A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer.

Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of α-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

Antigenicity and immunogenicity of allogeneic retinal transplants.

The transplantation of neuronal cells and tissues represents a promising approach for the treatment of incurable neurodegenerative diseases. Indeed, it has been reported recently that retinal transplantation can rescue photoreceptor cells and delay age-related changes in various retinal layers in rodents. However, retinal grafts deteriorate progressively after placement in recipients' eyes. Here we investigated whether a host's immune response elicited toward the graft contributes to its deterioration. Using an ELISA spot assay, we measured T cell responses to retinal tissues placed in the vitreous cavity of syngeneic and allogeneic mice. We found that allogeneic retinas induced potent alloimmune responses mediated by T cells secreting type 1 cytokines (IFN-gamma and IL-2). No response was found in mice engrafted with syngeneic retinas. In addition, all syngeneic retinal grafts displayed no signs of tissue damage (at 55 days), while the majority of allogeneic retinas deteriorated as early as 12 days after placement. Next, we showed that anti-donor responses occurred within two phenotypically and functionally distinct T cell subsets: CD4+ T cells secreting IL-2 and CD8+ T cells producing IFN-gamma. Importantly, CD4+ T cells were necessary and sufficient to cause graft deterioration, while CD8+ T cells did not contribute to this process.

Role of CD4+ and CD8+ T cells in allorecognition: lessons from corneal transplantation.

Corneal transplantation represents an interesting model to investigate the contribution of direct vs indirect Ag recognition pathways to the alloresponse. Corneal allografts are naturally devoid of MHC class II+ APCs. In addition, minor Ag-mismatched corneal grafts are more readily rejected than their MHC-mismatched counterparts. Accordingly, it has been hypothesized that these transplants do not trigger direct T cell alloresponse, but that donor Ags are presented by host APCs, i.e., in an indirect fashion. Here, we have determined the Ag specificity, frequency, and phenotype of T cells activated through direct and indirect pathways in BALB/c mice transplanted orthotopically with fully allogeneic C57BL/6 corneas. In this combination, only 60% of the corneas are rejected, while the remainder enjoy indefinite graft survival. In rejecting mice the T cell response was mediated by two T cell subsets: 1) CD4+ T cells that recognize alloantigens exclusively through indirect pathway and secrete IL-2, and 2) IFN-gamma-producing CD8+ T cells recognizing donor MHC in a direct fashion. Surprisingly, CD8+ T cells activated directly were not required for graft rejection. In nonrejecting mice, no T cell responses were detected. Strikingly, peripheral sensitization to allogeneic MHC molecules in these mice induced acute rejection of corneal grafts. We conclude that only CD4+ T cells activated via indirect allorecognition have the ability to reject allogeneic corneal grafts. Although alloreactive CD8+ T cells are activated via the direct pathway, they are not fully competent and cannot contribute to the rejection unless they receive an additional signal provided by professional APCs in the periphery.

Blockade of CD40-CD154 costimulatory pathway promotes survival of allogeneic corneal transplants.

PURPOSE: To determine the effect of systemic anti-CD154 monoclonal antibody on the survival of orthotopic murine corneal transplants. METHODS: BALB/c mice were used as recipients of syngeneic, multiple minor histocompatability (H)-disparate, or major histocompatibility complex MHC-mismatched corneal transplants. Recipient beds were either avascular (normal risk) or neovascularized (high risk). Mice were randomized to receive either anti-CD154 antibody or control immunoglobulin by intraperitoneal injection at surgery and once weekly after surgery. After orthotopic corneal transplantation, all grafts were evaluated for signs of rejection by slit lamp biomicroscopy over 8 weeks. The high-risk transplants were continuously observed until week 18 after the therapy was discontinued at week 8. Allospecific delayed-type hypersensitivity (DTH) was evaluated after transplantation in high-risk graft recipients. Frequency of interferon (IFN)-gamma-secreting T cells in the hosts was measured by enzyme-linked immunospot (ELISPOT) assay. RESULTS: In normal-risk transplantation, the 8-week survival rate improved from 25% in control mice to 88% in anti-CD154-treated hosts of minor H-disparate grafts (P = 0.0087) and from 78% in control mice to 100% in anti-CD154-treated recipients of MHC-mismatched transplants (P = 0.177). Of particular significance, in high-risk transplantation, anti-CD154 therapy dramatically enhanced the survival of both minor H- and MHC-disparate corneal transplants to 100% (P = 0.0001) and 92% (P = 0.0002), respectively. In addition, the anti-CD154-treated mice did not exhibit allospecific immunity. However, termination of anti-CD154 led to some loss in graft survival, especially among high-risk minor H-disparate grafts. The frequency of IFN-gamma-producing T cells was significantly reduced in anti-CD154-treated hosts. CONCLUSIONS: Continuous suppression of the CD40-CD154 costimulatory pathway promotes the acceptance of corneal transplants, regardless of the degree of allodisparity or preoperative risk. The beneficial effect of anti-CD154 treatment may be due in part to inhibition of Th1-mediated responses.

