Cross-Presentation of Skin-Targeted Recombinant Adeno-associated Virus 2/1 Transgene Induces Potent Resident Memory CD8+ T Cell Responses.

A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of nonrecirculating tissue-resident memory T cells (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated virus (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells. Using a model rAAV2/1-based vaccine, we determined that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared to be dispensable for the priming of transgene-specific skin TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell-inducing vaccine carriers.IMPORTANCE rAAVs display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing the ability of rAAV to induce protective T cell responses, however, are scarce. Notably, the potential to induce a tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrated that rAAV vectors can induce bona fide skin resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.

Intradermal Immunization with rAAV1 Vector Induces Robust Memory CD8+ T Cell Responses Independently of Transgene Expression in DCs.

Recombinant adeno-associated viral (rAAV) vectors exhibit interesting properties as vaccine carriers for their ability to induce long-lasting antibody responses. However, rAAV-based vaccines have been suggested to trigger functionally impaired long-term memory CD8+ T cell responses, in part due to poor dendritic cell (DC) transduction. Such results, albeit limited to intramuscular immunization, undermined the use of rAAV as vaccine vehicles against intracellular pathogens. We report here that intradermal immunization with a model rAAV2/1-based vaccine drives the development of bona fide long-term memory CD8+ T cell responses. The intradermal route of immunization and the presence of potent major histocompatibility complex (MHC) class II responses showed synergistic effects on the overall quantity and quality of systemic long-term effector memory transgene-specific CD8+ T cells being generated against the transgene. Of key interest, we found that the induction of memory cytotoxic T lymphocytes (CTLs) following intradermal immunization was solely dependent on the cross-presentation of skin-expressed transgene products, which appeared highly enhanced as compared to muscle-expressed transgene products. Overall our results highlight key tissue-specific differences in transgene presentation pathway requirements of importance for the design of rAAV-based T cell-inducing vaccines.

Intrinsic transgene immunogenicity gears CD8(+) T-cell priming after rAAV-mediated muscle gene transfer.

Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.

Extrathymic induction of Foxp3⁺ regulatory T cells declines with age in a T-cell intrinsic manner.

Extrathymically induced Foxp3⁺ regulatory T (Treg) cells contribute to the pool of Treg cells and are implicated in the maintenance of immune tolerance at environmental interfaces. The impact of T-cell senescence on their generation and function is, however, poorly characterized. We report here that steady-state induction of Foxp3 is impaired in aged T cells in vivo. In vitro assays further revealed that this defective generation of Treg cells was independent from the strength of TCR stimulation and arose before T-cell proliferation. Importantly, they also revealed that this impairment of Foxp3 induction is unrelated to known age-related T-cell defects, such as IL-2 secretion impairment, accumulation of activated T-cell populations, or narrowing of the T-cell repertoire. Finally, a loss of extrathymic induction of Foxp3 and tolerance to minor-mismatched skin graft were observed in aged mice treated by nondepleting anti-CD4 antibody. The T-cell intrinsic impairment of Treg-cell generation revealed here highlights age as a key factor to be considered in immune tolerance induction.

Mast Cell Interaction with Foxp3+ Regulatory T Cells Occur in the Dermis after Initiation of IgE-Mediated Cutaneous Anaphylaxis.

Mast cells (MCs) are well-known for their role in IgE-mediated cutaneous anaphylactic responses, but their regulatory functions in the skin are still under intense scrutiny. Using a Red MC and Basophil reporter (RMB) mouse allowing red fluorescent detection and diphtheria toxin mediated depletion of MCs, we investigated the interaction of MCs, Foxp3+ regulatory T lymphocytes (Tregs) and Langerhans cells (LCs) during passive cutaneous anaphylaxis (PCA) responses. Using intravital imaging we show that MCs are sessile at homeostasis and during PCA. Breeding RMB mice with Langerin-eGFP mice revealed that dermal MCs do not interact with epidermal-localized LCs, the latter showing constant sprouting of their dendrites at homeostasis and during PCA. When bred with Foxp3-eGFP mice, we found that, although a few Foxp3+ Tregs are present at homeostasis, many Tregs transiently infiltrated the skin during PCA. While their velocity during PCA was not altered, Tregs increased the duration of their contact time with MCs compared to PCA-control mice. Antibody-mediated depletion of Tregs had no effect on the intensity of PCA. Hence, the observed increase in Treg numbers and contact time with MCs, regardless of an effect on the intensity of PCA responses, suggests an anti-inflammatory role dedicated to prevent further MC activation.

Neuropilin-1 cooperates with PD-1 in CD8+ T cells predicting outcomes in melanoma patients treated with anti-PD1.

Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by restoring antitumor immune responses. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenesis embryonic development, also expressed in immune cells. We hypothesized that NRP1 could be an immune checkpoint co-receptor modulating CD8+ T cells activity in the context of the antitumor immune response. Here, we show that NRP1 is recruited in the cytolytic synapse of PD1+CD8+ T cells, cooperates and enhances PD-1 activity. In mice, CD8+ T cells specific deletion of Nrp1 improves anti-PD1 antibody antitumor immune responses. Likewise, in human metastatic melanoma, the expression of NRP1 in tumor infiltrating CD8+ T cells predicts poor outcome of patients treated with anti-PD1. NRP1 is a promising target to overcome resistance to anti-PD1 therapies.

Antigen-specific Treg impair CD8(+) T-cell priming by blocking early T-cell expansion.

Foxp3(+) Treg are crucial for the maintenance of self-tolerance and have been shown to control CD8(+) T-cell effector functions. In addition, Treg are thought to control the priming of CD8(+) T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA-specific Treg suppress antigen-linked CTL responses through early blockade of CD8(+) T-cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8(+) T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA-specific CD8(+) T-cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA-specific Treg are less effective but still reduce the frequency of newly primed HA-specific CD8(+) T cells and the ensuing frequency of memory CD8(+) T cells. Our results demonstrate that Treg control CTL priming in an antigen-specific manner at the level of T-cell expansion, highlighting how self-reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T-cell proliferation.

ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells.

Induction of cytotoxic T-cell immunity requires the phagocytosis of pathogens, virus-infected or dead tumour cells by dendritic cells. Peptides derived from phagocytosed antigens are then presented to CD8+ T lymphocytes on major histocompatibility complex (MHC) class I molecules, a process called "cross-presentation". After phagocytosis, antigens are exported into the cytosol and degraded by the proteasome. The resulting peptides are thought to be translocated into the lumen of the endoplasmic reticulum (ER) by specific transporters associated with antigen presentation (TAP), and loaded onto MHC class I molecules by a complex "loading machinery" (which includes tapasin, calreticulin and Erp57). Here we show that soon after or during formation, phagosomes fuse with the ER. After antigen export to the cytosol and degradation by the proteasome, peptides are translocated by TAP into the lumen of the same phagosomes, before loading on phagosomal MHC class I molecules. Therefore, cross-presentation in dendritic cells occurs in a specialized, self-sufficient, ER-phagosome mix compartment.

IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses.

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation.

Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation.

BACKGROUND: Gene modified dendritic cells (DC) are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC) are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. METHODS: We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV) pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG), the gibbon ape leukaemia virus envelope (GaLV) or the feline endogenous virus envelope (RD114). At the same time, we evaluated transgene expression (E-GFP reporter gene) under the control of different promoters. RESULTS: We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV) under the control of phoshoglycerate kinase (PGK) and elongation factor-1 (EF1alpha) promoters (28% to 90% of E-GFP+ cells, respectively) in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5-12) described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. CONCLUSION: Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

Transient immunomodulation allows repeated injections of AAV1 and correction of muscular dystrophy in multiple muscles.

Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (microKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in microKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge.

Foxp3+ Regulatory and Conventional CD4+ T Cells Display Similarly High Frequencies of Alloantigen-Reactive Cells.

Foxp3+ regulatory T cells (Tregs) play a major role in acquired immune tolerance to allogenic transplants. Their suppressive activity is thought to require T cell receptor (TCR)-driven antigen recognition; little, however, is known about the fraction of Tregs able to recognize alloantigens within this T cell subset primarily educated against self-antigens. Performing transfer experiments of Tregs or conventional T cells (Tconv) into both lymphoreplete and lymphopenic mice, we observed a similarly high proportion of cells signaling through their TCR and proliferating in allogenic hosts. Furthermore, using an in vivo proliferation assay with limited T cell numbers infused into lymphopenic mice, we found that the overall frequency of alloreactive Tregs was similar if not higher to that of alloreactive Tconv. Overall our study highlights a noticeably high level of alloreactive Foxp3+ regulatory T cells accounting for their predominant role in transplantation tolerance.

Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle.

The pro-tolerogenic environment of the liver makes this tissue an ideal target for gene replacement strategies. In other peripheral tissues such as the skeletal muscle, anti-transgene immune response can result in partial or complete clearance of the transduced fibers. Here, we characterized liver-induced transgene tolerance after simultaneous transduction of liver and muscle. A clinically relevant transgene, α-sarcoglycan, mutated in limb-girdle muscular dystrophy type 2D, was fused with the SIINFEKL epitope (hSGCA-SIIN) and expressed with adeno-associated virus vectors (AAV-hSGCA-SIIN). Intramuscular delivery of AAV-hSGCA-SIIN resulted in a strong inflammatory response, which could be prevented and reversed by concomitant liver expression of the same antigen. Regulatory T cells and upregulation of checkpoint inhibitor receptors were required to establish and maintain liver-mediated peripheral tolerance. This study identifies the fundamental role of the synergy between Tregs and upregulation of checkpoint inhibitor receptors in the liver-mediated control of anti-transgene immunity triggered by muscle-directed gene transfer.

Dual muscle-liver transduction imposes immune tolerance for muscle transgene engraftment despite preexisting immunity.

