Keri L. Csencsits

M cell-targeted DNA vaccination

DNA immunization, although attractive, is poor for inducing mucosal immunity, thus limiting its protective value against most infectious agents. To surmount this shortcoming, we devised a method for mucosal transgene vaccination by using an M cell ligand to direct the DNA vaccine to mucosal inductive tissues and the respiratory epithelium. This ligand, reovirus protein σ1, when conjugated to polylysine (PL), can bind the apical surface of M cells from nasal-associated lymphoid tissues. Intranasal immunizations with protein σ1-PL-DNA complexes produced antigen-specific serum IgG and prolonged mucosal IgA, as well as enhanced cell-mediated immunity, made evident by elevated pulmonary cytotoxic T lymphocyte responses. Therefore, targeted transgene vaccination represents an approach for enabling DNA vaccination of the mucosa.

CD8+ Th17 Mediate Costimulation Blockade-Resistant Allograft Rejection in T-bet-Deficient Mice

While studying Th responses induced by cardiac transplantation, we observed that mice deficient in the Th1 transcription factor T-bet (T-bet−/−) mount both Th1 and Th17 responses, whereas wild-type recipients mount only Th1 responses. Cells producing both IFN-γ and IL-17 were readily detectable within the rejecting graft of T-bet−/− recipients, but were absent from the spleen, indicating that the in vivo microenvironment influences Th function. In addition, disrupting CD40-CD40L costimulatory interactions was highly effective at prolonging allograft survival in WT mice, but ineffective in T-bet−/− recipients. In this study, we report that CD8+ Th17 mediate costimulation blockade-resistant rejection in T-bet−/− allograft recipients. Depleting CD8+ cells or neutralizing IL-17 or the Th17-inducing cytokine IL-6 ablated the Th17 response and reversed costimulation blockade-resistant graft rejection. Neutralizing IL-4 in IFN-γ−/− allograft recipients did not induce Th17, suggesting that T-bet, rather than IL-4 and IFN-γ (known inhibitors of Th17), plays a critical role in negatively regulating Th17 in the transplant setting.

Contrasting alloreactive CD4+ and CD8+ T cells: There's more to it than MHC restriction

Surface expression of CD4 or CD8 is commonly used to identify T‐cell subsets that recognize antigen presented by class II MHC or class I MHC, respectively. This holds true for T cells that respond to allogeneic MHC molecules that are directly recognized as foreign, as well as peptides from allogeneic MHC molecules that are indirectly presented by self MHC molecules. CD4 or CD8 expression was initially believed to define cytokine secreting helper T cells or cytotoxic cells, respectively. However, this association of phenotype and function is not absolute, in that CD4+ cells may possess lytic activity and CD8+ cells secrete cytokines, notably IFNγ. Recently, additional fundamental differences in the immunobiology of these T‐cell subsets have been identified. These include differences in costimulatory requirements, cytokine responsiveness, cytokine production, cell survival, and the maintenance of memory. This review will survey these differences, emphasizing alloreactive T‐cell responses as well as relevant observations that have been made in other systems.

Transforming growth factor beta-induced connective tissue growth factor and chronic allograft rejection.

Late loss of allograft function is primarily attributed to chronic rejection (CR). There are no effective treatments for CR and the underlying cause of the disease is unknown. This study compared events that occurred within cardiac allografts placed in mice that received either anti‐CD4 therapy and develop CR or anti‐CD40L therapy and do not develop CR. Both TGFβ and connective tissue growth factor (CTGF), which is induced by TGFβ, were expressed in grafts with CR but were not expressed in grafts without CR. TGFβ transfection of allografts in anti‐CD40L‐treated recipients resulted in CTGF expression and CR. However, TGFβ transfection of syngeneic grafts did not result in CTGF expression or CR. These data indicate that TGFβ alone is insufficient to induce CR and that CTGF is required. Further, antigenic stimulation is required for TGFβ induction of CTGF. Thus, CTGF may serve as a therapeutic target for CR.

Cutting Edge: Dichotomy of Homing Receptor Dependence by Mucosal Effector B Cells: αE Versus L-Selectin

The common mucosal immune system may be compartmentalized because lymphocyte homing to the upper respiratory tract appears to be mediated by L-selectin interactions rather than α4β7 interactions, as is the case for gut-associated lymphoreticular tissue. To assess the role of L-selectin in effector B cell immunity, L-selectin-deficient mice were intranasally immunized with cholera toxin (CT), and mucosal immune responses were compared with C57BL/6 mice. The absence of L-selectin correlated with a reduction in CT-specific secretory-IgA responses in nasal passages and reproductive tract, but not intestinal lamina propria. Cell sorting experiments showed that an L-selectin-dependent subset was responsible for CT-specific responses in nasal passages and reproductive tract, whereas an αEβ7+ B cell subset was responsible for L-selectin-independent intestinal immunity. This study provides evidence for compartmentalization of the common mucosal immune system into “intestinal” vs “nonintestinal” effector sites.