CD4+ T cell responses to self- and mutated p53 determinants during tumorigenesis in mice.

We analyzed CD4+ T helper responses to wild-type (wt) and mutated (mut) p53 protein in normal and tumor-bearing mice. In normal mice, we observed that although some self-p53 determinants induced negative selection of p53-reactive CD4+ T cells, other p53 determinants (cryptic) were immunogenic. Next, BALB/c mice were inoculated with J774 syngeneic tumor cell line expressing mut p53. BALB/c tumor-bearing mice mounted potent CD4+ T cell responses to two formerly cryptic peptides on self-p53. This response was characterized by massive production of IL-5, a Th2-type lymphokine. Interestingly, we found that T cell response was induced by different p53 peptides depending upon the stage of cancer. Mut p53 gene was shown to contain a single mutation resulting in the substitution of a tyrosine by a histidine at position 231 of the protein. Two peptides corresponding to wt and mutated sequences of this region were synthesized. Both peptides bound to the MHC class II-presenting molecule (Ed) with similar affinities. However, only mut p53.225-239 induced T cell responses in normal BALB/c mice, a result strongly suggesting that high-affinity wt p53.225-239 autoreactive T cells had been eliminated in these mice. Surprisingly, CD4+ T cell responses to both mut and wt p53.225-239 peptides were recorded in J774 tumor-bearing mice, a phenomenon attributed to the recruitment of low-avidity p53.225-239 self-reactive T cells.

Induction of T-cell response to cryptic MHC determinants during allograft rejection.

The presentation of MHC peptides by recipient and donor antigen presenting cells is an essential element in allorecognition and allograft rejection. MHC proteins contains two sets of determinants: the dominant determinants that are efficiently processed and presented to T cells, and the cryptic determinants that are not presented sufficiently enough to induce T-cell responses in vivo. In transplanted mice, initial T-cell response to MHC peptides is consistently limited to a single or a few immunodominant determinants on donor MHC molecule. However, in this article we show that under appropriate circumstances the hierarchy of determinants on MHC molecules can be disrupted. First, we observed that gamma IFN can trigger de novo presentation of cryptic self-MHC peptides by spleen cells. Moreover, we showed that allotransplantation is associated with induction of T-cell responses to formerly cryptic determinants on both syngeneic and allogeneic MHC molecules. Our results suggest that cross-reactivity and inflammation are responsible for the initiation of these auto- and alloimmune responses after transplantation.

Naturally processed peptides from HLA-DQ7 (alpha1*0501-beta1*0301): influence of both alpha and beta chain polymorphism in the HLA-DQ peptide binding specificity.

Self peptides bound to HLA-DQ7 (alpha1*0501-beta1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (alpha1*0501-beta1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (alpha1*0501-beta1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215-230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (alpha1*0501-beta1*0301); (2) when either the DQalpha or DQbeta chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the alpha and beta chains. (3) Unexpectedly, the TfR 215-230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.

Alteration of HLA-B27 peptide presentation after infection of transfected murine L cells by Shigella flexneri.

Shigella flexneri is a triggering agent for reactive arthritis in HLA-B27-susceptible individuals. Considering the intracellular multiplication of bacteria, it seems likely that bacterial peptides may be presented by the major histocompatibility complex (MHC) class I pathway. To examine this hypothesis, we infected HLA-B*2705- and/or human beta2-microglobulin-transfected murine L-cell lines with M90T, an invasive strain of S. flexneri. Bacterial infection induced no detectable modifications in the biosynthesis and expression level of HLA-B27, as assessed by immunoprecipitation, Northern blot analysis, and flow cytometry. Using confocal microscopy, we observed that bacterial infection induced a clustering of HLA-B27 molecules during macropinocytosis and before bacterial dissemination from cell to cell. Peptides naturally bound to HLA-B27 molecules were acid eluted from infected cells and separated by high-performance liquid chromatography. Major differences were observed in high-performance liquid chromatography profiles and in the nature of peptides presented following bacterial infection. Although most of the antigens presented were not accessed by Edman degradation, we obtained two sequences partially homologous to bacterial proteins. These peptides lacked the major HLA-B27 peptide anchor (Arg) at position 2, and one had an unusual length of 14 amino acids. These data suggest that alterations in the peptide presentation by HLA-B27 occur during infection, which could be relevant to the pathogenesis of HLA-B27-related arthritis.

