Impact of IgA isoforms on their ability to activate dendritic cells and to prime T cells.

Human IgA could be from different isotypes (IgA1/IgA2) and/or isoforms (monomeric, dimeric, or secretory). Monomeric IgA mainly IgA1 are considered as an anti-inflammatory isotype whereas dimeric/secretory IgA have clearly dual pro- and anti-inflammatory effects. Here, we show that IgA isotypes and isoforms display different binding abilities to FcαRI, Dectin-1, DC-SIGN, and CD71 on monocyte-derived dendritic cells (moDC). We describe that IgA regulate the expression of their own receptors and trigger modulation of moDC maturation. We also demonstrate that dimeric IgA2 and IgA1 induce different inflammatory responses leading to cytotoxic CD8+ T cells activation. moDC stimulation by dimeric IgA2 was followed by a strong pro-inflammatory effect. Our study highlights differences regarding IgA isotypes and isoforms in the context of DC conditioning. Further investigations are needed on the activation of adaptive immunity by IgA in the context of microbiota/IgA complexes during antibody-mediated immune selection.

Lipid-Based Particles: Versatile Delivery Systems for Mucosal Vaccination against Infection.

Vaccination is the process of administering immunogenic formulations in order to induce or harness antigen (Ag)-specific antibody and T cell responses in order to protect against infections. Important successes have been obtained in protecting individuals against many deleterious pathological situations after parenteral vaccination. However, one of the major limitations of the current vaccination strategies is the administration route that may not be optimal for the induction of immunity at the site of pathogen entry, i.e., mucosal surfaces. It is now well documented that immune responses along the genital, respiratory, or gastrointestinal tracts have to be elicited locally to ensure efficient trafficking of effector and memory B and T cells to mucosal tissues. Moreover, needle-free mucosal delivery of vaccines is advantageous in terms of safety, compliance, and ease of administration. However, the quest for mucosal vaccines is challenging due to (1) the fact that Ag sampling has to be performed across the epithelium through a relatively limited number of portals of entry; (2) the deleterious acidic and proteolytic environment of the mucosae that affect the stability, integrity, and retention time of the applied Ags; and (3) the tolerogenic environment of mucosae, which requires the addition of adjuvants to elicit efficient effector immune responses. Until now, only few mucosally applicable vaccine formulations have been developed and successfully tested. In animal models and clinical trials, the use of lipidic structures such as liposomes, virosomes, immune stimulating complexes, gas-filled microbubbles and emulsions has proven efficient for the mucosal delivery of associated Ags and the induction of local and systemic immune reponses. Such particles are suitable for mucosal delivery because they protect the associated payload from degradation and deliver concentrated amounts of Ags via specialized sampling cells (microfold cells) within the mucosal epithelium to underlying antigen-presenting cells. The review aims at summarizing recent development in the field of mucosal vaccination using lipid-based particles. The modularity ensured by tailoring the lipidic design and content of particles, and their known safety as already established in humans, make the continuing appraisal of these vaccine candidates a promising development in the field of targeted mucosal vaccination.

Oral Passive Immunization With Plasma-Derived Polyreactive Secretory-Like IgA/M Partially Protects Mice Against Experimental Salmonellosis.

Secretory immunoglobulins have a critical role in defense of the gastrointestinal tract and are known to act by preventing bacterial acquisition. A stringent murine model of bacterial infection with Salmonella enterica Typhimurium was used to examine protection mediated by oral passive immunization with human plasma-derived polyreactive IgA and IgM antibodies (Abs) reconstituted as secretory-like immunoglobulins (SCIgA/M). This reagent has been shown to trigger Salmonella agglutination and to limit the entry of bacterium into intestinal Peyer's patches via immune exclusion. We now demonstrate that upon administration into ligated intestinal loops, SCIgA/M properly anchors in the mucus and is protected from degradation to a better extent that IgA/M or IgG. Moreover, prophylactic oral administration of SCIgA/M before intragastric infection of mice with a virulent strain of S. enterica Typhimurium allows to protect infected animals, as reflected by reduced colonization of both mucosal and systemic compartments, and conserved integrity of intestinal tissues. In comparison with IgA/M or IgG administration, SCIgA/M provided the highest degree of protection. Moreover, such protective efficacy is also observed after therapeutic oral delivery of SCIgA/M. Either prophylactic or therapeutic treatment with passively delivered SCIgA/M ensured survival of up to 50% of infected mice, while untreated animals all died. Our findings unravel the potential of oral passive immunization with plasma-derived polyreactive SCIgA/M Abs to fight gastrointestinal infections.

