Autoimmune Disease Deconstruction:

We have implemented a disease deconstruction pipeline using single cell and spatial technologies applied to inflamed tissues in humans to discover new cell types, cell states and pathways that mediate autoimmune disease. We then use in vitro organoids, reductionist approaches, and genetically altered mouse models to define mechanisms and therapeutic insights for the new pathways and cell states. By shifting between humans and mice, our studies unravel the most relevant autoimmune and inflammatory abnormalities in vivo and then interrogate them in experimental systems. Over the past few years, we have identified new pathogenic T cell populations, macrophage states and stromal cell subsets that drive pathology in autoimmune disease. Below we described several examples.

New pathological T helper cell subset (Tph cells) drives autoantibody production:

We carried out mass cytometry (CyTOF) for deep immunophenotyping and unbiased clustering using single cell RNA sequencing of synovial tissues in rheumatoid arthritis (RA). From these approaches, we identified a new population of T helper cells that we have named T peripheral helper (Tph) cells, to distinguish them from T follicular helper (Tfh) cells. Tph cells are found in leukocyte aggregates in chronic inflammatory peripheral tissues where they drive autoantibody production. Tph cells express high levels of PD1 and ICOS, like Tfh cells, but have low expression of Bcl6 and instead of expressing CXCR5 and localizing in lymph node germinal centers, they express CCR2 and home to peripheral tissues where they secrete CXCL13, the ligand for CXCR5 and IL-21, and drive T-B clusters and provide help to B cells in peripheral autoimmune lesions. Since our initial studies, Tph cells have been found to be expanded in a range of inflammatory diseases including lupus, autoimmune hepatitis, Celiac disease, juvenile idiopathic arthritis (JIA) and other conditions where autoantibodies are present.

1. Rao DA, Gurish MF, Marshall JL, Slowikowski K, Fonseka CY, Liu Y, Donlin LT, Henderson LA, Wei K, Mizoguchi F, Teslovich NC, Weinblatt ME, Massarotti EM, Coblyn JS, Helfgott SM, Lee YC, Todd DJ, Bykerk VP, Goodman SM, Pernis AB, Ivashkiv LB, Karlson EW, Nigrovic PA, Filer A, Buckley CD, Lederer JA, Raychaudhuri S, Brenner MB. Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Natur 2017; 542:110-114. PMCID: PMC534932

Granzyme K (GzmK) CD8 T cells are the dominant tissue CD8 T cell phenotype in inflamed tissues. They drive a newly identified pathway of tissue complement activation.

While most previous studies on CD8 T cells have focused on the role of CTL (cytolytic T lymphocytes) that express granzyme B, using single cell RNA sequencing of inflamed tissue we found that the major phenotype of tissue CD8 T cells expressed high levels of granzyme K (GzmK) either without or with low levels of GzmB. In fact, GzmK CD8 T cells dominate in the inflamed tissues not only in the RA synovium, but also in Crohn’s Disease and ulcerative colitis gut, lupus nephritis kidney and even in viral infection such as COVID-19 pneumonia. This surprising finding led us to ask what GzmK+ CD8 T cells do. First, we found that rather than CD4+ T cells, these CD8 T cells are the dominant T cell source of IFN in RA synovium. More remarkably, we found that GzmK mediates a new pathway of complement activation. GzmK cleaves both complement factor 4 and factor 2 to generate an active C3 convertase (4b2a) which cleaves factor 3 to yield C3a (the anaphylatoxin) and CD3b (the opsonin) with consequent activation of downstream complement mediated inflammation. In inflamed tissues, stromal fibroblasts produce large amounts of C2, C3 and C4 which are the source of complement acted on by locally produced GzmK. This represents a tissue focused process that differs from the previously known pathways of complement activation. First, in contrast to serum complement derived from the liver, here complement is produced locally in tissues; and second, compared to the proteases that initiate the classical pathway (C1r, C1s), the alternative pathway (CFB) or the lectin pathway (MASP1, MASP2), it is lymphocyte derived GzmK that drives this new pathway of complement activation (Donado et al submitted).

