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Granzyme K activates the entire complement cascade

Nature volume 641, pages 211–221 (2025)Cite this article

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Abstract

Granzymes are a family of serine proteases that are mainly expressed by CD8+ T cells, natural killer cells and innate-like lymphocytes1. Although their primary function is thought to be the induction of cell death in virally infected cells and tumours, accumulating evidence indicates that some granzymes can elicit inflammation by acting on extracellular substrates1. We previously found that most tissue CD8+ T cells in rheumatoid arthritis synovium, and in inflamed organs for some other diseases, express granzyme K (GZMK)2, a tryptase-like protease with poorly defined function. Here, we show that GZMK can activate the complement cascade by cleaving the C2 and C4 proteins. The nascent C4b and C2b fragments form a C3 convertase that cleaves C3, enabling the assembly of a C5 convertase that cleaves C5. The resulting convertases generate all the effector molecules of the complement cascade: the anaphylatoxins C3a and C5a, the opsonins C4b and C3b, and the membrane attack complex. In rheumatoid arthritis synovium, GZMK is enriched in regions with abundant complement activation, and fibroblasts are the main producers of complement proteins that serve as substrates for GZMK-mediated complement activation. Furthermore, Gzmk-deficient mice are significantly protected from inflammatory disease, exhibiting reduced arthritis and dermatitis, with concomitant decreases in complement activation. Our findings describe the discovery of a previously unidentified mechanism of complement activation that is driven entirely by lymphocyte-derived GZMK. Given the widespread abundance of GZMK-expressing T cells in tissues in chronic inflammatory diseases, GZMK-mediated complement activation is likely to be an important contributor to tissue inflammation in multiple disease contexts.

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Fig. 1: GZMK is expressed by CD8+ T cells, NK cells and innate-like T cells in blood and tissues.
Fig. 2: GZMK cleaves the complement components C4 and C2 to generate a C3 convertase that cleaves C3 into C3a and C3b.
Fig. 3: Synovial fibroblasts are major producers of tissue-derived complement proteins that are substrates for GZMK.
Fig. 4: GZMK activates the entire complement cascade.
Fig. 5: GZMK drives complement activation and inflammatory disease in mouse models of arthritis and psoriasis.

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Data availability

CITE-seq and RNA-seq single-cell expression matrices were obtained from the following publicly available datasets: RA synovial cells from dbGaP13 (phs001457.v1.p1), dbGaP10 (phs001529.v1.p1), GEO2 (GSE202375) and Synapse7 (https://www.synapse.org/Synapse:syn26710600/datasets/); SLE kidney cells from dbGaP12 (phs001457.v1.p1); UC colon cells from Broad Institute Single Cell Portal8 (https://singlecell.broadinstitute.org/single_cell/study/SCP259); CD ileum cells from GEO9 (GSE134809); and COVID-19 and healthy bronchoalveolar lavage fluid cells from GEO11 (GSE145926). The bulk RNA-seq data from sorted synovial cell types are available at ImmPort13 (https://www.immport.org/shared/study/SDY998). Source data are provided with this paper.

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Acknowledgements

This work was supported by US NIH grants R01 AR073290 and R01 AR081792 (to M.B.B.), Rheumatology Research Foundation grant 889234 (M.B.B.), NIAMS K08 AR081412 (A.H.J.), Rheumatology Research Foundation Investigator Award (A.H.J.), US NIH grant 5T32AR007098-48 (E.T.) and the Dermatology Foundation Career Development Award (E.T.) We thank V. M. Holers for input and discussions. S.R. is supported by NIH grants 5P01AI148102-03, 5UC2AR081023-02 and 5R01AR063759-08. This work was supported by the Accelerating Medicines Partnership Rheumatoid Arthritis and Systemic Lupus Erythematosus (AMP RA/SLE). The AMP is a public–private partnership (AbbVie, the Arthritis Foundation, Bristol-Myers Squibb, the Foundation for the National Institutes of Health, GlaxoSmithKline, Janssen Research and Development, the Lupus Foundation of America, Lupus Research Alliance, Merck & Co., Sharp & Dohme Corporation, the National Institute of Allergy and Infectious Diseases, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, Pfizer, the Rheumatology Research Foundation, Sanofi and Takeda Pharmaceuticals International) created to develop new ways of identifying and validating promising biological targets for diagnostics and drug development. Funding was provided through grants from the National Institutes of Health (UH2-AR067676, UH2-AR067677, UH2-AR067679, UH2-AR067681, UH2-AR067685, UH2- AR067688, UH2-AR067689, UH2-AR067690, UH2-AR067691, UH2-AR067694 and UM2-AR067678). We thank the NIH Tetramer Core Facility at Emory University for the MR1 tetramers. The MR1 tetramer technology was developed jointly by J. McCluskey, J. Rossjohn and D. Fairlie, and the MR1 tetramers were produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne.