The Classical Complement Pathway in Transplantation: Unanticipated Protective Effects of C1q and Role in Inductive Antibody Therapy

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C‐mediated cytolysis may be necessary for the tolerance‐inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q‐deficient (C1q‐/‐) recipients rejected allografts at a faster rate than wild‐type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T‐cell responses in C1q‐/‐ recipients. However, the humoral response to donor alloantigens was accelerated in C1q‐/‐ mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q‐/‐ recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q‐/‐ recipients were treated with anti‐CD4 or anti‐CD40L mAb. The protective effects of anti‐CD4 mAb were reduced in C1q‐/‐ recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti‐CD40L mAb were less compromised in C1q‐/‐ recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.

Transforming Growth Factor-Beta1 Gene Transfer is Associated with the Development of Regulatory Cells

Adenovirus‐mediated transfection of mouse cardiac allografts with active human transforming growth factor‐beta 1 (TGF‐β1) prolongs transplant survival provided that recipients are initially depleted of CD8+ T cells. To test if graft survival was prolonged by persistent TGF‐β1 transgene expression, long‐term transfected allografts were re‐transplanted into naïve mice that were transiently depleted of CD8+ T cells. Re‐transplanted allografts were acutely rejected, indicating that TGF‐β1 transgene expression did not suppress effector cell function. We next asked whether TGF‐β1 gene transfer was associated with the development of regulatory cells. When splenocytes obtained from mice bearing long‐term TGF‐β1‐transfected allografts were adoptively transferred into recipients of non‐transfected cardiac allografts, prolonged allograft survival was observed, and increased levels of the regulatory T cell transcription factor Foxp3 were present. To further test for regulation, differentiated effector cells were obtained from mice that had rejected cardiac allografts and were adoptively transferred into mice bearing long‐term TGF‐β1 transfected cardiac allografts. The effector cells failed to mediate rejection in mice bearing TGF‐β1‐transfected allografts and we observed a significant increase in intra‐graft Foxp3 expression. These findings indicate that TGF‐β1 gene transfer allows for the development of regulatory cells that control graft‐reactive T cell responses once therapeutic levels of the transgene product are no longer produced.

Absence of L-Selectin Delays Mucosal B Cell Responses in Nonintestinal Effector Tissues

Previous studies suggest that lymphocyte trafficking to head and neck lymph nodes, also referred to as cranial-, oral-, nasal-associated lymphoid tissue (CONALT), is L-selectin (L-Sel) dependent, despite coexpression of α4β7, resulting in their marked reduction in L-Sel-deficient (L-Sel−/−) mice. Consequently, early phase (16 days) Ab responses to cholera toxin (CT) are diminished. The following studies reveal that lack of mucosal effector responses is not caused by loss of inductive immune responses in the L-Sel−/− CONALT. Indeed, there was an increased accumulation of total IgA, but not Ag-specific IgA Ab-forming cells (AFC) in L-Sel−/− CONALT. This increased accumulation was not evident in L-Sel+/+ CONALT. Identification of lymphocyte-homing receptors on L-Sel−/− and L-Sel+/+ CONALT lymphocytes revealed no significant differences in expression of α4β7, which might contribute to lymphocyte homing in the absence of L-Sel. Studies of CONALT responses during the late phase (6 wk post-intranasal immunization) revealed the number of lymphocytes recovered from L-Sel−/− CONALT was less than L-Sel+/+ CONALT; however, L-Sel−/− CT-specific and total AFC did not vary from 16-day responses, suggesting a defect in CT-specific B cell export. No significant differences in α4β7 expression between L-Sel−/− and L-Sel+/+ CONALT were noted. Yet, these increases in CONALT AFC correlated with restoration of immunity in L-Sel−/− nasal passages and reproductive tracts.

Gene transfer facilitated by a cellular targeting molecule, reovirus protein σ1

To facilitate eventual genetic vaccination of mucosal tissues, a receptor-mediated gene transfer system was devised using the reovirus adhesin, protein σ1. Highly efficient uptake and internalization of protein σ1 polylysine (PL) DNA complexes could be demonstrated by fluorescent microscopy. Successful cellular transfection of rodent and human cell lines was obtained with the recombinant protein σ1 as a PL–DNA complex, and could be shown to be receptor-specific. Transfection efficiency was dependent upon the ratio of DNA complexed to protein σ1-PL and chloroquine treatment improved transfection efficiency dramatically. To test its ability to bind a mucosal inductive tissue, recombinant protein σ1 was specifically bound to the nasal-associated lymphoid tissues (NALT). Thus, recombinant protein σ1-PL–DNA conjugates can efficiently bind and transfect cells that express the receptor for protein σ1.

Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4+ T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8+ T cells and transplanted with class II transactivator (CIITA)‐deficient cardiac allografts, which cannot directly present class II alloantigens to CD4+ T cells. In this manner, the rejection response by CD4+ cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8+ cells, both BALB/c and C57BL/6 mice rejected CIITA–/– allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA–/– allografts were depleted of CD8+ T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8‐depleted C57BL/6 recipients of CIITA–/– allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4+ T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2‐dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.