12, 24 and 28 (AAPP group). <b>A.</b> PBMCs were stimulated <i>in vitro</i> overnight with a pool of peptides covering the F4 antigen sequence at several time-points, and production of IL-2, IFN- and TNF- was measured by ICS. Frequencies of F4-specific CD8⁺ T cells were expressed as percentages of CD3⁺ CD8⁺ T cells expressing IFN-γ and/or TNF-α and/or IL2 over total CD3⁺ CD8⁺ T cells. Data are represented as group geometric mean frequencies. *: PPAA group: N = 8 at Weeks 0, 2 and 6, and N = 7 at all time-points thereafter. The dashed line indicates the assay cut-off value for CD8⁺ T cells (i.e., 0.14%). <b>B.</b> The cytokine co-expression profile of F4-specific CD8⁺ T cells at 2 weeks post last immunization is represented for each prime-boost regimen. Data are reported as median frequencies of responding CD8⁺ T cells expressing any combination of IL-2, IFN- and TNF-, with first and third quartiles measured. Pie charts show the group mean proportions of responding CD8⁺ T cells expressing (after <i>in vitro</i> stimulation) one, two or three cytokines (represented in light grey, dark grey and black, respectively) among IL-2, IFN- and TNF-. These group mean proportions were compared using Fisher’s exact test and a significance level of p<0.05. The number in the center of each pie represents the geometric mean of the total percentage of cytokine-expressing CD8⁺ T cells at 2 weeks post last immunization.</p

HIV-1 antigens targeted by CD4+/CD8+ T-cell responses in NHP (2 weeks post last immunization).

<p>Magnitudes of p24-, p17-, RT- and Nef-specific CD40L⁺ CD4⁺ T-cell responses (panel A) and CD8⁺ T-cell responses (panel B) expressing IL-2, IFN- and/or TNF- were assessed at 2 weeks post last immunization using ICS. PBMCs were stimulated <i>in vitro</i> overnight with a pool of peptides covering the p24, p17, RT or Nef sequences. Individual data and box plots, showing the medians, upper and lower interquartiles and minimum and maximum values, are represented for each group. Pie charts show the mean proportions of CD40L⁺ CD4⁺ or CD8⁺ T cells specific to Nef, p17, p24 and RT (represented in light grey, medium grey, dark grey and black, respectively). The number in the center of each pie represents the geometric mean of the total percentage of cytokine-expressing CD40L⁺ CD4⁺ or CD8⁺ T cells at 2 weeks post last immunization.</p