Central clonal deletion has been considered the critical factor responsible for the robust state of tolerance achieved by chimerism-based experimental protocols, but split-tolerance models and the clinical experience are calling this assumption into question. Although clone-size reduction through deletion has been shown to be universally required for achieving allotolerance, it remains undetermined whether it is sufficient by itself. Therapeutic Treg treatment induces chimerism and tolerance in a stringent murine BM transplantation model devoid of myelosuppressive recipient treatment. In contrast to irradiation chimeras, chronic rejection (CR) of skin and heart allografts in Treg chimeras was permanently prevented, even in the absence of complete clonal deletion of donor MHC-reactive T cells. We show that minor histocompatibility antigen mismatches account for CR in irradiation chimeras without global T cell depletion. Furthermore, we show that Treg therapy–induced tolerance prevents CR in a linked suppression–like fashion, which is maintained by active regulatory mechanisms involving recruitment of thymus-derived Tregs to the graft. These data suggest that highly efficient intrathymic and peripheral deletion of donor-reactive T cells for specificities expressed on hematopoietic cells preclude the expansion of donor-specific Tregs and, hence, do not allow for spreading of tolerance to minor specificities that are not expressed by donor BM.
Nina Pilat, Benedikt Mahr, Lukas Unger, Karin Hock, Christoph Schwarz, Andreas M. Farkas, Ulrike Baranyi, Fritz Wrba, Thomas Wekerle
Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and
Tao Huang, Meredith Hazen, Yonglei Shang, Meijuan Zhou, Xiumin Wu, Donghong Yan, Zhonghua Lin, Margaret Solon, Elizabeth Luis, Hai Ngu, Yongchang Shi, Arna Katewa, David F. Choy, Nandhini Ramamoorthi, Erick R. Castellanos, Mercedesz Balazs, Min Xu, Wyne P. Lee, Marissa L. Matsumoto, Jian Payandeh, Joseph R. Arron, Jo-Anne Hongo, Jianyong Wang, Isidro Hötzel, Cary D. Austin, Karin Reif
DC-based vaccines that initiate T cell responses are well tolerated and have demonstrated efficacy for tumor immunotherapy, with the potential to be combined with other therapies. Targeting vaccine antigens (Ag) directly to the DCs in vivo is more effective than cell-based therapies in mouse models and is therefore a promising strategy to translate to humans. The human CD141+ DCs are considered the most clinically relevant for initiating CD8+ T cell responses critical for killing tumors or infected cells, and they specifically express the C-type lectin-like receptor CLEC9A that facilitates presentation of Ag by these DCs. We have therefore developed a human chimeric Ab that specifically targets CLEC9A on CD141+ DCs in vitro and in vivo. These human chimeric Abs are highly effective at delivering Ag to DCs for recognition by both CD4+ and CD8+ T cells. Given the importance of these cellular responses for antitumor or antiviral immunity, and the superior specificity of anti-CLEC9A Abs for this DC subset, this approach warrants further development for vaccines.
Kirsteen M. Tullett, Ingrid M. Leal Rojas, Yoshihito Minoda, Peck S. Tan, Jian-Guo Zhang, Corey Smith, Rajiv Khanna, Ken Shortman, Irina Caminschi, Mireille H. Lahoud, Kristen J. Radford
To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.
Shereen Oon, Huy Huynh, Tsin Yee Tai, Milica Ng, Katherine Monaghan, Mark Biondo, Gino Vairo, Eugene Maraskovsky, Andrew D. Nash, Ian P. Wicks, Nicholas J. Wilson
BACKGROUND. Some adult patients presenting with unexplained pyrexia, serositis, skin rashes, arthralgia, myalgia, and other symptoms commonly found in autoinflammatory disorders may not fit a specific diagnosis, either because their clinical phenotype is nondiagnostic or genetic tests are negative. We used the term undifferentiated systemic autoinflammatory disorder (uSAID) to describe such cases. Given that well-defined autoinflammatory diseases show responses to IL-1 blockade, we evaluated whether anakinra was useful for both diagnosing and treating uSAID patients.
METHODS. We performed a retrospective analysis of consecutive patients presenting with uSAID between 2012–2015 who were treated with the recombinant IL-1 receptor antagonist anakinra. uSAID was diagnosed after excluding malignancy, infection, and pathogenic mutations in known hereditary fever syndromes (HFS) genes and where clinical criteria for adult onset Still’s disease (AOSD) were not met.