Plasma-Derived Polyreactive Secretory-Like IgA and IgM Opsonizing Salmonella enterica Typhimurium Reduces Invasion and Gut Tissue Inflammation through Agglutination.

Due to the increasing emergence of antibiotic-resistant strains of enteropathogenic bacteria, development of alternative treatments to fight against gut infections is a major health issue. While vaccination requires that a proper combination of antigen, adjuvant, and delivery route is defined to elicit protective immunity at mucosae, oral delivery of directly active antibody preparations, referred to as passive immunization, sounds like a valuable alternative. Along the gut, the strategy suffers, however, from the difficulty to obtain sufficient amounts of antibodies with the appropriate specificity and molecular structure for mucosal delivery. Physiologically, at the antibody level, the protection of gastrointestinal mucosal surfaces against enteropathogens is principally mediated by secretory IgA and secretory IgM. We previously demonstrated that purified human plasma-derived IgA and IgM can be associated with secretory component to generate biologically active secretory-like IgA and IgM (SCIgA/M) that can protect epithelial cells from infection by Shigella flexneri in vitro. In this study, we aimed at evaluating the protective potential of these antibody preparations in vivo. We now establish that such polyreactive preparations bind efficiently to Salmonella enterica Typhimurium and trigger bacterial agglutination, as observed by laser scanning confocal microscopy. Upon delivery into a mouse ligated intestinal loop, SCIgA/M-mediated aggregates persist in the intestinal environment and limit the entry of bacteria into intestinal Peyer's patches via immune exclusion. Moreover, oral administration to mice of immune complexes composed of S. Typhimurium and SCIgA/M reduces mucosal infection, systemic dissemination, and local inflammation. Altogether, our data provide valuable clues for the future appraisal of passive oral administration of polyreactive plasma-derived SCIgA/M to combat infection by a variety of enteropathogens.

Therapeutic intranasal instillation of allergen-loaded microbubbles suppresses experimental allergic asthma in mice.

Despite proven efficiency, subcutaneous immunotherapy for aeroallergens is impaired by the duration of the protocol, the repeated injections and potential side-effects associated with the doses of allergen administered. Intranasal delivery of immunotherapeutic agents may overcome several of these drawbacks, provided that an efficient allergen delivery vehicle can be identified. This study evaluates whether intranasally delivered gas-filled microbubble (MB)-associated ovalbumin (OVA), used as a model allergen, can serve as a therapeutic treatment in a mouse model of established allergic asthma. Lung and systemic production of pro-tolerogenic markers, including Foxp3+ CD4 T cells, IL-10, and TGF-ß, as well as the Th1-type cytokine IFN-γ, was observed after intranasal immunization with OVA-MB. Post-treatment, aerosol-sensitized mice exhibited the same pattern of markers. Moreover, decrease of eosinophils and neutrophils in BALs, lower frequencies of Th2 cytokine- and IL-17-producing CD4 T cells in lungs and reduced specific IgE in BALs and sera after allergen challenge were observed. Concomitantly, lung resistance and mucus production diminished in OVA-MB-treated animals. Thus, therapeutic intranasal administration of OVA-MBs in established experimental allergic asthma allows modulating pathology-associated immune and physiological parameters usually triggered after exposure to the allergen.

