Randomized phase I trial of antigen-specific tolerizing immunotherapy with peptide/calcitriol liposomes in ACPA+ rheumatoid arthritis

BACKGROUND. Antigen-specific regulation of autoimmune disease is a major goal. In seropositive rheumatoid arthritis (RA), T cell help to autoreactive B cells matures the citrullinated (Cit) antigen-specific immune response, generating RA-specific V domain glycosylated anti-Cit protein antibodies (ACPA VDG) before arthritis onset. Low or escalating antigen administration under “sub-immunogenic” conditions favors tolerance. We explored safety, pharmacokinetics, and immunological and clinical effects of s.c. DEN-181, comprising liposomes encapsulating self-peptide collagen II259-273 (CII) and NF-κB inhibitor 1,25-dihydroxycholecalciferol. METHODS. A double-blind, placebo-controlled, exploratory, single-ascending-dose, phase I trial assessed the impact of low, medium, and high DEN-181 doses on peripheral blood CII-specific and bystander Cit64vimentin59-71–specific (Cit-Vim–specific) autoreactive T cell responses, cytokines, and ACPA in 17 HLA-DRB1*04:01+ or *01:01+ ACPA+ RA patients on methotrexate. RESULTS. DEN-181 was well tolerated. Relative to placebo and normalized to baseline values, Cit-Vim–specific T cells decreased in patients administered medium and high doses of DEN-181. Relative to placebo, percentage of CII-specific programmed cell death 1+ T cells increased within 28 days of DEN-181. Exploratory analysis in DEN-181–treated patients suggested improved RA disease activity was associated with expansion of CII-specific and Cit-Vim–specific T cells; reduction in ACPA VDG, memory B cells, and inflammatory myeloid populations; and enrichment in CCR7+ and naive T cells. Single-cell sequencing identified T cell transcripts associated with tolerogenic TCR signaling and exhaustion after low or medium doses of DEN-181. CONCLUSION. The safety and immunomodulatory activity of low/medium DEN-181 doses provide rationale to further assess antigen-specific immunomodulatory therapy in ACPA+ RA

The role of posttranslational modifications in generating neo-epitopes that bind to rheumatoid arthritis-associated HLA-DR alleles and promote autoimmune T cell responses.

While antibodies to citrullinated proteins have become a diagnostic hallmark in rheumatoid arthritis (RA), we still do not understand how the autoimmune T cell response is influenced by these citrullinated proteins. To investigate the role of citrullinated antigens in HLA-DR1- and DR4-restricted T cell responses, we utilized mouse models that express these MHC-II alleles to determine the relationship between citrullinated peptide affinity for these DR molecules and the ability of these peptides to induce a T cell response. Using a set of peptides from proteins thought to be targeted by the autoimmune T cell responses in RA, aggrecan, vimentin, fibrinogen, and type II collagen, we found that while citrullination can enhance the binding affinity for these DR alleles, it does not always do so, even when in the critical P4 position. Moreover, if peptide citrullination does enhance HLA-DR binding affinity, it does not necessarily predict the generation of a T cell response. Conversely, citrullinated peptides can stimulate T cells without changing the peptide binding affinity for HLA-DR1 or DR4. Furthermore, citrullination of an autoantigen, type II collagen, which enhances binding affinity to HLA-DR1 did not enhance the severity of autoimmune arthritis in HLA-DR1 transgenic mice. Additional analysis of clonal T cell populations stimulated by these peptides indicated cross recognition of citrullinated and wild type peptides can occur in some instances, while in others cases the citrullination generates a novel T cell epitope. Finally, cytokine profiles of the wild type and citrullinated peptide stimulated T cells unveiled a significant disconnect between proliferation and cytokine production. Altogether, these data demonstrate the lack of support for a simplified model with universal correlation between affinity for HLA-DR alleles, immunogenicity and arthritogenicity of citrullinated peptides. Additionally they highlight the complexity of both T cell receptor recognition of citrulline as well as its potential conformational effects on the peptide:HLA-DR complex as recognized by a self-reactive cell receptor

