Elsevier

Journal of Autoimmunity

Volume 103, September 2019, 102276
Journal of Autoimmunity

HLA-DR15-specific inhibition attenuates autoreactivity to the Goodpasture antigen

https://doi.org/10.1016/j.jaut.2019.05.004Get rights and content

Highlights

  • PV-267 inhibits HLA-DR15 and blocks α3135-145-specific responses.

  • HLA-DR15 inhibition protects mice from autoimmune anti-GBM glomerulonephritis.

  • Blocking HLA-DR15 after autoimmunity induction stops disease development in mice.

  • Treating renal injury with HLA-DR15 inhibition limits disease severity in mice.

Abstract

Goodpasture's disease manifests as rapidly progressive glomerulonephritis. Current immunosuppressive treatments do not specifically target the pathological immune response and have significant side effects. Like most autoimmune diseases, the strongest genetic association is with the HLA alleles. Inheritance of HLA-DR15 confers susceptibility, and structure-function studies have shown that HLA-DR15 plays a causative role in activating autoreactive pro-inflammatory T cells. Thus, specific inhibition of HLA-DR15 would provide a targeted therapeutic approach. We hypothesised that PV-267, an HLA-DR15-specific inhibitor, would effectively block HLA-DR15 presentation of the dominant epitope, attenuate the activation of autoreactive T cells, and limit disease. Using humanised HLA-DR15 transgenic mice, α3135-145-specific, pro-inflammatory T cell recall responses were measured using IFN-γ and IL-17A ELISPOTs and by proliferation assay. To determine if PV-267 could limit disease, experimental autoimmune anti-GBM glomerulonephritis was induced in HLA-DR15 transgenic mice (on an Fcgr2b−/- background), and functional and histological disease endpoints were measured. PV-267 effectively inhibited α3135-145-specific immune responses and disease development. Mice treated prior to immunization with α3135-145 had reduced α3135-145-specific recall responses, and limited disease by albuminuria, histological glomerular injury, IgG deposition, and inflammatory cell infiltrates. PV-267 treatment commencing after the onset of active anti-α3(IV)NC1 autoimmunity attenuated functional and histological renal injury. When treatment was administered after disease was established, PV-267 limited the severity of histological injury. In conclusion, HLA-DR15 inhibition attenuates α3(IV)NC1-specific pro-inflammatory responses and could be used as an adjunct therapy for anti-GBM disease.

Introduction

Anti-glomerular basement membrane (GBM) disease, also known as Goodpasture's disease, results from autoimmunity against the non-collagenous domain of the α3 chain of type IV collagen, α3(IV)NC1, present in the GBM [1]. This disease manifests as rapidly progressive glomerulonephritis, with glomerular crescent formation and linear staining of glomerular antibody deposits [2]. Patients also develop pulmonary haemorrhage. Both humoral and cell mediated effectors contribute to the disease pathogenesis, with pathogenic anti- α3(IV)NC1 antibodies and autoreactive CD4+ T cells found in patients with anti-GBM disease and in experimental animal models of disease [[3], [4], [5], [6], [7], [8]]. Anti-GBM disease is strongly associated with the MHC class II allele HLA-DRB1*15:01 (HLA-DR15, previously known as HLA-DR2b), with an average odds ratio of 8.5 [9]. CD4+ T cells reactive to the immunodominant CD4+ T cell epitope, α3135-145 (135GWISLWKGFSF145), are expanded in HLA-DR15+ humans and induce disease in HLA-DR15 transgenic mice (HLA-DR15+, lacking mouse MHC class II), demonstrating the important contribution of HLA-DR15-mediated CD4+ T cell responses in this disease [10].

Treatments for autoimmune disease have remained largely unchanged for many years, with few advances that provide better options for patients. Current treatment for anti-GBM disease involves high dose corticosteroids, cyclophosphamide, and acute plasmapheresis to remove autoantibodies [2]. These toxic immunosuppressants often have severe side effects, including those that compromise protective immune function, risking life-threatening infections. There is a need for better treatments, in this and in other autoimmune kidney diseases.

