Eric Morand1, Rachel Cheong2, Peter Eggenhuizen2, Janet Chang2, Ashraf Broury2, Boaz Ng2, Khai Loh2, Elean Tay2, Chanjuan Shen3, Julie Monk2, Yong Zhong4, Steven Lim2, Jia Xi Chung2, Rangi Kandane-Rathnayake5, Rachel Koelmeyer2, Alberta Hoi6, Sarah Snelgrove2, Yi Tian Ting2 and Joshua Ooi2, 1Monash University, Centre for Inflammatory Diseases, Melbourne, Australia, 2Monash University, Clayton, Australia, 3Central South University, Changsha, China, 4Xiangya Hospital, Central South University, Changsha, China, 5Monash University, Department of Medicine, Sub-faculty of Clinical and Molecular Medicine, Clayton, Australia, 6Monash University, Department of Medicine, Sub-faculty of Clinical and Molecular Medicine, Melbourne, Australia
Background/Purpose: Regulatory T cells (Tregs) play an important role in maintaining immune system homeostasis. Antigen-specific Tregs potently and specifically suppress autoreactivity, suggesting their potential to be engineered to treat autoimmune diseases. Lupus nephritis (LN) is a severe manifestation of SLE associated with the presence of anti-Smith (Sm) autoantibodies as well as the HLA haplotypes DR15 and DR3, with the majority of patients DR15. Due to the strong association of LN with anti-Sm and HLA-DR15, we developed Sm-specific Tregs for potential treatment of LN.
Methods: We identified DR15-restricted Sm T cell epitopes using a physical affinity binding assay. T cell receptors (TCRs) specific for Sm epitopes were identified by co-culturing CD4+ T cells with dendritic cells from HLA-DR15+ healthy donors and Sm epitopes, and sequencing proliferating CD4+ T cells using high-throughput 10X single cell V(D)J sequencing. TCRs were ranked based on clonal expansion, cloned into a lentiviral vector then transduced onto primary SLE patient Tregs. Sm-specific Tregs were evaluated for suppression of T effector cell function in vitro and in a humanised model of LN created by the transfer of PBMCs from anti-Sm positive HLA-DR15+ SLE patient into NSG-MHC-null mice.
Results: The top three Sm epitopes identified were SmB/B'58-72, SmB/B'1-15, and SmD343-57; the respective stability indices (SI) were 16.0, 1.4 and 1.2. Highly reactive TCRs specific for SmB/B'58-72 were identified and the highest ranked SmB/B'58-72 reactive TCR was cloned into a lentiviral vector then transduced onto primary SLE patient Tregs, resulting in Sm-Tregs. In in vitro co-cultures of SLE patient PBMCs with SmB/B'58-72, Sm-Tregs, but not polyclonal Tregs induced 90% suppression of pro-inflammatory cytokine secretion (IFN-g (pg/mL): control 39.7±11.4, polyclonal Tregs 37.3±12.6, Sm-Tregs 2.7±1.0, p< 0.05; IL-17A (pg/mL): control 26.3±2.9, polyclonal Tregs 17.0±2.7, Sm-Tregs 2.4±0.6, p< 0.01) while Sm-Tregs produced significantly more IL-10 (p< 0.01). Transfer into NSG-MHC-null mice of Sm+ HLA-DR15+SLE patient PBMCs, but not healthy HLA-DR15+ PBMC, induced an LN phenotype characterised by proteinuria and histological glomerular necrosis. Treatment with Sm-Tregs halted the progression of functional and histological injury in this model (proteinuria: control 3.6±0.2, polyclonal Tregs 3.2±0.2, Sm-Tregs 1.2±0.2, p< 0.01; glomerular segmental necrosis: control 75.2±2.9%, polyclonal Tregs 61.2±6.3%, Sm-Tregs 14.4±1.9%, p< 0.001).
Conclusion: Antigen-specific Tregs generated against an immunodominant Sm peptide were highly efficient at suppressing Sm-specific T effector responses. HLDA-DR15+Sm+ SLE patient PBMC induced a model of LN, in which Sm-Tregs were therapeutic. Autologous Sm-Treg cell therapy is a promising treatment for LN, and other antigen-specific autoimmune diseases could be similarly targeted.
E. Morand: AbbVie, 2, 5, Amgen, 5, AstraZeneca, 2, 5, 6, Biogen, 2, 5, Bristol Myers Squibb, 2, 5, Eli Lilly, 2, 5, EMD Serono, 2, 5, Galapagos, 2, Genentech, 2, 5, GlaxoSmithKline, 2, 5, IgM, 2, Janssen, 2, 5, Novartis, 2, Servier, 2, Takeda, 2, UCB, 5; R. Cheong: None; P. Eggenhuizen: Amgen, 5; J. Chang: None; A. Broury: None; B. Ng: None; K. Loh: None; E. Tay: None; C. Shen: None; J. Monk: None; Y. Zhong: None; S. Lim: Bio-Rad, 6, Sony Biotechnology, 6; J. Chung: None; R. Kandane-Rathnayake: None; R. Koelmeyer: None; A. Hoi: Abbvie, 6, AstraZeneca, 5, Australian Rheumatology Association, 4, Eli Lilly, 6, EUSA Pharma (UK) Limited, 2, Limbic, 6, Moose Republic, 6, Novartis, 6; S. Snelgrove: None; Y. Ting: None; J. Ooi: Amgen, 5, 10.
.png "Eric Morand, MD, PhD photo")
