In vitro expansion of alloantigen-specific regulatory T cells and their use in prevention of allograft rejection.

Regulatory T lymphocytes expressing CD4, high levels of CD25, and the transcription factor Foxp3 play a crucial role in the control of immune responses to self and nonself antigens. In contrast to immunosuppressive drugs currently used to treat immunopathology, these cells act in a very specific manner. Consequently, their clinical potential in the treatment of autoimmune disorders, inflammatory diseases, graft-versus-host disease, and allograft rejection is currently extensively studied in experimental animal models as well as in clinical trials. We have previously shown that appropriately in vitro stimulated CD4(+)CD25(high) regulatory T cells can be used to prevent rejection of bone marrow, skin, and heart allografts in the Mouse. We here describe the protocols used in our laboratory to isolate mouse regulatory T cells, to stimulate them in vitro in order to enrich in cells specific for donor-antigens, and to transplant bone marrow under cover of regulatory T cells. Thus, generated hematopoietic chimeras may subsequently be transplanted with solid tissues and organs from the same donor.

Immobilized cytokines as biomaterials for manufacturing immune cell based vaccines.

Manufacturing of bioactive cell culture substrates represents a major challenge for the development of cell therapy for tissue repair and immune treatment of cancers, infectious diseases, or immunodeficiencies. In this context, we evaluated the capacity of several differentiation factors, including Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF), to drive differentiation of primary cell cultures, once immobilized on surfaces. We show that covalently immobilized signal factors fully retain their biological properties and efficiently promote differentiation of mouse and/or human precursor cells leading to the production of dendritic cells and macrophages. For GM-CSF, we also show that the efficiency of receptor signaling is comparable using either soluble or tethered molecules. Such artificial bioactive interfaces are suitable for the development and automated production of cell-based vaccines and therapies.

Eradication of large tumors in mice by a tritherapy targeting the innate, adaptive, and regulatory components of the immune system.

Targeting the human papillomavirus (HPV) E7 antigen to dendritic cells with the adenylate cyclase (CyaA) of Bordetella pertussis as a vaccine vector led to potent therapeutic immune responses against TC-1 tumors in a murine model of cervical carcinoma induced by HPV. However, as the time between tumor graft and vaccination increased, the antitumor efficacy of the CyaA-E7 vaccine gradually decreased. The vaccine had no effect if the tumor diameter was >8 mm. Analyses of regulatory cells recruited during TC-1 tumor growth revealed a high number of splenic MDSCs and a large percentage of regulatory T cells, particularly in the tumor. Administration of a tritherapy including CpG complexed with a cationic lipid, low-dose cyclophosphamide, and the CyaA-E7 vaccine completely overcame tumor-associated immunosuppression and eradicated large, established tumors in almost all treated animals. This strong antitumor response was followed by a large expansion of regulatory T cells in tumor, spleen, and tumor-draining lymph nodes and of splenic neutrophils. These findings indicate that immunotherapeutic strategies that simultaneously target innate, adaptive, and regulatory components of the immune system are effective in the eradication of large tumors.

Inhibition of phagosome maturation by mycobacteria does not interfere with presentation of mycobacterial antigens by MHC molecules.

Pathogenic mycobacteria escape host innate immune responses by surviving within phagosomes of host macrophages and blocking their delivery to lysosomes. Avoiding lysosomal delivery may also be involved in the capacity of living mycobacteria to modulate MHC class I- or II-dependent T cell responses, which may contribute to their pathogenicity in vivo. In this study, we show that the presentation of mycobacterial Ags is independent of the site of intracellular residence inside professional APCs. Infection of mouse macrophages or dendritic cells in vitro with mycobacterial mutants that are unable to escape lysosomal transfer resulted in an identical efficiency of Ag presentation compared with wild-type mycobacteria. Moreover, in vivo, such mutants induced CD4(+) Th1 or CD8(+) CTL responses in mice against various mycobacterial Ags that were comparable to those induced by their wild-type counterparts. These results suggest that the limiting factor for the generation of an adaptive immune response against mycobacteria is not the degree of lysosomal delivery. These findings are important in the rational design of improved vaccines to combat mycobacterial diseases.

High frequency of CD4+ T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection.

TB10.4 is a newly identified antigen of Mycobacterium tuberculosis recognized by human and murine T cells upon mycobacterial infection. Here, we show that immunization with Mycobacterium bovis BCG induces a strong, genetically controlled, Th1 immune response against TB10.4 in mice. BALB/c and C57BL/6 strains behave as high and low responders to TB10.4 protein, respectively. The TB10.4:74-88 peptide was identified as an immunodominant CD4+ T-cell epitope for H-2d mice. Since recent results, as well as the present study, have raised interest in TB10.4 as a subunit vaccine, we analyzed immune responses induced by this antigen delivered by a new vector, the adenylate cyclase (CyaA) of Bordetella pertussis. CyaA is able to target dendritic cells and to deliver CD4+ or CD8+ T-cell epitopes to the major histocompatibility complex class II/I molecule presentation pathways, triggering specific Th1 or cytotoxic T-lymphocyte (CTL) responses. Several CyaA harboring either the entire TB10.4 protein or various subfragments containing the TB10.4:20-28 CTL epitope were shown to induce TB10.4-specific Th1 CD4+ and CD8+ T-cell responses. However, none of the recombinant CyaA, injected in the absence of adjuvant, was able to induce protection against M. tuberculosis infection. In contrast, TB10.4 protein administered with a cocktail of strong adjuvants that triggered a strong Th1 CD4+ T-cell response induced significant protection against M. tuberculosis challenge. These results confirm the potential value of the TB10.4 protein as a candidate vaccine and show that the presence of high frequencies of CD4+ T cells specific to this strong immunogen correlates with protection against M. tuberculosis infection.

An increase in antimycobacterial Th1-cell responses by prime-boost protocols of immunization does not enhance protection against tuberculosis.

Bordetella pertussis adenylate cyclase (CyaA) toxoid is a powerful nonreplicative immunization vector targeting dendritic cells, which has already been used successfully in prophylactic and therapeutic vaccination in various preclinical animal models. Here, we investigated the potential of CyaA, harboring strong mycobacterial immunogens, i.e., the immunodominant regions of antigen 85A or the complete sequence of the 6-kDa early secreted antigenic target (ESAT-6) protein, to induce antimycobacterial immunity. By generating T-cell hybridomas or by using T cells from mice infected with mycobacteria, we first demonstrated that the in vitro delivery of 85A or ESAT-6 to antigen-presenting cells by CyaA leads to processing and presentation, by major histocompatibility complex class II molecules, of the same epitopes as those displayed upon mycobacterial infection. Importantly, compared to the recombinant protein alone, the presentation of ESAT-6 in vitro was 100 times more efficient upon its delivery to antigen-presenting cells in fusion to CyaA. Immunization with CyaA-85A or CyaA-ESAT-6 in the absence of any adjuvant induced strong antigen-specific lymphoproliferative, interleukin-2 (IL-2) and gamma interferon (IFN-gamma) cytokine responses, in the absence of any IL-4 or IL-5 production. When used as boosters after priming with a BCG expressing ESAT-6, the CyaA-85A and CyaA-ESAT-6 proteins were able to strikingly increase the sensitivity and intensity of proliferative and Th1-polarized responses and notably the frequency of antigen-specific IFN-gamma-producing CD4+ T cells. However, immunization with these CyaA constructs as subunit vaccines alone or as boosters did not allow induction or improvement of protection against Mycobacterium tuberculosis infection. These results question the broadly admitted correlation between the frequency of IFN-gamma-producing CD4+ T cells and the level of protection against tuberculosis.