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Increased cathepsin S in Prdm1−/− dendritic cells alters the TFH cell repertoire and contributes to lupus

Nature Immunology volume 18, pages 1016–1024 (2017)Cite this article

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Abstract

Aberrant population expansion of follicular helper T cells (TFH cells) occurs in patients with lupus. An unanswered question is whether an altered repertoire of T cell antigen receptors (TCRs) is associated with such expansion. Here we found that the transcription factor Blimp-1 (encoded by Prdm1) repressed expression of the gene encoding cathepsin S (Ctss), a cysteine protease that cleaves invariant chains and produces antigenic peptides for loading onto major histocompatibility complex (MHC) class II molecules. The increased CTSS expression in dendritic cells (DCs) from female mice with dendritic cell–specific conditional knockout of Prdm1 (CKO mice) altered the presentation of antigen to CD4+ T cells. Analysis of complementarity-determining region 3 (CDR3) regions containing the β-chain variable region (Vβ) demonstrated a more diverse repertoire of TFH cells from female CKO mice than of those from wild-type mice. In vivo treatment of CKO mice with a CTSS inhibitor abolished the lupus-related phenotype and reduced the diversity of the TFH cell TCR repertoire. Thus, Blimp-1 deficiency in DCs led to loss of appropriate regulation of Ctss expression in female mice and thereby modulated antigen presentation and the TFH cell repertoire to contribute to autoimmunity.

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Figure 1: Increased expression of Ctss in DCs from female CKO mice.
Figure 2: Blimp-1 regulates Ctss expression in DCs.
Figure 3: IL-6 has a role in Ctss expression in DCs.
Figure 4: 'Preferential' differentiation of IL-21-producing T cells by co-culture with Blimp-1-deficient DCs in vitro.
Figure 5: The TFH cells of female CKO mice harbor a diverse CDR3 repertoire.
Figure 6: The CTSS inhibitor RO5461111 prevents development of the lupus-like phenotype in female CKO mice.
Figure 7: RO5461111 suppresses the population expansion of TFH cells and germinal-center B cells in CKO mice.

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Acknowledgements

We thank H. Borrero and C. Colon for assistance with the flow cytometry; G. Klein and M. DeFranco RN for recruiting PRDM1-genotyped subjects; and C. Chrysostomou for discussions and assistance in bioinformatics analysis. Supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases, the US National Institutes of Health (R01 AR065209 to S.J.K. and B.D.), the Alliance of Lupus Research (B.D.) and the Defense Threat Reduction Agency (HDTRA 1-12-C-0105 for G.G.).

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Authors and Affiliations

  1. Center for Autoimmune and Musculoskeletal Diseases, The Feinstein Institute for Medical Research, Manhasset, New York, USA

    Sun Jung Kim, Su Hwa Jang & Betty Diamond

  2. Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA

    Sebastian Schätzle & George Georgiou

  3. Immunology, Inflammation, and Infectious Diseases (Disease and Therapeutic Area), Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Basel, Switzerland

    S Sohail Ahmed & Wolfgang Haap

  4. Center for Genomics and Human Genetics, The Feinstein Institute for Medical Research, Manhasset, New York, USA

    Peter K Gregersen

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Contributions

S.J.K. designed and performed experiments, analyzed data and interpreted results; S.S. performed the sequencing experiments and analyzed data; S.S.A. and W.H. provided RO5461111, and critically reviewed the manuscript; S.H.J. performed the ChIP experiments and Il6 promoter assay; P.K.G. provided the samples for the human study; G.G. designed the sequencing experiments and interpreted data; B.D. designed the study and analyzed and interpreted results; S.J.K., S.S., G.G. and B.D. participated in the interpretation of the study and wrote the manuscript, and also provided critical review of the paper; and all contributing authors agreed to the submission of this manuscript for publication.

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Correspondence to Betty Diamond.

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S.S.A. and W.H. are employed by and hold stock in Roche Pharmaceuticals.

Integrated supplementary information

Supplementary Figure 1 Expression of cathepsin-encoding genes in splenic DCs from wild-type or CKO mice.

DCs were purified from the spleens of age matched female wild-type or CKO mice, and RNA was purified for qPCR. Gene specific primers were used for each cathepsin gene and the relative expression of each was calculated by normalization to the level of Hprt. Each dot represents an individual mouse and the bar represents the mean ± SEM (n=5).