Author information

Author notes

  1. These authors contributed equally: Carlos A. Donado, Erin Theisen

  2. These authors jointly supervised this work: A. Helena Jonsson, Michael B. Brenner

Authors and Affiliations

  1. Division of Rheumatology, Inflammation, and Immunity, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Carlos A. Donado, Erin Theisen, Aparna Nathan, Madison L. Fairfield, Karishma Vijay Rupani, Dominique Jones, Adam Chicoine, Ellen M. Gravallese, Deepak Rao, Joyce B. Kang, Gregory Keras, Yuhong Li, Katherine Liao, Kathryne E. Marks, Joseph R. Mears, Nghia Millard, Yakir Reshef, Laurie Rumker, Saori Sakaue, Kamil Slowikowski, Kathryn Weinand, Dana Weisenfeld, Qian Xiao, Zhu Zhu, Zhihan J. Li, Soumya Raychaudhuri, A. Helena Jonsson & Michael B. Brenner

  2. Department of Dermatology, Brigham and Women’s Hospital, Boston, MA, USA

    Erin Theisen

  3. Division of Rheumatology and Department of Biomedical Informatics, University of Colorado School of Medicine, Aurora, CO, USA

    Fan Zhang

  4. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Aparna Nathan, Michelle Curtis, Maria Gutierrez-Arcelus, Joyce B. Kang, Joseph R. Mears, Nghia Millard, Yakir Reshef, Laurie Rumker, Saori Sakaue, Kamil Slowikowski, Kathryn Weinand, Qian Xiao & Soumya Raychaudhuri

  5. Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA

    Aparna Nathan, Michelle Curtis, Maria Gutierrez-Arcelus, Joyce B. Kang, Joseph R. Mears, Nghia Millard, Yakir Reshef, Laurie Rumker, Saori Sakaue, Kamil Slowikowski, Kathryn Weinand, Qian Xiao & Soumya Raychaudhuri

  6. Center for Data Sciences, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Aparna Nathan, Michelle Curtis, Maria Gutierrez-Arcelus, Joyce B. Kang, Joseph R. Mears, Nghia Millard, Yakir Reshef, Laurie Rumker, Saori Sakaue, Kamil Slowikowski, Kathryn Weinand, Qian Xiao & Soumya Raychaudhuri

  7. Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Aparna Nathan & Soumya Raychaudhuri

  8. Division of Rheumatology, University of Colorado School of Medicine, Aurora, CO, USA

    Kellsey P. Johannes, Michelle Curtis, Kevin D. Deane, V. Michael Holers, Larry Moreland, Jennifer A. Seifert & A. Helena Jonsson

  9. Division of Allergy and Clinical Immunology, Jeff and Penny Vinik Center for Allergic Disease Research, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Daniel F. Dwyer

  10. Division of Allergy, Immunology and Rheumatology, Department of Medicine, University of Rochester Medical Center, Rochester, NY, USA

    Jennifer Albrecht, Jennifer H. Anolik, Jennifer L. Barnas, Brendan F. Boyce, Debbie Campbell, Nida Meednu, Javier Rangel-Moreno, Christopher Ritchlin & Darren Tabechian

  11. Accelerating Medicines Partnership: RA/SLE Network, Bethesda, MD, USA

    William Apruzzese

  12. Division of Rheumatology, Columbia University College of Physicians and Surgeons, New York, NY, USA

    Joan M. Bathon & Laura Geraldino-Pardilla

  13. Division of Rheumatology, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Ami Ben-Artzi, Lindsy Forbess & Michael H. Weisman