SIgA-Shigella Immune Complexes Interact with Dectin-1 and SIGNR3 to Differentially Regulate Mouse Peyer's Patch and Mesenteric Lymph Node Dendritic Cell's Responsiveness.

In addition to contributing to immune exclusion at mucosal surfaces, secretory IgA (SIgA) made of polymeric IgA and secretory component is able to selectively reenter via microfold cells into Peyer's patches (PPs) present along the intestine and to associate with dendritic cells (DCs) of the CD11c+CD11b+MHCII+F4/80-CD8-phenotype in the subepithelial dome region and the draining mesenteric lymph nodes (MLNs). However, the nature of the receptor(s) for SIgA on murine PP and MLN DCs is unknown. We find that glycosylated secretory component moiety and polymeric IgA are both involved in the specific interaction with these cells. Using blocking antibodies and competition experiments, we identify Dectin-1 and specific intercellular adhesion molecule-3 grabbing non-integrin receptor 3 (SIGNR3) as receptors for SIgA. While SIgA-commensal immune complexes (ICs) contribute to local homeostasis upon interaction with mucosal DCs, the picture is less clear for pathogenic agents. We find that in comparison with incubation of Shigella flexneri alone, association of the enteropathogen with SIgA prompts freshly isolated DCs from PPs and MLNs to invert the production of pro- versus non-inflammatory cytokines/chemokines. The sum of the data suggests that in contrast to IgG-based ICs boosting immune reactivity of antigen-presenting cells, SIgA produced during an ongoing immune response can, in addition to its known function of immune exclusion, modulate mucosal DC conditioning via specific interaction with Dectin-1 and SIGNR3.

Vaccination against Salmonella Infection: the Mucosal Way.

Salmonella enterica subspecies enterica includes several serovars infecting both humans and other animals and leading to typhoid fever or gastroenteritis. The high prevalence of associated morbidity and mortality, together with an increased emergence of multidrug-resistant strains, is a current global health issue that has prompted the development of vaccination strategies that confer protection against most serovars. Currently available systemic vaccine approaches have major limitations, including a reduced effectiveness in young children and a lack of cross-protection among different strains. Having studied host-pathogen interactions, microbiologists and immunologists argue in favor of topical gastrointestinal administration for improvement in vaccine efficacy. Here, recent advances in this field are summarized, including mechanisms of bacterial uptake at the intestinal epithelium, the assessment of protective host immunity, and improved animal models that closely mimic infection in humans. The pros and cons of existing vaccines are presented, along with recent progress made with novel formulations. Finally, new candidate antigens and their relevance in the refined design of anti-Salmonella vaccines are discussed, along with antigen vectorization strategies such as nanoparticles or secretory immunoglobulins, with a focus on potentiating mucosal vaccine efficacy.

Gas-filled microbubbles: Novel mucosal antigen-delivery system for induction of anti-pathogen's immune responses in the gut.

Despite important success in protecting individuals against many pathogenic infections, parenteral vaccination is not optimal to induce immunity at the site of pathogen entry, i.e. mucosal surfaces. Moreover, designing adequate delivery systems and safe adjuvants to overcome the inherent tolerogenic environment of the mucosal tissue is challenging, in particular in the gastrointestinal tract prone to antigen degradation. We recently demonstrated that intranasal administration of a Salmonella-derived antigen associated with gas-filled microbubbles induced specific Ab and T cell responses in the gut and was associated with a reduction in local and systemic bacterial load after oral Salmonella infection. Building on these promising data, the adequate choice of antigen(s) to be administered and how to make it suitable for possible human application are discussed. We additionally present novel data dealing with oral administration of microbubbles and describe research strategies to direct them to mucosal sampling/inductive sites.

Intranasal Vaccination With Salmonella-Derived Serodominant Secreted Effector Protein B Associated With Gas-Filled Microbubbles Partially Protects Against Gut Infection in Mice.