1. Jonsson AH, Zhang F, Dunlap G, Gomez-Rivas E, Watts GFM, Faust HJ, Vijay Rupani K, Mears JR, Meednu N, Wang R, Keras G, Coblyn JS, Massarotti EM, Todd DJ, Anolik H, McDavid A, Accelerating Medicines Partnership (AMP): RA/SLE Network, Wei K, Rao DA, Raychaudhuri S, Brenner MB. Granzyme K+ CD8 T cells form a core population in inflamed human tissue. Science Translational Medicine 2022; PMID: 35704599 DOI: 10.1126/scitranslmed.abo0686

Super-activated macrophages:

Macrophages regulate protective immune responses to infectious microbes, but aberrant macrophage activation frequently drives pathological inflammation. To identify regulators of pathological macrophage activation, we analyzed RNA-seq data from human autoimmune disease tissues and identified SLAMF7 as a receptor associated with a super-activated macrophage state in rheumatoid arthritis. This occurs by a two-step process in which IFN exposure upregulates SLAMF7 expression following which homotypic engagement of SLAMF7 by SLAMF7 on other cells, drives an exuberant wave of inflammatory cytokine expression exceeding that of typically activated macrophages that we call the super-activated macrophage state. We observed this SLAMF7-induced super-activated macrophage state across a range of diseases including rheumatoid arthritis, Crohn’s disease, and severe COVID-19 pneumonia patients. This suggests a central role for SLAMF7 in macrophage super-activation with broad implications in pathology.

1. Simmons DP, Nguyen HN, Gomez-Rivas E, Jeong Y, Jonsson AH, Chen AF, Lange JK, Dyer GS, Blazar P, Earp BE, Coblyn JS, Massarotti EM, Sparks JA, Todd DJ; Accelerating Medicines Partnership (AMP) RA/SLE Network, Rao DA, Kim EY, Brenner MB. SLAMF7 engagement superactivates macrophages in acute and chronic inflammation. Sci. Immunol. 2022; 7:eabf2846. PMCID: PMC8991457

Pathologically expanded fibroblastic stromal cells in RA drive inflammation and tissue damage.

While fibroblastic stromal cells were known to play key roles in regulating immune responses in lymph nodes and tumors, our studies are defining their central roles in chronic inflammation in peripheral tissues. Using RA as an example, we defined subsets of synovial fibroblasts that are inflammatory and are expanded in RA that express high levels of MHC class II. The expanded population accounts for over half of all fibroblasts in the disease, and they are primarily found in the sublining region of the synovium. These fibroblasts produce nearly all the IL-6 and most of the chemokines made in the inflamed tissue, higher levels than produced by leukocytes (Mizoguchi et al. Nat. Comm. (2019), Fan et al. Nat. Immunol. (2019). Importantly, we noted that a particular inflammatory subpopulation of fibroblasts was oriented around blood vessels. We showed that blood vessel endothelial cell-derived Notch ligands drive Notch signaling on the fibroblasts which imparts their sublining phenotype and that this pathway is essential for inflammatory arthritis as either deletion or blockade of the pathway abrogated inflammatory arthritis in mouse models (Wei et al. Nature (2020). We then defined the inflammatory fibroblast populations that are shared across multiple tissues and diseases (including RA, IBD, and Sjogren’s Disease) and found the Notch signaled fibroblast populations to be shared across diseases (Korsunsky et al Med 3: 481 (2022).

In identifying the pathways that activate stromal cell populations in autoimmune diseases, we defined signal specific gene expression modules in fibroblasts that regulate their secretion of IL-6 in chronically inflamed tissues. IL-6 is part of a larger program of inflammatory factors and transcription factor regulators that are co-expressed as an inflammatory module dependent upon an autocrine feedback loop mediated by cell surface IL-6 receptor family member, LIFR (leukemia inhibitory factor receptor. No matter what exogenous factors activate fibroblasts (e.g. TNF, IL-17, IL-1, LPS) the full activation response is dependent on autocrine induction of LIF which acts on the LIFR to profoundly amplify the activation response. Blocking the LIF-LIFR axis prevents fibroblast activation across a range of stimuli. We are now altering autocrine amplification pathways in therapeutic approaches to target fibroblast induced inflammation and pathology.

