No abstract

Survival of various immune cell populations has been proposed to preferentially rely on a particular anti-apoptotic BCL-2 family member, for example, naive T cells require BCL-2, while regulatory T cells require MCL-1. Here we examined the survival requirements of multiple immune cell subsets in vitro and in vivo, using both genetic and pharmacological approaches. Our findings support a model in which survival is determined by quantitative participation of multiple anti-apoptotic proteins rather than by a single anti-apoptotic protein. This model provides both an insight into how the sum of relative levels of anti-apoptotic proteins BCL-2, MCL-1 and A1 influence survival of T cells, B cells and dendritic cells, and a framework for ascertaining how these different immune cells can be optimally targeted in treatment of immunopathology, transplantation rejection or hematological cancers.

SUMMARY CD40L on CD4+ T cells has been shown to license dendritic cells (DC) via CD40 to prime CTL responses. Surprisingly, we found that the converse (CD40L on DC) was also important. Anti-CD40L treatment decreases endogenous CTL responses to both OVA and influenza infection even in the absence of CD4+ T cells. DC express CD40L upon stimulation with agonists to TLR 3 and 9. Moreover, influenza infection, which stimulates CTL without help upregulates CD40L on DC, but herpes simplex infection, which elicits CTL through help, does not. CD40L−/− DC are suboptimal both in vivo in bone marrow chimera experiments and in vitro in mixed lymphocyte reactions. In contrast, CD40L−/− CD8+ T cells kill as effectively as wildtype. We conclude that CD40L upregulation on DC promotes optimal priming of CD8+ T cells without CD4+ T cells, providing a mechanism by which pathogens may elicit helper-independent CTL immunity.

Although multiple dendritic cell (DC) subsets have the potential to induce Th17 differentiation in vitro, the key DC that is critical in Th17 induction and Th17-mediated disease remains moot. In this study, we revealed that CCR2+ monocyte-derived DCs (moDCs), but not conventional DCs, were critical for in vivo Th17 induction and autoimmune inflammation. Functional comparison in vitro indicated that moDCs are the most potent type of Th17-inducing DCs compared with conventional DCs and plasmacytoid DCs. Furthermore, we demonstrated that the importance of GM-CSF in Th17 induction and Th17-mediated disease is its endowment of moDCs to induce Th17 differentiation in vivo, although it has little effect on moDC numbers. Our findings identify the in vivo cellular targets that can be selectively manipulated to ameliorate Th17-mediated inflammatory diseases, as well as the mechanism of GM-CSF antagonism in such diseases.

Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80-like receptor) or CIRE (C-type lectin receptor), two molecules expressed on the surface of immature CD8 -DC in the mouse, increases Ab production 100-1000-fold over a non-targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8 + DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRc -/-mice, and with the use of F(ab') 2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8 + and CD8 -DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any "danger" or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization inf luenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA-and proteinimmunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100-and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA-or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon ␥ production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.Injection of mammalian expression plasmid DNAs directly into muscle (1) and skin (2) or facilitated with biolistic systems (3) results in the uptake of DNA and expression of the encoded proteins. Expression levels of the encoded protein are often below detection and have been estimated by reporter enzymes such as luciferase (lux) to be in the nanogram range (1). However, even this relatively low dose of protein is sufficient to produce long-lasting immune responses (4) to encoded antigens that are capable of protecting animals from a wide range of pathogens (for review see ref. 5).The mechanisms underlying the induction of immune responses after DNA immunization are unclear. I.m. injection results in low-level transfection of myocytes (1) whereas intradermal (i.d.) injection may directly transfect antigenpresenting cells (APCs) (2, 6). Because myocytes express major histocompatibility complex class I at low levels and do not constitutively express class II or costimulatory molecules such as B7 (7), they appear unlikely candidates for the induction of antibody (Ab) or cytotoxic T cell (CTL) responses after i.m. DNA injection. The immunological consequences resulting from priming with different cell types and perhaps location of immune induction have not been fully resolved for DNA immunization, because the majority of the research to date has concentrated on vaccine efficacy. We sought to examine the effect of immunization with DNA as compared with soluble protein and to ascertain the contribution of route and dose on differences observed. In...

Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines. Immunization with DNA vaccines encoding antigen (Ag) has been used to induce both cellular and humoral immune responses and holds enormous potential for developing vaccines to a variety of pathogens. Recent reports of the first human clinical trials have shown that DNA vaccines were well tolerated, and the responses [especially cytotoxic T lymphocytes (CTLs)] were generally encouraging (1-3). However, there are still some questions concerning the potency of DNA vaccines, particularly with regard to Ab responses.There is a low level of protein expression after DNA immunization (e.g., nanograms produced). We sought to enhance the effectiveness of this dose by delivering what small amount of Ag is made to the cells relevant to immune induction [i.e., Agpresenting cells (APCs)]. We showed that increased Ab and T cell responses to a model Ag [human IgG (hIgG)] could be achieved by fusion of the Ag to CTLA4, which directs it to APCs through binding to the surface receptors B7-1 and B7-2 (4). Targeting APCs through the CD11c receptor has also been shown recently to enhance Ab responses (5). In this study, we sought to assess the efficacy of our targeting strategy in a viral challenge system. A plasmid expressing the influenza hemagglutinin (HA) molecule fused with CTLA4 was constructed and examined for its ability to enhance the immune response after DNA immunization and to reduce the amount of virus present in the lungs of mice after a nonlethal influenza challenge. Materials and MethodsPlasmids. The backbone of all the plasmids used for immunization was the same, namely pCI (Promega), which contains a human cytomegalovirus promoter plus intron. Inf luenza A͞Puerto Rico͞8͞34 H1 (PR8) HA was fused to hIg or CTLA4-hIg [the nont...

Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.apoptosis | dendritic cells | BH3-mimetic