Citrullination only infrequently impacts peptide binding to HLA class II MHC

<div><p>It has been hypothesized that HLA class II alleles associated with rheumatoid arthritis (RA) preferentially present self-antigens altered by post-translational modification, such as citrullination. To understand the role of citrullination we tested four RA-associated citrullinated epitopes and their corresponding wild-type version for binding to 28 common HLA class II. Binding patterns were variable, and no consistent impact of citrullination was identified. Indeed, in one case citrullination significantly increased binding compared to the WT peptide, in another citrullination was associated with a reduction in promiscuity by 40%. For a more comprehensive analysis, we tested over 200 citrullinated peptides derived from vimentin and collagen II for their capacity to bind the RA-associated shared epitope alleles DRB1*01:01 and DRB1*04:01. The overall effect of citrullination on binding was found to be relatively minor, and only rarely associated with 3-fold increases or decreases in affinity. Previous studies have suggested that citrullination of MHC anchor residues, in particular P4, is associated with generation of novel RA-associated epitopes. However, analysis of the predicted MHC-binding cores of all peptides tested found that in modified peptides with increased binding affinity the citrullinated residue was predicted to occupy an anchor position in only a minority of cases. Finally, we also show that identification of citrullinated peptide binders could be facilitated by using the NetMHCIIpan 3.1 algorithm, representing citrullination as a wildcard. Our studies identify a total of 117 citrullinated peptides that bound RA-associated alleles with an affinity of 1000 nM or better.</p></div

Prediction of the DRB1*01:01 and DRB1*04:01 binding capacity of citrullinated peptides.

<p>The DRB1*01:01 (left panel) and DRB1*04:01 (right panel) binding capacity of citrullinated peptides were predicted using NetMHCIIpan version 3.1 predictions by substituting the citrullinated residues with the wildcard “X”. Trend lines are show in red.</p

Predicted HLA DR binding cores of four known citrullinated RA-associated epitopes.

<p>Predicted HLA DR binding cores of four known citrullinated RA-associated epitopes.</p

Adaptor Protein SLAT Modulates Fcγ Receptor-mediated Phagocytosis in Murine Macrophages*

SLAT (SWAP-70-like adaptor protein of T cells) is an adaptor protein expressed in cells of the hematopoietic system. SLAT interacts with and alters the function of small GTPase Rac1 in fibroblasts. In these nonhematopoietic models, the SLAT-Rac interaction leads to changes in F-actin and causes cytoskeletal reorganization. In T cells, SLAT expression regulates the development of T helper cells through Cdc42- and Rac1-mediated activation of the NF-AT transcription factor. Here we show that SLAT is expressed in macrophages. Overexpression of SLAT in a macrophage cell line inhibits the IgG Fcγ receptor-mediated phagocytic ability of THP1 cells. In bone marrow-derived macrophages, SLAT protein is recruited to the early phagosomes formed via Fcγ receptor engagement. SLAT recruitment to the phagosome was most efficient when the macrophages express at least one isoform of Rac (Rac1 or Rac2), because SLAT recruitment was reduced in macrophages of Rac-deficient mice. Macrophages derived from animals lacking SLAT show an elevation in the rate of Fcγ receptor-mediated phagocytosis. The absence of SLAT is associated with an increase in the amount of F-actin formed around these phagosomes as well as an increase in the amount of Rac1 protein recruited to the phagosome. Our results suggest that SLAT acts as a gatekeeper for the amount of Rac recruited to the phagosomes formed by Fcγ receptor engagement and thus is able to regulate F-actin re-organization and consequently phagocytosis

HLA class II binding of RA-associated epitopes¹.

<p>HLA class II binding of RA-associated epitopes<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177140#t001fn001" target="_blank">¹</a>.</p

Comparison of binding affinity of wild-type and citrullinated versions of vimentin peptides.

<p>Each data point indicates the DRB1*01:01 (left panel) or DRB1*04:01 (right panel) binding capacity of WT vimentin peptides with the corresponding citrullinated version. Effects greater or less than 3-fold are demarcated by the diagonal dashed red lines and highlighted by red fill. Points to the lower right indicate instances where the citrullinated peptide binds with higher affinity that the WT peptide, and vice versa.</p