T cell receptor (TCR) sequences are exceptionally diverse and can now be comprehensively measured with next generation sequencing technologies. However, a thorough investigation of longitudinal TCR repertoires throughout childhood in health and during development of a common childhood disease, type 1 diabetes (T1D), has not been undertaken. Here, we deep-sequenced the TCR beta chain (TCRβ) repertoires from longitudinal peripheral blood DNA samples at four time points beginning early in life (median age of 1.4 years) from children that progressed to T1D (n=29) and age/sex-matched islet autoantibody negative controls (n=25). From 53 million TCRβ sequences, we show that the repertoire is extraordinarily diverse early in life and narrows with age independent of disease. We demonstrate the ability to identify specific TCR sequences, including those known to recognize influenza A and separately those specific for insulin and its precursor, preproinsulin. Insulin-reactive TCRβ sequences are more common and frequent in number as the disease progressed in those who developed T1D compared to genetically at-risk non-diabetic children, which was not the case for influenza-reactive sequences. As an independent validation, we sequenced and analyzed TCRβ repertoires from a cohort of new-onset T1D patients (n=143), identifying the same preproinsulin-reactive TCRs. These results demonstrate an enrichment of preproinsulin-reactive TCR sequences during the progression to T1D, highlighting the importance of using disease-relevant TCR sequences as powerful biomarkers in autoimmune disorders.
Angela M. Mitchell, Erin E. Baschal, Kristen A. McDaniel, Kimber M. Simmons, Laura Pyle, Kathleen Waugh, Andrea K. Steck, Liping Yu, Peter A. Gottlieb, Marian J. Rewers, Maki Nakayama, Aaron W. Michels
Cub domain-containing protein 1 (CDCP1) is a surface protein highly expressed on the surface of many cancer cells, however, the distribution of CDCP1 in normal tissues and its potential roles in non-tumor cells are poorly understood. We previously reported that CDCP1 interacts with CD6, a surface marker of T cells, suggesting that it is a novel immunoregulator, but the physiological significance of the newly discovered CDCP1-CD6 interaction remains unclear. In this report, we found that CDCP1 is present on both human and mouse retinal pigmented epithelial cells (RPEs), a component of the blood-retina barrier (BRB), using a new anti-CDCP1 monoclonal antibody that we developed. CDCP1 knockout (KO) mice on two different genetic backgrounds both developed significantly attenuated retinal T cell infiltration and uveitis after adoptive transfer of pre-activated pathogenic T cells in a model of autoimmune uveitis. We also found that tight junctions were severely disrupted with infiltrating T cells detected in the RPE flat mounts prepared from the WT but not CDCP1 KO mice during EAU development. Mechanistically, we discovered that CDCP1 on RPE was upregulated by IFNγ in vitro and after EAU induction in vivo. CD6 stimulation induced significantly increased RPE barrier permeability of WT, but not CDCP1 knockdown (KD) RPE, and activated T cells migrated through the WT RPE monolayes more efficiently than the CDCP1 KD RPE monolayers. In addition, CD6 stimulation of WT, but not the CDCP1 KD RPEs, induced massive stress fiber formation and focal adhesion disruption to reduce cell barrier tight junctions. These data suggest that CDCP1 on RPEs interacts with CD6 on T cells to induce RPE cytoskeleton remodeling and focal adhesion disruption, which open up the tight junctions to facilitate T cell infiltration for the development of uveitis.
Lingjun Zhang, Nozha Borjini, Yu Lun, Sweta Parab, Gospel Enyindah-Asonye, Rupesh Singh, Brent A. Bell, Vera L. Bonilha, Andrei I. Ivanov, David A. Fox, Rachel R. Caspi, Feng Lin
Checkpoint inhibitors (CPIs) targeting programmed death-1(PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have revolutionized cancer treatment but can trigger autoimmune complications including CPI-induced diabetes (CPI-DM), which occurs preferentially with PD-1 blockade. We found evidence of pancreatic inflammation in patients with CPI-DM with shrinkage of pancreases, increased pancreatic enzymes, and in a case from a patient who died with CPI-DM, peri-islet lymphocytic infiltration. In the NOD mouse model, anti-PD-L1 but not anti-CTLA-4 induces diabetes rapidly. RNA sequencing revealed that cytolytic IFNγ+ CD8+ T cells infiltrated islets with anti-PD-L1. Changes in β cells were predominantly driven by IFNγ and TNFα and included induction of a potentially novel β cell population with transcriptional changes suggesting dedifferentiation. IFNγ increased checkpoint ligand expression and activated apoptosis pathways in human β cells in vitro. Treatment with anti-IFNγ and anti-TNFα prevented CPI-DM in anti-PD-L1 treated NOD mice. CPIs targeting the PD-1/PD-L1 pathway result in transcriptional changes in β cells and immune infiltrates that may lead to the development of diabetes. Inhibition of inflammatory cytokines can prevent CPI-DM, suggesting a strategy for clinical application to prevent this complication.