Salmonella infection is an increasingly important public health problem owing to the emergence of multidrug resistance and the lack of broadly efficient vaccines. Novel strategies of vaccination are required to induce protective immune responses at mucosal surfaces and in the circulation, to limit bacteria entry and dissemination. To this aim, intranasal anti-Salmonella vaccination with an innovative formulation composed of gas-filled microbubbles and the pathogen-derived protective protein serodominant secreted effector protein B (SseB-MB) was evaluated in a mouse infection model. Intranasal application of SseB-MB induced gut and systemic immunoglobulin A, T-helper type 17 cell (Th17), and Th1 responses, all of which are associated with natural immunity against Salmonella In vaccinated mice, a significant reduction in bacterial load was observed in intestinal tissues and the spleen after an otherwise lethal oral infection. Therefore, MB serve as an efficient carrier for nasal delivery of a Salmonella antigen that results in protection upon activation of the common mucosal immune system.

Long-term persistence of immunity induced by OVA-coupled gas-filled microbubble vaccination partially protects mice against infection by OVA-expressing Listeria.

Vaccination aims at generating memory immune responses able to protect individuals against pathogenic challenges over long periods of time. Subunit vaccine formulations based on safe, but poorly immunogenic, antigenic entities must be combined with adjuvant molecules to make them efficient against infections. We have previously shown that gas-filled microbubbles (MB) are potent antigen-delivery systems. This study compares the ability of various ovalbumin-associated MB (OVA-MB) formulations to induce antigen-specific memory immune responses and evaluates long-term protection toward bacterial infections. When initially testing dendritic cells reactivity to MB constituents, palmitic acid exhibited the highest degree of activation. Subcutaneous immunization of naïve wild-type mice with the OVA-MB formulation comprising the highest palmitic acid content and devoid of PEG2000 was found to trigger the more pronounced Th1-type response, as reflected by robust IFN-γ and IL-2 production. Both T cell and antibody responses persisted for at least 6 months after immunization. At that time, systemic infection with OVA-expressing Listeria monocytgenes was performed. Partial protection of vaccinated mice was demonstrated by reduction of the bacterial load in both the spleen and liver. We conclude that antigen-bound MB exhibit promising properties as a vaccine candidate ensuring prolonged maintenance of protective immunity.

The effect of vaccines based on ovalbumin coupled to gas-filled microbubbles for reducing infection by ovalbumin-expressing Listeria monocytogenes.

Gas-filled microbubbles (MB) are a very promising alternative to the currently evaluated lipid- or polymer-based particulate Ag delivery systems. We recently demonstrated the ability of MB to deliver associated Ag to DC, to activate them and thereby induce both humoral and cellular immune responses. We now extended the characterization of MB as antigen-delivery system by appraising the efficiency of MB-associated ovalbumin (OVA-MB) at protecting mice against pathogen infection. Ultrasound-mediated imaging demonstrated that the administration of OVA via MB generates a depot at the injection site that lasts for several hours. We found that OVA-MB injected subcutaneously is far more effective at inducing specific Ab and T cell immunity than immunization with free OVA. Moreover, a covalent link between MB and OVA causes a stronger bias towards a Th1-type of immune response than adsorption of the Ag or its covalent link to liposomes of the same lipid composition. Finally, vaccination of mice with OVA-MB partially protects against a systemic infection with OVA-expressing Listeria monocytogenes. The vaccine induces specific effector CD8 T cell responses capable of decreasing more than 100 fold the bacterial load. MB thus represent a potent Ag delivery system for vaccination against intracellular infectious agents.

Gas-filled microbubble-mediated delivery of antigen and the induction of immune responses.