1. Mizoguchi F, Slowikowski K, Wei K, Marshall JL, Rao DA, Chang SK, Nguyen HN, Noss EH, Turner JD, Earp BE, Blazar PE, Wright J, Simmons BP, Donlin LT, Kalliolias GD, Goodman SM, Bykerk VP, Ivashkiv LB, Lederer JA, Hacohen N, Nigrovic PA, Filer A, Buckley CD, Raychaudhuri S, Brenner MB. Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis. Nat Commun. 2018; 9:789. PMCID: PMC5824882
2. Wei K, Korsunsky I, Marshall JL, Gao A, Watts GFM, Major T, Croft AP, Watts J, Blazar PE, Lange JK, Thornhill TS, Filer A, Raza K, Donlin LT; Accelerating Medicines Partnership Rheumatoid Arthritis & Systemic Lupus Erythematosus (AMP RA/SLE) Consortium, Siebel CW, Buckley CD, Raychaudhuri S, Brenner MB. Notch signalling drives synovial fibroblast identity and arthritis pathology. Nature 2020; 582:259-264. PMCID: PMC7841716
3. Nguyen HN, Noss EH, Mizoguchi F, Huppertz C, Wei KS, Watts GF, Brenner MB. Autocrine loop involving IL-6 family member LIF, LIF receptor, and STAT4 drives sustained fibroblast production of inflammatory mediators. Immunity 2017; 46:220-232. PMCID: PMC5567864
4. Zhang F, Wei K, Slowikowski K, Fonseka CY, Rao DA, … Donlin LT, Anolik JH, Brenner MB, Raychaudhuri S. (* co-first, co-last authors) Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nat. Immunol. 2019; 20:928–942. PMID: 31061532 PMCID: PMC6602051

Earlier classical discoveries from the Brenner lab:

Discovery of T cells and some of their functional roles:

Discovery of the T cell receptor (TCR) proteins and genes was one of the most important advances in immunology. During that time, a third rearranging gene, initially thought to be TCR, but later renamed TCR, was identified. It was initially considered a vestigial gene as all the rearrangements in T cells were defective. The Brenner lab revealed that an entirely separate lineage of T cells existed that expressed a TCR distinct from TCR. We found that one of the CD3-associated chains on these T cells was the functional protein product of the TCR gene, and we found another protein associated in the complex that we named TCR. We showed that T cells were a separate population of mature effector T cells that lacked expression of functionally rearranged TCR or TCR genes. We defined many of key functional characteristics of these cells including the fact that they did not recognize peptide-MHC presented antigens. We were the first to propose and identify that their recognition focused on nonpeptide antigens, an idea that has now grown to be a common theme for all 3 groups of innate T cells (T cells, CD1 restricted T cells (below) and MAIT cells). Most recently we found that an invariant subpopulation of T cells that produces IL-17 and expresses PLZF is highly enriched in adipose tissue where it critically regulates Treg cell numbers and adaptive thermogenesis. Mice lacking these T cells cannot maintain body temperature and die in the cold. Example publications below:

1. Brenner MB, McLean J, Dialynas DP, Strominger JL, Smith JA, Owen FL, Seidman JG, Ip S, Rosen F, Krangel MS. Identification of a putative second T-cell receptor. Nature 1986; 322:145-149.
2. Brenner MB, McLean J, Scheft H, Riberdy J, Ang S-L, Seidman JG, Devlin P, Krangel MS. Two forms of the T-cell receptor protein found on peripheral blood cytotoxic T lymphocytes. Nature 1987; 325:689-694.
3. Band H, Hochstenbach F, McLean J, Hata S, Krangel MS, Brenner MB. Immunochemical proof that a novel rearranging gene encodes the T cell receptor subunit. Science 1987; 238:682-684.
4. Kohlgruber AC, Gal-Oz S-T, LaMarche NM, Shimazaki M, Duquette D, Nguyen HN, Mina AI, Paras T, Tavakkoli A, von Andrian U, Banks AS, Shay T, Brenner MB* and Lynch L* T cells producing interleukin-17A regulate adipose regulatory T cell homeostasis and thermogenesis Nat. Immunol. 2018;19, 464-474. PMID: 29670241 (*authors contributed equally)