Protein transport and processing by human HT29-19A intestinal cells: effect of interferon gamma.

BACKGROUND: The nature of the breakdown products produced in enterocytes during epithelial transport of intact proteins may be critical in determining the functional consequences of protein absorption. AIM: (a) To measure the transepithelial transport of horseradish peroxidase (HRP) and to identify the nature of HRP breakdown products released on the basal side of enterocytes and (b) to assess the role of interferon gamma (IFN gamma) on HRP transport and processing. METHODS: HT29-19A intestinal cells were used to assess transepithelial transport of HRP in Ussing chambers, and the nature of breakdown products in the basal compartment was analysed by high performance liquid chromatography (HPLC). RESULTS: (1) In control conditions, [3H]HRP equivalent fluxes (3135 (219) ng/h per cm2; mean (SEM) comprised 50% amino acids, 40% peptides, and 10% intact HRP. Steric exclusion HPLC of the breakdown products indicated a wide range of molecular masses including a major peptide of about 1150 Da. Lysosomal aspartyl and thiol proteases were expressed but no HLA-DR surface expression was noted, (2) At 48 to 72 hours after IFN gamma stimulation, [3H]HRP equivalent fluxes increased significantly (7392 (1433) ng/h per cm2) without modification of the relative proportions of amino acids, peptides, and intact HRP, and without modification of the distribution of breakdown products in HPLC. Lysosomal protease activities were not modified by IFN gamma but HLA-DR expression was increased. CONCLUSION: Intestinal cells are able to process HRP into peptides potentially capable of stimulating the immune system. IFN gamma stimulates the transport and processing of HRP thus increasing the antigenic load in the intestinal mucosa.

[Subtypes of the HLA-B27 molecule and association with spondylarthropathies]. / Sous-types de la molécule HLA-B27 et association aux spondylarthropathies.

HLA-B27 subtypes differ in their ethnic distribution and in their susceptibility to spondylarthropathies (SA). B*2705 and B*2702 are the most frequent disease-associated subtypes in Caucasians as well as B*2704 and B*2707 in Asia while B*2706 in Asia and B*2709 in Sardinia have been reported not to be associated to SA. Differences in antigenic peptide presentation could underlie such behavior. Several studies suggested that a Tyr C-terminal peptide anchor could be found preferentially in disease-associated subtypes and could be therefore one of the criteria in the search of putative arthritogenic peptide(s). We analyzed by HPLC and Edman sequencing peptides eluted from immunopurified HLA-B27 molecules expressed on B-lymphoblastoid cell lines or C1R transfectans of human origin. We focused our work on B*2707, associated with SA in the same geographical area where B*2706 is not. We found the same preference for Leu at the C-terminus in the peptides bound by both subtypes without any significant signal for Tyr. In the same experimental conditions a Tyr C-terminal anchor was found for B*2705, B*2702, B*2704, B*2703 and also for B*2701 and B*2708, 2 rare subtypes for which binding specificity was previously unknown. Comparison of the F-pocket aminoacid composition in these various subtypes showed a correlation between Asp at position 116 and Tyr at the peptide C-terminus. Asp116 is changed for Tyr in B*2706, B*2707 and His in B*2709, all subtypes allowing a Leu C-terminal anchor. Therefore a Tyr C-terminal anchor correlates with the HLA-B27 F-pocket composition rather than with susceptibility to SA.

Differences in endogenous peptides presented by HLA-B*2705 and B*2703 allelic variants. Implications for susceptibility to spondylarthropathies.

The association between HLA-B27 and spondylarthropathies is currently being reinvestigated in the light of HLA-B27 subtyping. At least 11 different subtypes have been described among which B*2703, B*2706, and B*2709 could be less closely associated with disease at the population level. Differences in the presentation of antigenic peptides by these subtypes could be related to differences in disease susceptibility. We focused our work on the comparison of B*2705 and B*2703 which differ at a single position at residue 59 in pocket A of the peptide binding groove. Endogenous peptides from the human C1R line transfected by B*2705 or B*2703 were acid-eluted and separated by HPLC. Major individual fractions were sequenced by Edman NH2-terminal degradation. Differences observed between B*2705 versus B*2703 individual ligands were confirmed in an in vitro stabilization assay with T2-B*2705 or B*2703 transfected cells in the presence of synthetic peptides. One B*2705 associated peptide is derived from the sequence 169-179 in the second extracellular domain of several HLA class I molecules including HLA-B27. This sequence (RRYLENGKETL) is highly homologous to a previously reported sequence (LRRYLENGK) sharing similarities with proteins from enteric bacteria. We show here that it is naturally presented as a major endogenous peptide by B*2705 and B*2702 disease-associated subtypes and not by B*2703.

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.