  14. Division of Rheumatology, Allergy and Immunology, University of California, San Diego, La Jolla, CA, USA

    David L. Boyle, Arnold Ceponis & Gary S. Firestein

  15. Hospital for Special Surgery, New York, NY, USA

    S. Louis Bridges Jr, Vivian P. Bykerk, Laura T. Donlin, Susan M. Goodman, Lionel B. Ivashkiv, Amit Lakhanpal, Ian Mantel, Dana E. Orange & Anvita Singaraju

  16. Weill Cornell Medicine, New York, NY, USA

    S. Louis Bridges Jr, Vivian P. Bykerk, Laura T. Donlin, Susan M. Goodman, Lionel B. Ivashkiv, Amit Lakhanpal, Ian Mantel, Anvita Singaraju & Melanie Smith

  17. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Michelle Curtis, Maria Gutierrez-Arcelus, Joyce B. Kang, Joseph R. Mears, Nghia Millard, Yakir Reshef, Laurie Rumker, Saori Sakaue, Kamil Slowikowski, Kathryn Weinand & Qian Xiao

  18. Department of Pathology and Laboratory Medicine, Hospital for Special Surgery, New York, NY, USA

    Edward DiCarlo

  19. Division of Allergy, Immunology and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Patrick Dunn

  20. Northrop Grumman Health Solutions, Rockville, MD, USA

    Patrick Dunn

  21. Rheumatology Research Group, Institute for Inflammation and Ageing, University of Birmingham, Birmingham, UK

    Andrew Filer, Hayley Carr, Mark Maybury, Saba Nayar, Karim Raza, Ilfita Sahbudin & Dagmar Scheel-Toellner

  22. Birmingham Tissue Analytics, Institute of Translational Medicine, University of Birmingham, Birmingham, UK

    Andrew Filer, Saba Nayar & Karim Raza

  23. NIHR Birmingham Biomedical Research Center and Clinical Research Facility, University of Birmingham, Queen Elizabeth Hospital, Birmingham, UK

    Andrew Filer, Hayley Carr, Mark Maybury, Ilfita Sahbudin & Dagmar Scheel-Toellner

  24. Feinstein Institute for Medical Research, Northwell Health, Manhasset, New York, NY, USA

    Peter K. Gregersen & Diane Horowitz

  25. Department of Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

    Joel M. Guthridge & Judith A. James

  26. Division of Immunology, Department of Pediatrics, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Maria Gutierrez-Arcelus

  27. Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

    Laura B. Hughes & Felice Rivellese

  28. Laboratory for Human Immunogenetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Kazuyoshi Ishigaki & Felice Rivellese

  29. Department of Surgery, Brigham and Women’s Hospital, Boston, MA, USA

    James A. Lederer

  30. Centre for Experimental Medicine and Rheumatology, EULAR Centre of Excellence, William Harvey Research Institute, Queen Mary University of London, London, UK

    Miles J. Lewis, Alessandra Nerviani & Costantino Pitzalis

  31. Barts Health NHS Trust, Barts Biomedical Research Centre (BRC), National Institute for Health and Care Research (NIHR), London, UK

    Miles J. Lewis, Alessandra Nerviani & Costantino Pitzalis

  32. Division of Rheumatology, Department of Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Arthur M. Mandelin II & Harris Perlman