Most autoimmune diseases have genetic associations with HLA alleles, although their mechanistic contribution to autoimmunity is unclear [11]. In anti-GBM disease, however, a mechanism by which HLA polymorphisms influence disease risk has been defined. HLA-DR15 increases disease susceptibility by presenting α3135-145 in a conformation that activates pro-inflammatory T cells; in contrast, the negatively associated HLA-DR1 confers protection by presenting α3135-145 in a conformation that activates regulatory T cells [8]. Thus, blocking the ability of HLA-DR15 to present α3135-145 could specifically inhibit pro-inflammatory α3135-145-specific responses while allowing protective immunity to be effected by the other unaffected HLA class II allomorphs found in each human (HLA-DQ, HLA-DP, and if not homozygous for DR15, the other HLA-DR). PV-267 is one such HLA-DR15-specific inhibitor, a small molecule that binds with high affinity and specificity to HLA-DR15 [12]. Thus, we hypothesize that HLA-DR15 inhibition with PV-267 will block the activation of α3135-145-specific T cells and attenuate experimental autoimmune anti-GBM disease. We tested these hypotheses using humanised HLA-DR15 transgenic mice.

Section snippets

Peptides and PV-267

The α3135-145 peptide (GWISLWKGFSF) and OVA323-339 peptide (ISQAVHAAHAEINEAGR) were synthesised to at least 95% purity (Mimotopes, Clayton, Australia). The PV-267 peptide [Ac–V(Chg)R (Tic)F–NH2] was designed and synthesised by Provid Pharmaceuticals (Monmouth Junction, NJ, USA). PV-267 was dissolved in 0.1 M sodium phosphate buffer (pH 7.4, with 0.02% Tween 80).

Mice

HLA-DR15 transgenic mice (mouse MHC class II−/, HLA-DRA1*01:01 transgenic, HLA-DRB1*15:01 transgenic, Fcgr2b+/+ or Fcgr2b−/-) were

Blocking HLA-DR15 inhibits α3135-145-specific immune responses

To determine whether PV-267 could limit the activation of α3135-145-specific T cells, HLA-DR15 transgenic (DR15+Fcgr2b+/+) mice were administered either vehicle or PV-267 (30 mg/kg, intraperitoneally) daily from one day prior to α3135-145 immunization (Fig. 1A). Ten days after α3135-145 immunization, α3135-145-specific responses were measured on cells from draining lymph nodes by proliferation using [3H]-thymidine incorporation, and IFN-γ and IL-17A ELISPOTs after ex vivo stimulation with α3

Discussion

Specifically inhibiting HLA allomorphs in autoimmune renal disease may offer a more targeted approach than current broadly immunosuppressive treatments. In the current studies, we explored the possibility of using a small molecule inhibitor, PV-267, to block HLA-DR15 mediated antigen presentation in an HLA transgenic mouse model of autoimmune anti-GBM glomerulonephritis. PV-267 effectively suppressed inflammatory responses to α3135-145 (Fig. 1) and protecteding mice from developing disease (

Conclusion

This study has shown that specific HLA-DR15 inhibition by PV-267 can attenuate renal injury, demonstrating antigen-specific therapeutic potential. If used as an adjunct treatment for anti-GBM disease, PV-267 may be able to provide a less toxic alternative and reduce adverse outcomes, for example by allowing lower doses of current therapies to be used. Furthermore, these results support the use of selective HLA class II inhibition in other autoimmune diseases, where specific HLA allomorphs

Author contributions

M.H., G.L.O., N.B.R., C.R.S., Y.S., S.R.H., A.R.K. and J.D.O. designed the experiments; M.H., P.E. and J.D.O. performed the experiments and collected the data; M.H., A.R.K. and J.D.O. drafted and revised the paper; all authors approved the final version of the paper.

Acknowledgments and disclosures

This work was supported by NHMRC Project grants (APP1084869 and APP1145105). J.D.O. is supported by an Al and Val Rosenstrauss Fellowship by the Rebecca Cooper Medical Research Foundation. G.L.O. is the chief executive officer of Provid; N.B.R., C.R.S. and Y.S. are employees of Provid.

References (18)

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