RESULTS. A total of 11 patients presented with uSAID (5 males and 6 females), with a mean time to diagnosis of 3.5 years (1–8 years). Patients were unresponsive or only partially controlled on disease-modifying antirheumatic drug (DMARD)/steroid treatment. Anakinra controlled symptoms within 4–6 weeks of starting treatment in 9 of 11 cases. Two patients discontinued therapy — one due to incomplete response and another due to severe injection-site reactions.
CONCLUSION. This retrospective case series demonstrates that the spectrum of poorly defined autoinflammatory disorders that show responsiveness to anakinra is considerable. Anakinra seems a viable treatment option for these patients, who are unresponsive to standard steroid/DMARD treatments. Moreover, given the mechanisms of action, response to anakinra implicates underlying IL-1 dysregulation in the disease pathogenesis of responding uSAIDs patients.
Stephanie R. Harrison, Dennis McGonagle, Sharmin Nizam, Stephen Jarrett, Jeroen van der Hilst, Michael F. McDermott, Sinisa Savic
Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA–transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT.
Wei Li, Liangyi Liu, Aurelie Gomez, Jilu Zhang, Abdulraouf Ramadan, Qing Zhang, Sung W. Choi, Peng Zhang, Joel K. Greenson, Chen Liu, Di Jiang, Elizabeth Virts, Stephanie L. Kelich, Hong Wei Chu, Ryan Flynn, Bruce R. Blazar, Helmut Hanenberg, Samir Hanash, Sophie Paczesny
Rheumatoid arthritis (RA) is a debilitating disease characterized by persistent accumulation of leukocytes within the articular cavity and synovial tissue. Metabololipidomic profiling of arthritic joints from omega-3 supplemented mice identified elevated levels of specialized proresolving lipid mediators (SPM) including resolvin D1 (RvD1). Profiling of human RA synovial fluid revealed physiological levels of RvD1, which — once applied to human neutrophils — attenuated chemotaxis. These results prompted analyses of the antiarthritic properties of RvD1 in a model of murine inflammatory arthritis. The stable epimer 17
Lucy V. Norling, Sarah E. Headland, Jesmond Dalli, Hildur H. Arnardottir, Oliver Haworth, Hefin R. Jones, Daniel Irimia, Charles N. Serhan, Mauro Perretti
Immunotherapies targeting the immune checkpoint receptor programmed cell death protein 1 (PD-1) have shown remarkable efficacy in treating cancer. CD4+CD25hiFoxP3+ Tregs are critical regulators of immune responses in autoimmunity and malignancies, but the functional status of human Tregs expressing PD-1 remains unclear. We examined functional and molecular features of PD-1hi Tregs in healthy subjects and patients with glioblastoma multiforme (GBM), combining functional assays, RNA sequencing, and cytometry by time of flight (CyTOF). In both patients with GBM and healthy subjects, circulating PD-1hi Tregs displayed reduced suppression of CD4+ effector T cells, production of IFN-γ, and molecular signatures of exhaustion. Transcriptional profiling of tumor-resident Tregs revealed that several genes coexpressed with PD-1 and associated with IFN-γ production and exhaustion as well as enrichment in exhaustion signatures compared with circulating PD-1hi Tregs. CyTOF analysis of circulating and tumor-infiltrating Tregs from patients with GBM treated with PD-1-blocking antibodies revealed that treatment shifts the profile of circulating Tregs toward a more exhausted phenotype reminiscent of that of tumor-infiltrating Tregs, further increasing IFN-γ production. Thus, high PD-1 expression on human Tregs identifies dysfunctional, exhausted Tregs secreting IFN-γ that exist in healthy individuals and are enriched in tumor infiltrates, possibly losing function as they attempt to modulate the antitumoral immune responses.
