Linda Klavinskis

Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1

In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α4β7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.

Cutting Edge: CD8+ T Cell Priming in the Absence of NK Cells Leads to Enhanced Memory Responses

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8+ T cell fate by killing recently activated CD8+ T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8+ T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8+ T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8+ T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.

Bacillus subtilis spores: A novel microparticle adjuvant which can instruct a balanced Th1 and Th2 immune response to specific antigen

There is a current need for safe, cheap, and effective vaccine adjuvants, to combine with sub‐unit antigens to enhance their immunogenicity. In this study we have used probiotic Bacillus subtilis spores, known to be safe and fully tolerated by ingestion in man, and explored their ability to influence the magnitude and diversity of immune responses induced against two model antigens, tetanus toxoid fragment C (TT) and ovalbumin (OVA) in mice. The results show that B. subtilis spores not only increased antibody and T cell responses to a co‐administered soluble antigen, but also broadened them, to include both antigen‐specific CD4+ and CD8+ T cell responses as well as complement and non‐complement fixing antibody isotypes. Furthermore, following intranasal immunization, spores augmented specific IgA to co‐administered antigen both in the local respiratory and distal vaginal mucosa, as well as increased antigen‐specific IgG antibody in draining LN and blood. Collectively, these data demonstrate that naturally occurring, non‐pathogenic, non‐commensal spores of B. subtilis both instruct and augment polyvalent immune responses and highlight their clinical potential in future vaccines to generate broad‐based immunity.

Plasmacytoid Dendritic Cells Do Not Migrate in Intestinal or Hepatic Lymph

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRαβ or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.

Mucosal immunization with DNA vaccines

DNA vaccines represent a new approach to the control of infectious disease. Both cellular and humoral immune responses are induced without the attendant concerns associated with live, attenuated vaccines. The vast majority of DNA vaccines are delivered by parental routes, which rarely elicit immune responses at the mucosal epithelia, the primary sites of pathogen transmission. In view of the importance of mucosal and regional lymph node immunity in the control of pathogens transmitted across the mucosal epithelia, a number of groups, including our own, have developed immunization strategies that target plasmid DNA to mucosal inductive sites associated with the lymphoid tissues of the respiratory, gastrointestinal, and genital tracts. Here, we describe the procedures for the formulation and delivery of plasmid DNA to mucosal inductive tissues and address the theoretical basis to selection of particular mucosal locations for the induction of effective immune responses.

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs.

The Relative Efficiency of Acquisition of MHC:Peptide Complexes and Cross-Presentation Depends on Dendritic Cell Type

Intercellular exchange of MHC molecules has been reported between many cells, including professional and nonprofessional APCs. This phenomenon may contribute to T cell immunity to pathogens. In this study, we addressed whether the transfer of MHC class I:peptide complexes between cells plays a role in T cell responses and compare this to conventional cross-presentation. We observed that dsRNA-matured bone marrow-derived dendritic cells (BMDCs) acquired peptide:MHC complexes from other BMDCs either pulsed with OVA257–264 peptide, soluble OVA, or infected with a recombinant adenovirus expressing OVA. In addition, BMDCs were capable of acquiring MHC:peptide complexes from epithelial cells. Spleen-derived CD8α+ and CD8α− dendritic cells (DCs) also acquired MHC:peptide complexes from BMDCs pulsed with OVA257–264 peptide. However, the efficiency of acquisition by these ex vivo derived DCs is much lower than acquisition by BMDC. In all cases, the acquired MHC:peptide complexes were functional in that they induced Ag-specific CD8+ T cell proliferation. The efficiency of MHC transfer was compared with cross-presentation for splenic CD8α+ and CD8α− as well as BMDCs. CD8α+ DCs were more efficient at inducing T cell proliferation when they acquired Ag via cross-presentation, the opposite was observed for BMDCs and splenic CD8α− DCs. We conclude from these observations that the relative efficiency of MHC transfer vs cross-presentation differs markedly between different DC subsets.

A distinct subset of intestinal dendritic cells responds selectively to oral TLR7/8 stimulation

The intestinal innate immune system continually interacts with commensal bacteria, thus oral vaccines should induce extra/alternative activation of DC, potentially through TLR. To examine this we collected intestinal lymph DC (iL‐DC) under steady‐state conditions and after feeding resiquimod (R‐848), a synthetic TLR7/8 ligand, which we showed induces complete emptying of gut DC into lymph. iL‐DC are heterogeneous with subset‐specific functions. In this study we determined the kinetics of iL‐DC subset release, activation and cytokine secretion induced by R‐848. We show that L‐DC comprise three distinct subsets (CD172ahigh, CD172aint and CD172alow) present with similar frequencies in intestinal but not hepatic lymph. No iL‐DC express TLR7 mRNA, and only CD172a+ iL‐DC express TLR8. However, after oral R‐848 administration, output of all three subsets increases dramatically. CD172ahigh DC release precedes that of CD172alow DC, and the increased frequency of CD25high iL‐DC is restricted to the two CD172a+ subsets. After feeding R‐848 only CD172ahigh iL‐DC secrete IL‐6 and IL‐12p40. However, CD172aint and CD172ahigh DC secrete similar but markedly lower amounts when stimulated in vitro. These results highlight the importance of in vivo approaches to assess adjuvant effects on DC and give novel insights into the subset‐specific effects of an oral TLR ligand on intestinal DC.

Hyporesponsiveness of Intestinal Dendritic Cells to TLR Stimulation Is Limited to TLR4

Dendritic cells (DCs) are crucial to intestinal immune regulation because of their roles in inducing protective immunity against pathogens while maintaining tolerance to commensal bacteria. Nonetheless, relatively little is known about intestinal DC responsiveness to innate immune stimuli via TLRs. We have previously shown that DCs migrating from the rat intestine in lymph (iLDCs) are hyporesponsive to LPS stimulation, thus possibly preventing harmful immune responses being induced to commensal flora. In this study, to understand how iLDC function is regulated by innate immune stimuli, we have characterized the expression and function of TLRs in iLDCs isolated from the thoracic duct lymph of mesenteric lymphadenectomized rats and compared these with DCs grown from bone marrow in the presence of Flt3 ligand. We show that iLDCs express mRNAs for all TLRs, but express significantly less TLR4 mRNA than bone marrow-derived DCs. Functionally, iLDCs could be activated by TLR agonists representing intestinal pathogen-associated molecular patterns, with the important exception of the TLR4 agonist LPS. Furthermore, we show that DCs in the intestinal wall interact directly with noninvasive bacteria (Bacillus subtilis spores), leading to an increase in the output of activated iLDCs into lymph, and that DCs containing spores are activated selectively. These data highlight a functional difference between TLR4 and other TLRs. As iLDCs can respond to TLR stimulation in vitro, there must be other mechanisms that prevent their activation by commensal bacteria under steady-state conditions.

Acquisition of MHC:Peptide Complexes by Dendritic Cells Contributes to the Generation of Antiviral CD8+ T Cell Immunity In Vivo

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8+ T cells in host defense. However, although it has been shown that memory CD8+ T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8+ T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8+ T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α− DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8+ T cell effectors with cytolytic function. As CD8α− DCs are poor cross-presenters, this may represent the main mechanism by which CD8α− DCs present exogenously encountered Ag to CD8+ T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.