Ana Luisa Perdigoto, Songyan Deng, Katherine C. Du, Manik Kuchroo, Daniel B. Burkhardt, Alexander Tong, Gary Israel, Marie E. Robert, Stuart P. Weisberg, Nancy Kirkiles-Smith, Angeliki M. Stamatouli, Harriet M. Kluger, Zoe Quandt, Arabella Young, Mei-Ling Yang, Mark J. Mamula, Jordan S. Pober, Mark S. Anderson, Smita Krishnaswamy, Kevan C. Herold
Gut microbiota (GM) dysbiosis is associated with inflammatory bowel diseases and also with cardiometabolic, neurologic, and autoimmune diseases. GM composition has a direct effect on the immune system, and vice versa, and particularly on regulatory T cell (Treg) homeostasis. Low-dose interleukin-2 (IL-2LD) stimulates Tregs and is a promising treatment for autoimmune and inflammatory diseases. We aimed to evaluate the impacts of IL-2LD on GM, and correlatively on the immune system. We used 16S ribosomal RNA profiling and metagenomics to characterize GM of mice and humans treated or not with IL-2LD. We performed faecal microbiota transplantation (FMT) from IL-2LD-treated to naïve recipient mice and evaluated its effects in models of gut inflammation and diabetes. IL-2LD markedly affects GM composition in mice and humans. Transfer of an IL-2-tuned microbiota by FMT protected C57BL/6J mice from dextran sulphate sodium-induced colitis and prevented diabetes in NOD mice. Metagenomic analyses highlighted a role for several species impacted by IL-2LD and for microbial pathways involved in the biosynthesis of amino acids, short-chain fatty acids, and L-arginine. Our results demonstrate that IL-2LD induces changes in GM that are involved in the immunoregulatory effects of IL-2LD and suggest a cross-talk between Tregs and GM. These results provide novel insights for understanding the mode of action of Treg-directed therapies.
Nicolas Tchitchek, Otriv Nguekap Tchoumba, Gabriel Pires, Sarah Dandou, Julien Campagne, Guillaume Churlaud, Gwladys Fourcade, Thomas W. Hoffmann, Francesco Strozzi, Camille Gaal, Christophe Bonny, Emmanuelle Le Chatelier, Stanislav Dusko Erlich, Harry Sokol, David Klatzmann
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a post-translational regulator of the low-density lipoprotein receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-sequencing-based variant discovery, we identified a novel psoriasis susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C>T). This finding was verified in independently acquired genomic and RNA-sequencing datasets. Single-cell RNA-sequencing (scRNA-seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-seq and immunohistochemistry demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL-36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression including, IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C>T was associated with lower PCSK9 expression and higher IL36G expression, when compared to heterozygous skin or cell lines. Together these results support PCSK9 as a novel psoriasis susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.
Alexander Merleev, Antonio Ji-Xu, Atrin Toussi, Lam C. Tsoi, Stephanie T. Le, Guillaume Luxardi, Xianying Xing, Rachael Wasikowski, William Liakos, Marie-Charlotte Brüggen, James T. Elder, Iannis E. Adamopoulos, Yoshihiro Izumiya, Annie Riera-Leal, Qinyuan Li, Nikolay Yu Kuzminykh, Amanda Kirane, Alina I. Marusina, Johann E. Gudjonsson, Emanual Maverakis
Recent clinical trials show promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. In contrast to BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells of relapsing and secondary progressive MS patients versus controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells of clinical responders to natalizumab (anti-VLA-4 antibody) treatment. Under in vitro TFH-like conditions, BTK phosphorylation was enhanced by T-bet-inducing stimuli IFN-γ and CpG-ODN, whilst the expression of T-bet and T-bet-associated molecules CXCR3, CD21 and CD11c were affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course in MS patients.
Liza Rijvers, Jamie van Langelaar, Laurens Bogers, Marie-José Melief, Steven C. Koetzier, Katelijn M. Blok, Annet F. Wierenga-Wolf, Helga E. De Vries, Jasper Rip, Odilia B.J. Corneth, Rudi W. Hendriks, Roland Grenningloh, Ursula Boschert, Joost Smolders, Marvin M. van Luijn
IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type–specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1β or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1β–dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.