Daniel E. Lowther, Brittany A. Goods, Liliana E. Lucca, Benjamin A. Lerner, Khadir Raddassi, David van Dijk, Amanda L. Hernandez, Xiangguo Duan, Murat Gunel, Vlad Coric, Smita Krishnaswamy, J. Christopher Love, David A. Hafler
Posttransplantation cyclophosphamide (PTCy) effectively prevents graft-versus-host disease (GVHD), but its immunologic impact is poorly understood. We assessed lymphocyte reconstitution via flow cytometry (
Christopher G. Kanakry, David G. Coffey, Andrea M.H. Towlerton, Ante Vulic, Barry E. Storer, Jeffrey Chou, Cecilia C.S. Yeung, Christopher D. Gocke, Harlan S. Robins, Paul V. O’Donnell, Leo Luznik, Edus H. Warren
Xenografting primary tumor cells allows modeling of the heterogeneous natures of malignant diseases and the influences of the tissue microenvironment. Here, we demonstrate that xenografting primary chronic lymphocytic leukemia (CLL) B lymphocytes with activated autologous T cells into alymphoid mice results in considerable CLL B cell division and sizable T cell expansion. Nevertheless, most/all CD5+CD19+ cells are eventually lost, due in part to differentiation into antibody-secreting plasmablasts/plasma cells. CLL B cell differentiation is associated with isotype class switching and development of new
Piers E.M. Patten, Gerardo Ferrer, Shih-Shih Chen, Rita Simone, Sonia Marsilio, Xiao-Jie Yan, Zachary Gitto, Chaohui Yuan, Jonathan E. Kolitz, Jacqueline Barrientos, Steven L. Allen, Kanti R. Rai, Thomas MacCarthy, Charles C. Chu, Nicholas Chiorazzi
We recently identified a genome-wide genetic association of eosinophilic esophagitis (EoE) at 2p23 spanning the calpain 14 (
Benjamin P. Davis, Emily M. Stucke, M. Eyad Khorki, Vladislav A. Litosh, Jeffrey K. Rymer, Mark Rochman, Jared Travers, Leah C. Kottyan, Marc E. Rothenberg
Despite the rare appearance of potent HIV-neutralizing mAbs in infected individuals requiring prolonged affinity maturation, little is known regarding this process in the majority of viremic individuals. HIV-infected individuals with chronic HIV viremia have elevated numbers of nonconventional tissue-like memory (TLM) B cells that predominate in blood over conventional resting memory (RM) B cells. Accordingly, we investigated affinity maturation in these 2 memory B cell populations. Analysis of IgG-expressing TLM B cells revealed a higher number of cell divisions compared with RM B cells; however, TLM B cells paradoxically displayed significantly lower frequencies of somatic hypermutation (SHM). To assess Ab reactivity in TLM and RM B cells, single-cell cloning was performed on HIV envelope CD4–binding site–sorted (CD4bs-sorted) B cells from 3 individuals with chronic HIV viremia. Several clonal families were present among the 127 cloned recombinant mAbs, with evidence of crosstalk between TLM and RM B cell populations that was largely restricted to non-VH4 families. Despite evidence of common origins, SHM frequencies were significantly decreased in TLM-derived mAbs compared with SHM frequencies in RM-derived mAbs. However, both cell populations had lower frequencies of SHMs than did broadly neutralizing CD4bs–specific mAbs. There was a significant correlation between SHM frequencies and the HIV-neutralizing capacities of the mAbs. Furthermore, HIV neutralization was significantly higher in the RM-derived mAbs compared with that seen in the TLM-derived mAbs, and both SHM frequencies and neutralizing capacity were lowest in TLM-derived mAbs with high polyreactivity. Thus, deficiencies in memory B cells that arise during chronic HIV viremia provide insight into the inadequacy of the Ab response in viremic individuals.
Eric Meffre, Aaron Louie, Jason Bannock, Leo J.Y. Kim, Jason Ho, Cody C. Frear, Lela Kardava, Wei Wang, Clarisa M. Buckner, Yimeng Wang, Olivia R. Fankuchen, Kathleen R. Gittens, Tae-Wook Chun, Yuxing Li, Anthony S. Fauci, Susan Moir
Ab-producing plasma cells (PCs) serve as key participants in countering pathogenic challenges as well as being contributors to autoimmune and malignant disorders. Thus far, only a limited number of PC–specific markers have been identified. The characterization of the unique variable lymphocyte receptor (VLR) Abs that are made by evolutionarily distant jawless vertebrates prompted us to investigate whether VLR Abs could detect novel PC antigens that have not been recognized by conventional Abs. Here, we describe a monoclonal lamprey Ab, VLRB MM3, that was raised against primary multiple myeloma cells. VLRB MM3 recognizes a unique epitope of the CD38 ectoenzyme that is present on plasmablasts and PCs from healthy individuals and on most, but not all, multiple myelomas. Binding by the VLRB MM3 Ab coincides with CD38 dimerization and NAD glycohydrolase activity. Our data demonstrate that the lamprey VLRB MM3 Ab is a unique reagent for the identification of plasmablasts and PCs, with potential applications in the diagnosis and therapeutic intervention of PC or autoimmune disorders.