Discovery of the pathway of CD1-based presentation of lipid antigens to T cells and some of their functions:

The paradigm for T cell recognition was based on the premise that MHC Class I and Class II antigen presenting molecules present peptides for recognition by TCRs. Thus, it seemed almost impossible when we reported a series of papers first indicating that (and) T cells could recognize foreign antigens in the context of CD1 proteins (which are not encoded in the MHC). Even more remarkable was our finding that the antigens presented were lipids, not proteins. We defined many of the mechanisms that allow the CD1 proteins to intersect with and bind lipid antigens in endosomes. CD1a localizes to and surveys the early endocytic compartment, CD1b and d localize and survey late endosomes and lysosomes, while CD1c promiscuously surveys to bind lipid antigens throughout the endocytic system. We showed how the saposins use different mechanisms to load lipids into CD1 proteins. Together, these and related studies outlined the existence of an independent antigen presentation system for T cells where the universe of lipid rather than peptide antigens are recognized by T cells.

In more recent studies on iNKT cells, we found that self-lipid antigens activate iNKT cells to upregulate FASL which signals IL-1 release from macrophages without inflammasome activation. This important 2-cell pathway enables IL-1 release from infected APCs when microbes can evade inflammasome activation (Donado et al. Cell Reports 2020; 31,107466). Furter, we defined a regulatory population of iNKT cells that controls inflammation in adipose tissue by regulating Tregs and M2/M1 macrophage homeostatsis and regulates thermogenesis and obesity (Lynch et al 2015) and then found the remarkable ability of adipose iNKT cells to regulate fat burning, weight loss and thermogenesis (Lynch et al 2016). We have defined distinct subsets of iNKT cells in adipose tissue that 1) produce IL-10 as a result of an XBP1s ER stress pathway and 2) produce INF which activates NK cells to kill macrophages and control inflammation at steady state (LaMarche et al 2020). Example publications below:

1. Beckman EM, Porcelli SA, Morita CT, Behar SM, Furlong ST, and Brenner MB. Recognition of a lipid antigen by CD1-restricted + T cells. Nature 1994; 372:691-694.
2. Moody DB, Young DC, Cheng T-Y, Rosat J-P, Roura-mir C, O’Connor PB, Zajonc DM, Walz A, Miller MJ, Levery SB, Wilson IA, Costello CE, Brenner MB. T cell activation by lipopeptide antigens. Science 2004; 303:527-531.
3. van den Elzen P, Garg S, León L, Brigl M, Leadbetter EA, Gumperz JE, Dascher CC, Cheng T-Y, Sacks FM, Illarionov PA, Besra GS, Kent SC, Moody DB, and Brenner MB. Apolipoprotein-mediated pathways of lipid antigen presentation. Nature 2005; 437:906-910.
4. Lynch L, Michelet X, Zhang S, Brennan PJ, Moseman A, Lester C, Besra G, Vomhof-Dekrey EE, Tighe M, Koay H-F, Godfrey DI, Leadbetter EA, Sant'Angelo DB, von Andrian U, Brenner MB. Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of Treg cells and macrophages in adipose tissue. Nat Immunol. 2015; 16:85-95. PMCID: PMC4343194
5. Lynch L, Hogan AE, Duquette D, Lester C, Banks A, LeClair K, Cohen DE, Ghosh A, Lu B, Corrigan M, Stevanovic D, Maratos-Flier E, Drucker DJ, O'Shea D, Brenner M. iNKT Cells Induce FGF21 for Thermogenesis and Are Required for Maximal Weight Loss in GLP1 Therapy. Cell Metab. 2016 24:510-519. PMCID: PMC5061124
6. Donado CA, Cao AB, Simmons DP, Croker BA, Brennan PJ, Brenner MB. A Two-Cell Model for IL-1 Release Mediated by Death-Receptor Signaling. Cell Rep. 2020; 31:107466. PMCID: PMC7192215

LaMarche NM, Kane H, Kohlgruber AC, Dong H, Lynch L, Brenner MB. Distinct iNKT Cell Populations Use IFN or ER Stress-Induced IL-10 to Control Adipose Tissue Homeostasis. Cell Metab. 2020; 32:243-258. PMCID: PMC8234787