Immune responses to therapeutic transgenes are a potential hurdle to treat monogenic muscle disorders. These responses result from the neutralizing activity of transgene-specific B cells and cytotoxic T cells recruited upon gene transfer. We explored here how dual muscle-liver expression of a foreign transgene allows muscle transgene engraftment after adenoassociated viral vector delivery. We found in particular that induction of transgene-specific tolerance is imposed by concurrent muscle and liver targeting, resulting in the absence of CD8+ T cell responses to the transgene. This tolerance can be temporally decoupled, because transgene engraftment can be achieved in muscle weeks after liver transduction. Importantly, transgene-specific CD8+ T cell tolerance can be established despite preexisting immunity to the transgene. Whenever preexisting, transgene-specific CD4+ and CD8+ memory T cell responses are present, dual muscle-liver transduction turns polyclonal, transgene-specific CD8+ T cells into typically exhausted T cells with high programmed cell death 1 (PD-1) expression and lack of IFN-γ production. Our results demonstrate that successful transduction of muscle tissue can be achieved through liver-mediated control of humoral and cytotoxic T cell responses, even in the presence of preexisting immunity to the muscle-associated transgene.

Mast Cell Degranulation Exacerbates Skin Rejection by Enhancing Neutrophil Recruitment.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy.

Most of the human tumor-associated antigens (TAAs) characterized thus far are derived from nonmutated "self"-proteins. Numerous strategies have been developed to break tolerance to TAAs, combining various forms of antigens with different vectors and adjuvants. However, no study has yet determined how to select epitopes within a given TAA to induce the highest antitumor effector response. We addressed this question by evaluating in HLA-A*0201-transgenic HHD mice the antitumor vaccination efficacy of high- and low-affinity epitopes from the naturally expressed murine telomerase reverse transcriptase (mTERT). Immunity against low-affinity epitopes was induced with heteroclitical variants. We show here that the CTL repertoire against high-affinity epitopes is partially tolerized, while that against low-affinity epitopes is composed of frequent CTLs with high avidity. The high-affinity p797 and p545 mTERT epitopes are not able to protect mice from a lethal challenge with the mTERT-expressing EL4-HHD tumor. In contrast, mice developing CTL responses against the p572 and p988 low-affinity epitopes exhibit potent antitumor immunity and no sign of autoimmune reactivity against TERT-expressing normal tissues. Our results strongly argue for new TAA epitope selection and modification strategies in antitumor immunotherapy applications in humans.

Imaging calpain protease activity by multiphoton FRET in living mice.

Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions.

Selective accumulation of mature DC-Lamp+ dendritic cells in tumor sites is associated with efficient T-cell-mediated antitumor response and control of metastatic dissemination in melanoma.

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

Lateral Mobility of the Cell Adhesion Molecule L1 Within the Surface Membrane of Morphologically Undifferentiated and Differentiated Neuroblastoma Cells.

Lateral mobility and localization in the surface membrane of the adhesion molecule L1 was studied in morphologically undifferentiated and differentiated neuroblastoma cells to gain insight into its possible association with the different molecular forms of N-CAM. In undifferentiated cells, the fraction of mobile L1 molecules is high and similar to that of N-CAM 140. Upon long-term morphological differentiation, the fraction of mobile L1 molecules is reduced by a factor of three and is similar to that of N-CAM 180, the predominant molecular form of N-CAM in differentiated neuroblastoma cells. Comparable to N-CAM 180, L1 is also preferentially accumulated at contact sites between these cells as seen by indirect immunofluorescence. These observations raise the question of whether at least part of the L1 molecules may be directly or indirectly (e.g. via N-CAM 180) linked to the cytoskeleton, thus stabilizing cell contacts between differentiated cells.

Mast cells aggravate sepsis by inhibiting peritoneal macrophage phagocytosis.

Controlling the overwhelming inflammatory reaction associated with polymicrobial sepsis remains a prevalent clinical challenge with few treatment options. In septic peritonitis, blood neutrophils and monocytes are rapidly recruited into the peritoneal cavity to control infection, but the role of resident sentinel cells during the early phase of infection is less clear. In particular, the influence of mast cells on other tissue-resident cells remains poorly understood. Here, we developed a mouse model that allows both visualization and conditional ablation of mast cells and basophils to investigate the role of mast cells in severe septic peritonitis. Specific depletion of mast cells led to increased survival rates in mice with acute sepsis. Furthermore, we determined that mast cells impair the phagocytic action of resident macrophages, thereby allowing local and systemic bacterial proliferation. Mast cells did not influence local recruitment of neutrophils and monocytes or the release of inflammatory cytokines. Phagocytosis inhibition by mast cells involved their ability to release prestored IL-4 within 15 minutes after bacterial encounter, and treatment with an IL-4-neutralizing antibody prevented this inhibitory effect and improved survival of septic mice. Our study uncovers a local crosstalk between mast cells and macrophages during the early phase of sepsis development that aggravates the outcome of severe bacterial infection.