Cuiling Yu, Yanling Liu, Justin Tze Ho Chan, Jiefei Tong, Zhihua Li, Mengyao Shi, Dariush Davani, Marion Parsons, Srijit Khan, Wei Zhan, Shuya Kyu, Eyal Grunebaum, Paolo Campisi, Evan J. Propst, David L. Jaye, Suzanne Trudel, Michael F. Moran, Mario Ostrowski, Brantley R. Herrin, F. Eun-Hyung Lee, Ignacio Sanz, Max D. Cooper, Götz R.A. Ehrhardt
Progressive HIV-1 infection leads to both profound immune suppression and pathologic inflammation in the majority of infected individuals. While adaptive immune dysfunction, as evidenced by CD4+ T cell depletion and exhaustion, has been extensively studied, less is known about the functional capacity of innate immune cell populations in the context of HIV-1 infection. Given the broad susceptibility to opportunistic infections and the dysregulated inflammation observed in progressive disease, we hypothesized that there would be significant changes in the innate cellular responses. Using a cohort of patients with multiple samplings before and after antiretroviral therapy (ART) initiation, we demonstrated increased responses to innate immune stimuli following viral suppression, as measured by the production of inflammatory cytokines. Plasma viral load itself had the strongest association with this change in innate functional capacity. We further identified epigenetic modifications in the
Eileen P. Scully, Ainsley Lockhart, Wilfredo Garcia-Beltran, Christine D. Palmer, Chelsey Musante, Eric Rosenberg, Todd M. Allen, J. Judy Chang, Ronald J. Bosch, Marcus Altfeld
Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.
Konrad Gabrusiewicz, Benjamin Rodriguez, Jun Wei, Yuuri Hashimoto, Luke M. Healy, Sourindra N. Maiti, Ginu Thomas, Shouhao Zhou, Qianghu Wang, Ahmed Elakkad, Brandon D. Liebelt, Nasser K. Yaghi, Ravesanker Ezhilarasan, Neal Huang, Jeffrey S. Weinberg, Sujit S. Prabhu, Ganesh Rao, Raymond Sawaya, Lauren A. Langford, Janet M. Bruner, Gregory N. Fuller, Amit Bar-Or, Wei Li, Rivka R. Colen, Michael A. Curran, Krishna P. Bhat, Jack P. Antel, Laurence J. Cooper, Erik P. Sulman, Amy B. Heimberger
Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia–derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/β-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.
Giuseppina Federico, Michael Meister, Daniel Mathow, Gunnar H. Heine, Gerhard Moldenhauer, Zoran V. Popovic, Viola Nordström, Annette Kopp-Schneider, Thomas Hielscher, Peter J. Nelson, Franz Schaefer, Stefan Porubsky, Danilo Fliser, Bernd Arnold, Hermann-Josef Gröne
Acute allograft rejection is mediated by host CD8+ cytotoxic T lymphocytes (CTL) targeting graft class I major histocompatibility complex (MHC) molecules. In experimental rodent models, rejection requires differentiation of naive CD8+ T cells into alloreactive CTL within secondary lymphoid organs, whereas in humans, CTL may alternatively develop within the graft from circulating CD8+ effector memory T cells (TEM) that recognize class I MHC molecules on graft endothelial cells (EC). This latter pathway is poorly understood. Here, we show that host CD4+ TEM, activated by EC class II MHC molecules, provide critical help for this process. First, blocking HLA-DR on EC lining human artery grafts in immunodeficient mice reduces CD8+ CTL development within and acute rejection of the artery by adoptively transferred allogeneic human lymphocytes. Second, siRNA knockdown or CRISPR/Cas9 ablation of class II MHC molecules on EC prevents CD4+ TEM from helping CD8+ TEM to develop into CTL in vitro. Finally, implanted synthetic microvessels, formed from CRISPR/Cas9-modified EC lacking class II MHC molecules, are significantly protected from CD8+ T cell–mediated destruction in vivo. We conclude that human CD8+ TEM–mediated rejection targeting graft EC class I MHC molecules requires help from CD4+ TEM cells activated by recognition of class II MHC molecules.
Parwiz Abrahimi, Lingfeng Qin, William G. Chang, Alfred L.M. Bothwell, George Tellides, W. Mark Saltzman, Jordan S. Pober
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