The use of well characterized recombinant or purified protein antigens (Ag) for vaccination is of interest for safety reasons and in the case where inactivated pathogens are not available (cancer, allergy). However it requires the addition of adjuvants such as Ag carrier or immune stimulators to potentiate their immunogenicity. In this study, we demonstrated that gas-filled microbubbles (MB) can serve as an efficient Ag delivery system to promote phagocytosis of the model Ag ovalbumin (OVA) without the need of ultrasound application. Once internalized by DC, OVA was processed and presented to both CD4 and CD8 T cells in vitro; such observations were coupled with the capacity of MB to activate DC. In vivo administration of MB-associated OVA in naïve wild-type Balb/c mice resulted in the induction of OVA-specific antibody and T cell responses. Detailed characterization of the generated immune response demonstrated the production of both IgG1 and IgG2a serum antibodies, as well as the secretion of IFN-γ and IL-10 by splenocytes. Interestingly, similar results were obtained with human DC in regards of Ag delivery and cell activation. Therefore, the data presented here settle the proof of principle for the further evaluation of MB-based immunomodulation studies.

The phagocytosis of gas-filled microbubbles by human and murine antigen-presenting cells.

This study was designed to evaluate the potential of gas-filled microbubbles (MB) to be internalized by antigen-presenting cells (APC). Fluorescently labeled MB were prepared, thus permitting to track binding to, and internalization in, APC. Both human and mouse cells, including monocytes and dendritic cells (DC), prove capable to phagocyte MB in vitro. Observation by confocal laser scanning microscopy showed that interaction between MB and target cells resulted in a rapid internalization in cellular compartments and to a lesser extent in the cytoplasm. Capture of MB by APC resulted in phagolysosomal targeting as verified by double staining with anti-lysosome-associated membrane protein-1 monoclonal antibody and decrease of internalization by phagocytosis inhibitors. Fluorescent MB injected subcutaneously (s.c.) in mice were found to be associated with CD11c(+)DC in lymph nodes draining the injection sites 24 h after administration. Altogether, our study demonstrates that MB can successfully target APC both in vitro and in vivo, and thus may serve as a potent Ag delivery system without requirement for ultrasound-based sonoporation. This adds to the potential of applications of MB already extensively used for diagnostic imaging in humans.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Vaccination with recombinant NY-ESO-1 protein elicits immunodominant HLA-DR52b-restricted CD4+ T cell responses with a conserved T cell receptor repertoire.

PURPOSE: ESO is a tumor-specific antigen with wide expression in human tumors of different histologic types and remarkable spontaneous immunogenicity. We have previously shown that specific T(H)1 and antibody responses can be elicited in patients with no detectable preexisting immune responses by vaccination with rESO administered with Montanide ISA-51 and CpG ODN 7909. The purpose of the present study was to characterize vaccine-induced ESO-specific CD4(+) T cell responses. EXPERIMENTAL DESIGN: We generated CD4(+) T cell clones from patient C2, who had the highest CD4(+) T cell response to the vaccine, and analyzed their fine specificity and HLA class II restriction to determine the recognized epitope. We then assessed the response to the identified epitope in all vaccinated patients expressing the corresponding HLA class II allele. RESULTS: We found that ESO-specific CD4(+) T cell clones from patient C2 recognize peptide ESO(119-143) (core region 123-137) presented by HLA-DR52b (HLA-DRB3*0202), a MHC class II allele expressed by about half of Caucasians. Importantly, following vaccination, all patients expressing DR52b developed significant responses to the identified epitope, accounting for, on average, half of the total CD4(+) T cell responses to the 119-143 immunodominant region. In addition, analysis of ESO-specific DR52b-restricted CD4(+) T cells at the clonal level revealed significant conservation of T cell receptor usage among different individuals. CONCLUSIONS: The identification of a DR52b-restricted epitope from ESO that is immunodominant in the context of vaccine-elicited immune responses is instrumental for the immunologic monitoring of vaccination trials targeting this important tumor antigen.

Human memory FOXP3+ Tregs secrete IL-17 ex vivo and constitutively express the T(H)17 lineage-specific transcription factor RORgamma t.