  33. Department of Biostatistics and Computational Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

    Andrew McDavid

  34. Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Mandy J. McGeachy

  35. Department of Biomedical Sciences, Humanitas University and Humanitas Research Hospital, Milan, Italy

    Costantino Pitzalis

  36. Division of Immunology and Rheumatology, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA

    William H. Robinson & Paul J. Utz

  37. Department of Radiology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

    Andrew Cordle & Aaron Wyse

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Consortia

Accelerating Medicines Partnership RA/SLE Network

  • Jennifer Albrecht
  • , Jennifer H. Anolik
  • , William Apruzzese
  • , Jennifer L. Barnas
  • , Joan M. Bathon
  • , Ami Ben-Artzi
  • , Brendan F. Boyce
  • , David L. Boyle
  • , S. Louis Bridges Jr
  • , Vivian P. Bykerk
  • , Debbie Campbell
  • , Arnold Ceponis
  • , Adam Chicoine
  • , Michelle Curtis
  • , Kevin D. Deane
  • , Edward DiCarlo
  • , Laura T. Donlin
  • , Patrick Dunn
  • , Andrew Filer
  • , Hayley Carr
  • , Gary S. Firestein
  • , Lindsy Forbess
  • , Laura Geraldino-Pardilla
  • , Susan M. Goodman
  • , Ellen M. Gravallese
  • , Deepak Rao
  • , Peter K. Gregersen
  • , Joel M. Guthridge
  • , Maria Gutierrez-Arcelus
  • , V. Michael Holers
  • , Diane Horowitz
  • , Laura B. Hughes
  • , Lionel B. Ivashkiv
  • , Kazuyoshi Ishigaki
  • , Judith A. James
  • , Joyce B. Kang
  • , Gregory Keras
  • , Amit Lakhanpal
  • , James A. Lederer
  • , Miles J. Lewis
  • , Yuhong Li
  • , Katherine Liao
  • , Arthur M. Mandelin II
  • , Ian Mantel
  • , Kathryne E. Marks
  • , Mark Maybury
  • , Andrew McDavid
  • , Mandy J. McGeachy
  • , Joseph R. Mears
  • , Nida Meednu
  • , Nghia Millard
  • , Larry Moreland
  • , Saba Nayar
  • , Alessandra Nerviani
  • , Dana E. Orange
  • , Harris Perlman
  • , Costantino Pitzalis
  • , Javier Rangel-Moreno
  • , Karim Raza
  • , Yakir Reshef
  • , Christopher Ritchlin
  • , Felice Rivellese
  • , William H. Robinson
  • , Laurie Rumker
  • , Ilfita Sahbudin
  • , Saori Sakaue
  • , Jennifer A. Seifert
  • , Dagmar Scheel-Toellner
  • , Anvita Singaraju
  • , Kamil Slowikowski
  • , Melanie Smith
  • , Darren Tabechian
  • , Paul J. Utz
  • , Kathryn Weinand
  • , Dana Weisenfeld
  • , Michael H. Weisman
  • , Qian Xiao
  • , Zhu Zhu
  • , Zhihan J. Li
  • , Andrew Cordle
  •  & Aaron Wyse

Contributions

C.A.D., A.H.J. and M.B.B. conceived the study. C.A.D. designed, performed and analysed the complement cleavage assays, the C3a-mediated degranulation assays, the C3b and C4d deposition and TCC formation assays, the C5a quantification and bioactivity assays and the fibroblast assays. A.H.J. designed and performed the GZMK expression analysis of lymphocytes, the T cell stimulation flow-cytometric assays and the synovial tissue microscopy studies. E.T. performed C3b deposition and TCC formation assays. C.A.D., E.T., A.H.J. and K.P.J. conducted GZMK surface binding studies. C.A.D. and K.V.R. performed and analysed T cell stimulation assays. M.L.F., E.T. and D.J. performed and analysed tissue microscopy studies. E.T. performed arthritis and IMQ dermatitis experiments. A.N. performed computational data analysis of T and NK cell subsets in RA. F.Z. performed computational data analysis of T cells across disease states. The AMP provided unpublished single-cell transcriptional and proteomic data from a cohort of patients with RA and osteoarthritis. S.R. supervised the computational analyses, including the processing and analysis of unpublished data from the AMP. D.F.D. provided cell lines and advice on experimental design and analysis. C.A.D., E.T., A.H.J. and M.B.B. wrote the manuscript with assistance from all other authors. M.B.B. supervised experiments and data analysis.

Corresponding author

Correspondence to Michael B. Brenner.

Ethics declarations

Competing interests

M.B.B. is a consultant to GSK, Third Rock Ventures, 4FO Ventures and Moderna and a consultant to and founder of Mestag Therapeutics. S.R. is a founder of Mestag Therapeutics, a scientific adviser for Janssen and Pfizer, and a consultant to Gilead and Rheos Medicine. D.F.D. is a consultant to Celldex Therapeutics. The remaining authors declare no competing interests.

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Nature thanks Paul Barlow, Peter A. Ward and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 GZMK is expressed by CD8+ T cells in many different inflamed and non-inflamed tissues.

a, Expression of T cell subset markers CCR7 and CD45RA by GZMK+ CD4+ and CD8+ T cells, respectively. Data are mean ± s.d. b, Expression of selected markers in UMAP space for the integrative dataset presented in Fig. 1g. c, Contribution of each of the six publicly available single-cell RNA-seq datasets to the integrated dataset of CD4+, CD8+, and NK cell profiles. d, Expression levels of selected genes by cell profiles the integrative dataset in UMAP space. e, Percentage of cells in all CD4+ T cell clusters (gray columns) or all CD8+T cell clusters (blue columns) with detectable GZMB gene expression, stratified by tissue and disease source. f, Aggregate data showing frequency of intracellular GZMK and GZMB staining among purified primary human CD8+ T cells cultured either in media alone (unstimulated) or with anti-CD3/CD28 antibody-coated beads for four days. Data in f show mean ± s.d. of four donors from a representative out of four independent experiments.