Supplementary Figure 2 Increased expression of Il6 by estrogen in BM-DCs.

BM-DCs were differentiated with GM-CSF (200 ng/ml) in hormone-free culture system without or with E2, ICI or E2 and ICI together for 7 days. On day 7, BM-DCs were counted and further cultured with or without LPS (1 μg/ml) for overnight. Total RNA was purified and Il6 transcript was assessed by qPCR. Relative level of Il6 was normalized to housekeeping gene Polr2a. In the box-and-whisker plot, horizontal bars indicate the median, boxes indicate 25th to 75th percentile, and whiskers indicate 10th and 90th percentile (n=7).

Supplementary Figure 3 Diagram of Ctss reporter construct.

Supplementary Figure 4 Gene expression in TFH and non-TFH cells.

Spleens were collected from age-matched female wild-type (open circle) or CKO (closed circle) mice. TFH cells and non-TFH cells were identified as represented in the flow image (left panel), and each population was purified. Total RNA was purified and gene expression was measured by qPCR. Relative level of each gene was normalized to housekeeping gene Polr2a. Each dot represents an individual mouse and the bar represents the mean ± SEM (n=3).

Supplementary Figure 5 Increased IL-21 protein in the supernatant from co-culture of OT-II CD4+ T cells and Blimp-1-deficient DCs.

CD4+ T cells from OT-II mice and splenic DCs (open box is wild-type mice and gray box is CKO mice) were co-cultured in various conditions for 4 days. To measure the secreted cytokines, IL-2 and IL-21, supernatant was collected and cytokines were detected by a multiplex assay. In the box-and-whisker plot, horizontal bars indicate the median, boxes indicate 25th to 75th percentile, and whiskers indicate 10th and 90th percentile (n=6).

Supplementary Figure 6 Cytokine production in the supernatant from day 3 co-cultures of OT-II T cells and DCs.

DCs were purified from the spleens of female wild-type (open box) or CKO (gray box) mice, and CD4+ T cells were purified from female OT-II mice. DCs and T cells were co-cultured for 3 days at a 1:10 ratio (DC:T) with either OVA323-339 peptide (100 ng/ml) or OVA protein (10 μg/ml). Cytokine levels were measured in the supernatant by multiplex assay. In the box-and-whisker plot, horizontal bars indicate the median, boxes indicate 25th to 75th percentile, and whiskers indicate 10th and 90th percentile (n=6).

Supplementary Figure 7 Proliferation and IL-21 production of OT-II T cells by DCs presenting OVA peptide (amino acids 323–339).

OT-II T cells were co-cultured with splenic DCs for 3 days at 1:10 ratio (DC:T cells) in the presence of OVA323-339 peptide. To assess proliferation, T cells were labeled with CFSE (10 mM) before the culture and proliferation was measured by calculation of the division index (left panel). In the box-and-whisker plot, horizontal bars indicate the median, boxes indicate 25th to 75th percentile, and whiskers indicate 10th and 90th percentile (n=3). To assess IL-21 production, culture supernatant was harvested and measured by Multiplex assay (right panel). In the box-and-whisker plot, horizontal bars indicate the median, boxes indicate 25th to 75th percentile, and whiskers indicate 10th and 90th percentile (n=3).

Supplementary Figure 8 Protein level of cytokines, IL-2 and IL-21, measured by intracellular staining of T cells.

OT-II T cells were co-cultured with splenic DCs that had been incubated with HEL (10 μ/ml) or OVA (10 μg/ml). CTSB inhibitor (1 nM) was added during processing of OVA protein antigen. After co-culture for 4 days, T cells were stimulated with PMA (100 ng/ml)/ionomycin (1 μg/ml) for 6 hours. BFA (20 μg/ml) was added during the last 4 hours of stimulation. Cytokine-positive cells were identified by flow cytometry and frequency was calculated and presented in the graph. Each dot represents an individual experiment and the bar represents the mean ± SEM (n=4).

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Kim, S., Schätzle, S., Ahmed, S. et al. Increased cathepsin S in Prdm1−/− dendritic cells alters the TFH cell repertoire and contributes to lupus. Nat Immunol 18, 1016–1024 (2017). https://doi.org/10.1038/ni.3793

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