Recent studies have suggested a close relationship between CD4(+)FOXP3(+) regulatory T cells (Tregs) and proinflammatory IL-17-producing T helper cells (T(H)17) expressing the lineage-specific transcription factor RORgamma t. We report here the unexpected finding that human memory Tregs secrete IL-17 ex vivo and constitutively express RORgamma t. IL-17-secreting Tregs share some phenotypic and functional features with conventional T(H)17 cells, expressing high levels of CCR4 and CCR6 and low levels of CXCR3. However, unlike conventional T(H)17 cells, they express low levels of CD161 and mostly fail to cosecrete IL-22 and TNF-alpha ex vivo. Ex vivo secretion of IL-17 and constitutive expression of RORgamma t by human memory Tregs suggest that, in addition to their well-known suppressive functions, these cells likely play additional, as yet undescribed, proinflammatory functions.

IL-1beta and IL-2 convert human Treg into T(H)17 cells.

Natural CD4(+)CD25(+) regulatory T cells (Treg) and interleukin 17 (IL-17)-producing T helper cells (T(H)17) carry out opposite functions, the former maintaining self-tolerance and the latter being involved in inflammation and autoimmunity. We report here that stimulation of human Natural Treg under T(H)17 polarizing conditions in the presence of IL-2 converts them into T(H)17 cells. Conversion of Tregs into T(H)17 cells occurs both from natural naïve Tregs (NnTregs) and, to a higher extent, from memory Tregs (MTregs). Among antigen presenting cells, fresh monocytes activated by microbial stimuli were the most efficient inducers of T(H)17 cells from Tregs. Conversion of Treg into T(H)17 cells was induced by IL-1beta and involved down-regulation of the Treg lineage transcription factor FOXP3 and suppressive functions. Our results indicate that, under inflammatory conditions, in the presence of IL-2, Treg can be converted into pro-inflammatory T(H)17 cells and establish a functional link between inflammation and autoimmunity.

Interleukin 2-mediated conversion of ovarian cancer-associated CD4+ regulatory T cells into proinflammatory interleukin 17-producing helper T cells.

Epithelial ovarian cancer (EOC) is a highly inflammatory malignancy, characterized by the presence, at the tumor site, of regulatory T cells (Treg) that suppress antitumor immunity. Recently, a new lineage of CD4+ T cells producing the proinflammatory cytokine interleukin (IL)-17 [T helper (TH) 17] has been identified as a major player in some autoimmune diseases. The role of TH17 cells in cancer, however, and their relationship with coexisting Treg populations, whose differentiation is partially controlled by the same mediators (ie, transforming growth factor-beta), are yet unclear. Here, we show that EOC-associated/infiltrating lymphocytes derived by culturing tumor samples in the presence of IL-2 contain significant frequencies of TH17 cells, coproducing interferon-gamma (IFN)-gamma and tumor necrosis factor (TNF)-alpha, which represent, in some cases, up to 40% of total CD4+ T cells. TH17 cells were also detected ex vivo, but at lower proportions than in cultured tumor-infiltrating lymphocytes/tumor-associated lymphocytes, and were confined to the CD4+CD25- fraction. Remarkably, analysis of EOC-associated conventional CD4CD25 T cell and Treg populations isolated ex vivo from tumor samples by cell sorting and cultured with tumor-associated CD3- cells in the presence of IL-2 revealed that EOC Treg stimulated under these conditions were rapidly converted into TH17 cells, down-regulated FOXP3 expression, and lost their suppressive capacity. Thus, although the impact of TH17 cells on the evolution of EOC remains to be established, our data suggest that local IL-2 treatment in ovarian cancer may result in the conversion of tumor-associated Treg into TH17 cells, relieve Treg-mediated suppression, and contribute to enhance antitumor immunity.

Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

HLA class I - associated immunodominance affects CTL responsiveness to an ESO recombinant protein tumor antigen vaccine.

PURPOSE: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. EXPERIMENTAL DESIGN: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. RESULTS: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. CONCLUSIONS: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.