Thomas Hoyler, Bettina Bannert, Cédric André, Damian Beck, Thomas Boulay, David Buffet, Nadja Caesar, Thomas Calzascia, Janet Dawson, Diego Kyburz, Robert Hennze, Christine Huppertz, Amanda Littlewood-Evans, Pius Loetscher, Kirsten D. Mertz, Satoru Niwa, Gautier Robert, James S. Rush, Giulia Ruzzante, Sophie Sarret, Thomas Stein, Ismahane Touil, Grazyna Wieczorek, Geraldine Zipfel, Stuart Hawtin, Tobias Junt
Follicular Th (Tfh) cells are key CD4+ Th cells involved in regulating B cell differentiation in the germinal center. Mice with conditional KO (CKO) of B lymphocyte-induced maturation protein-1 (BLIMP-1) expression in CD11chi DCs (Prdm1 CKO) spontaneously develop a lupus-like disease. We found an increase in the number of Tfh cells in secondary lymphoid organs in lupus-prone Prdm1-CKO mice. B cells stimulated with Tfh cells become Ab-secreting cells (ASCs); there was an increase in ASC differentiation mediated by Tfh cells from Prdm1-CKO mice compared with Tfh cells from control mice. This was mainly due to the increased expression of molecules within the IL-23/IL-17 pathway in Tfh cells from Prdm1-CKO mice. There was an increased frequency of IL-17A+ Tfh cells in Prdm1-CKO mice. These Tfh cells secrete IL-17A and an increased amount of IL-21. Furthermore, neutralizing IL-17A and IL-21 reduced ASC differentiation induced by Tfh cells. Lastly, we found the expression of IL-1β and IL-6 was increased in BLIMP-1–deficient DCs, which are key for Th17 induction. Altogether, the lack of BLIMP-1 expression in DCs induces not only an increased number of Tfh cells, but also functionally distinct Tfh cells that lead to increased ASC differentiation.
Vera Kim, Kyungwoo Lee, Hong Tian, Su Hwa Jang, Betty Diamond, Sun Jung Kim
Vitiligo is an autoimmune skin disease characterized by the destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells but why white patches in stable disease persist is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. We combine non-invasive multiphoton microscopy (MPM) imaging and single-cell RNA sequencing (scRNA-seq) to identify subpopulations of keratinocytes in stable vitiligo patients. We show that, as compared to non-lesional skin, these keratinocytes are enriched in lesional vitiligo skin and shift their energy utilization towards oxidative phosphorylation. Systematic investigation of cell-cell communication networks show that this small population of keratinocyte secrete CXCL9 and CXCL10 to potentially drive vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. Further MPM imaging of patients undergoing punch grafting treatment showed that keratinocytes favoring oxidative phosphorylation persist in non-responders but normalize in responders. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cell-cell communication networks and keratinocyte cell states that can perpetuate inflammation and prevent repigmentation.
Jessica Shiu, Lihua Zhang, Griffin Lentsch, Jessica L. Flesher, Suoqin Jin, Christopher M. Polleys, Seong Jin Jo, Craig Mizzoni, Pezhman Mobasher, Jasmine Kwan, Francisca Rius-Diaz, Bruce J. Tromberg, Irene Georgakoudi, Qing Nie, Mihaela Balu, Anand K. Ganesan
We describe a new mechanism responsible for Systemic Lupus Erythematosus (SLE). In humans with SLE and in two SLE murine models, there is marked enrichment of isolevuglandin (isoLG)-adducted proteins in monocytes and dendritic cells (DCs). We found that antibodies form against isoLG adducts in both SLE-prone mice and humans with SLE. In addition, isoLG ligation of the transcription factor PU.1 at a critical DNA binding site markedly reduces transcription of all C1q subunits. Treatment of SLE prone mice with the specific isoLG scavenger 2-hydroxybenzlyamine (2HOBA) ameliorates parameters of autoimmunity including plasma cell expansion, circulating IgG levels, and anti-dsDNA antibody titers. 2-HOBA also lowers blood pressure, attenuates renal injury, and reduces inflammatory gene expression uniquely in C1q expressing dendritic cells. Thus, isoLG adducts play an essential role in the genesis and maintenance of systemic autoimmunity and hypertension in SLE.
David M. Patrick, Néstor de la Visitación, Jaya Krishnan, Wei Chen, Michelle J. Ormseth, C. Michael Stein, Sean S. Davies, Venkataraman Amarnath, Leslie J. Crofford, Jonathan M. Williams, Shilin Zhao, Charles D. Smart, Sergey Dikalov, Anna Dikalova, Liang Xiao, Justin P. Van Beusecum, Mingfang Ao, Agnes B. Fogo, Annet Kirabo, David G. Harrison
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