Extended Data Fig. 2 GZMK is highly homologous to complement factor D, C1s, and MASP1 but does not cleave factor B.

a, Results of a protein blast comparing the protein sequence of GZMK to all human protein sequences. b,d,e, Structural alignments showing the structural similarity between GZMK (cyan) and (b) complement factor D (CFD) (gray), (d) C1s (yellow, catalytic domain) and (e) MASP1 (green, catalytic domain). Alignments on the bottom show a close up of the catalytic triad residues in the active site of GZMK, CFD, C1s and MASP1. The catalytic residues in GZMK (H67, D116, S214) are labeled, while the corresponding catalytic triad residues in CFD (H66, D14, S208), C1s (H475, D529, S632) and MASP1 (H490, D552, S646) are shown but not labeled. Note that the catalytic serine in the structures of GZMK and CFD is mutated to alanine. c, Serum-purified complement factor B (CFB) was incubated with either CFD or increasing concentrations of GZMK in the presence or absence of C3b and cleavage products were analyzed by immunoblot. Serum-purified Bb was used as a control to identify cleavage of CFB into Bb. (c) Data are representative of three independent experiments.

Extended Data Fig. 3 GZMK cleaves C4 and C2 into C4b and C2b but does not directly cleave C3.

a, Densitometric analysis of the immunoblot shown in Fig. 2b depicts a dose-dependent increase in the cleavage of C4 into C4b as more GZMK is incubated with C4. b, Densitometric analysis of the immunoblot shown in Fig. 2c depicts a dose-dependent increase in the cleavage of C2 into C2b as more C4 is incubated with GZMK and C2. c,d, Densitometric analysis of the immunoblots shown in Fig. 2d and Fig. 2e showing a dose-dependent increase in the generation of (c) C3a and (d) C3b as more GZMK is incubated with C2 + C3 + C4. e, Serum-purified C3 was incubated with increasing concentrations of GZMK and cleavage products were assessed by immunoblot. As a positive control for C3 cleavage, C3 was incubated with C3b + CFB + CFD in the presence of increasing concentrations of properdin. Serum-purified C3b was used to confirm the presence of the C3b cleaved fragment. (a-e) Data are representative of at least three independent experiments.

Extended Data Fig. 4 GZMK binds plasma membranes to trigger formation of membrane-bound C3 convertases.

a, HUVEC cells, synovial fibroblasts and THP-1 monocytes were left untreated or were treated with an isotype control or a cell-type specific sensitizing antibody (anti-HLA-A,B,C for fibroblasts, anti-CD31 for HUVEC and THP-1 cells). Cells were then incubated with either C1s or the C1 complex and surface C1s was measured by flow cytometry. b, HUVEC cells were incubated for 4 h with serum-purified C2 + C3 + C4 alone or in combination with GZMK or GZMA, and C3b deposition was measured by flow cytometry. Histograms depict representative data. Aggregate data is shown in Fig. 4c. c, Association plots of the geometric mean fluorescence intensity of surface staining of anti-heparan sulfate and anti-granzyme K antibodies from three HUVEC donors and cell subsets from four PBMC donors. Graph on the right shows PBMC cell subsets only. Statistics by Spearman correlation. d, Histograms of anti-heparan sulfate (left) and anti-granzyme K (right) antibody binding to the surfaces of unfixed, live cells of the indicated subset from a representative donor. Gray histograms depict cells in media; red histograms depict cells incubated with exogenous recombinant GZMK. e, HUVEC cells were incubated for 4 h with serum-purified C2 + C3 + C4 alone or in combination with GZMK in the presence or absence of heparin and surface-bound GZMK and C3b were measured by flow cytometry. Aggregate data is shown in Fig. 4d. f, Densitometric analysis of the immunoblots shown in Fig. 4e depicting that GZMK is more efficient at cleaving C4, C2 and eliciting the generation of C3a when it is bound to membranes than when it is in the fluid phase. (a,b,e,f) Data are representative of at least three independent experiments.

Extended Data Fig. 5 GZMK triggers formation of C5 convertases that generate bioactive C5a and the terminal complement complex (TCC).

a, C5aR1-expressing Chem-1 reporter cells labeled with Fluo-5F were incubated with C5, C5a or the supernatants obtained after incubating HUVEC cells with C2 + C3 + C4 + C5 with or without GZMA or GZMK. Calcium flux was immediately assessed by flow cytometry. Aggregate data is shown in Fig. 4g. b, HUVEC cells were incubated with serum-purified C2 + C3 + C4 + C5 + C6 + C7 + C8 + C9 with increasing amounts of GZMK or GZMA in the presence of dynasore to inhibit endocytosis. C5b,6-9 was used as the positive control. Terminal complement complex formation (TCC) was then measured by flow cytometry. Data are mean ± s.d of three independent experiments. P values were calculated using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons tests. NS = not significant. c, Surface deposition of C3b (left), C4d (middle) and TCC formation (right) on HUVEC cells after incubation with C1q-depleted serum and increasing concentrations of GZMK, as measured by flow cytometry. NHS was used as a positive control. Histograms depict representative data. Aggregate data is shown in Fig. 4i. All data are representative of at least three independent experiments.

Extended Data Fig. 6 In RA synovial tissue complement activation products are abundant in areas rich in GZMK.

a, Representative image showing immunofluorescence staining of RA synovial tissue with antibodies against C3d (clone C3D/2891, yellow in top composite), C5a (clone 2952, yellow in bottom composite), and GZMK (red) as well as Hoechst nuclear stain (blue). The area inside the dashed box is shown enlarged in Fig. 5a. Scale bar is 50 microns. b, Representative image showing immunofluorescence staining of RA synovial tissue with antibodies against GZMK (red) and C3d (clone 7C10, yellow) as well as Hoechst nuclear stain (blue). Scale bar is 50 microns. c, Enlarged view of area inside box in panel a. Scale bar is 25 microns. d, Representative image showing immunofluorescence staining of RA synovial tissue with antibodies against GZMK (red) and C5a (clone 2942, yellow) as well as Hoechst nuclear stain (blue). Scale bar is 50 microns. e, Enlarged view of area inside box in panel a. Scale bar is 25 microns. All images in this panel are tiled images collected on a confocal microscope. Data are representative of (a-c) three and (d,e) two independent experiments.

Extended Data Fig. 7 Gzmk-deficient mice have reduced swelling and complement activation in the wrists following induction of arthritis.

a, Littermate Gzmk+/− animals have less severe wrist swelling following treatment with mBSA than Gzmk−/− animals. b, Representative entire wrist joint H&E and immunofluorescence images with antibody staining for C3d (magenta), C4d (yellow) and Hoechst nuclear stain (blue) of Gzmk+/+ (top) or Gzmk−/− (bottom) wrist synovium at day 3 post intra-articular mBSA injection. White and yellow boxes depict enlarged images shown in Fig. 5d. ROIs for quantification of C3d and C4d were made by outlining synovium and surrounding inflammation on the Hoechst channel and overlaying a grid before being applied to corresponding C3d and C4d channels. Scale bar represents 500 um. (a) Data is mean ± of 7 mice per genotype and are representative of 3 independent experiments with significance calculated by area under the curve analysis. (b) Data are representative of 3 mice per genotype.

Source Data

Extended Data Fig. 8 Gzmk-deficient mice have reduced swelling and complement activation in the knees following induction of arthritis.

a, Gzmk+/+ mice have more severe swelling in mBSA-treated knees than Gzmk−/− mice. b, Quantification of C3d and C4d deposition in knee synovium at day 3 post intra-articular mBSA injection. c, Representative enlarged H&E and immunofluorescence images with antibody staining for C3d (magenta), C4d (yellow) and Hoechst nuclear stain (blue) of Gzmk+/+ (top) or Gzmk−/− (bottom) knee synovium at day 3 post intra-articular mBSA injection. Scale bar represents 50um. d, Representative entire knee joint H&E and immunofluorescence images with antibody staining for C3d (magenta), C4d (yellow) and Hoechst nuclear stain (blue) of Gzmk+/+ (top) or Gzmk−/− (bottom) knee synovium at day 3 post intra-articular mBSA injection. White and yellow boxes depict enlarged images shown in (c). ROIs for quantification of C3d and C4d were made by outlining synovium and surrounding inflammation on the Hoechst channel and overlaying a grid before being applied to corresponding C3d and C4d channels. Scale bar represents 500 um. (a) Data are mean ± s.e.m of 6 mice per genotype and are representative of two independent experiments. Significance was calculated using area under the curve analysis. (b) Data are the combination of 3 mice per genotype, with the dashed line depicting the median and dotted lines representing the 25th and 75th quartiles. Significance was calculated using a two-way t test with Welch’s correction. (c,d) Data are representative of 3 mice per genotype. ns = not significant.

Source Data

Extended Data Fig. 9 Gzmk-deficient mice are protected from IMQ-induced dermatitis and complement activation.

a, Littermate IMQ-treated Gzmk−/− mice have less severe total clinical scores, erythema, scaling, and thickness scores, and decreased thickening of back skin as measured by caliper compared to IMQ-treated Gzmk+/− mice. b, Representative skin H&E and immunofluorescence images with antibody staining for C3d (magenta), C4d (yellow) and Hoechst nuclear stain (blue) of Gzmk+/+ (top) or Gzmk−/− (bottom) after 5 daily applications of IMQ. White and yellow boxes represent enlarged images shown in Fig. 5h. ROIs for quantification of C3d and C4d were made by outlining the skin, overlaying a grid, and selecting squares that contained dermal-epidermal junction based on the Hoechst channel before being applied to corresponding C3d and C4d channels. Scale bars represent 500 um. (a) Data shown are mean ± s.e.m. and are representative of 4 independent experiments. Significance was calculated by multiple two-tailed t-tests with a false discovery rate of 5% using the method of Benjamini, Krieger and Yekutiel. (b) Data are representative of 3 mice per genotype.

Source Data

Extended Data Fig. 10 GZMK activates the entire complement cascade.

a. Model of GZMK-mediated complement activation. CD8+ T cells constitutively release GZMK independently of TCR stimulation. GZMK binds plasma membranes via interactions with heparan sulfate glycosaminoglycans (HSGAGs), where it cleaves C4 and C2 to generate C4b and C2b. The proximity of newly cleaved C4b to the membrane enables its covalent attachment via an exposed thioester, facilitating the formation of a membrane-bound C3 convertase upon association with C2b. This convertase cleaves C3 into C3a and C3b, with C3b either serving as an opsonin or associating with membrane-bound C3 convertases to form C5 convertases. These C5 convertases cleave C5 into C5a and C5b, the latter initiating the sequential recruitment of C6, C7, C8 and C9 to assemble the terminal complement complex (TCC). b. Comparison between the alternative, classical, lectin and GZMK-mediated complement activation pathways. Activation of the classical and lectin pathways unfolds in three sequential phases: recognition, initiation and execution. Soluble pattern recognition receptors, such as C1q and mannose-binding lectin, spearhead this process by detecting danger signals on target surfaces. This recognition event triggers the activation of initiator proteases, including C1s and MASP1, which cleave C4 and C2 to assemble C3 and C5 convertases. These convertases drive the execution phase, cleaving C3 and C5 to generate the effector molecules of the complement cascade. In contrast to these canonical mechanisms of complement activation, GZMK independently orchestrates both recognition and initiation, bypassing the need for soluble pattern recognition receptors. Through its intrinsic affinity for HSGAGs, GZMK binds directly to target surfaces. Like C1s and MASP1/2, GZMK functions as an initiator protease that cleaves C4 and C2 into C4b and C2b, assembling C3 and C5 convertases that propagate the cascade. This process enables recruitment of the alternative complement pathway, amplifying the response and enhancing its functional impact. Schematics were made using BioRender with the following publication licences: https://biorender.com/q53k807 (a) and https://biorender.com/w71n868 (b).

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Supplementary Figures 1–7 show representative flow-cytometry gating and uncropped images of western blots.

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Donado, C.A., Theisen, E., Zhang, F. et al. Granzyme K activates the entire complement cascade. Nature 641, 211–221 (2025). https://doi.org/10.1038/s41586-025-08713-9

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