Origin and evolution of the T cell repertoire after posttransplantation cyclophosphamide

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Abstract

Posttransplantation cyclophosphamide (PTCy) effectively prevents graft-versus-host disease (GVHD), but its immunologic impact is poorly understood. We assessed lymphocyte reconstitution via flow cytometry (n = 74) and antigen receptor sequencing (n = 35) in recipients of myeloablative, HLA-matched allogeneic BM transplantation using PTCy. Recovering T cells were primarily phenotypically effector memory with lower T cell receptor β (TRB) repertoire diversity than input donor repertoires. Recovering B cells were predominantly naive with immunoglobulin heavy chain locus (IGH) repertoire diversity similar to donors. Numerical T cell reconstitution and TRB diversity were strongly associated with recipient cytomegalovirus seropositivity. Global similarity between input donor and recipient posttransplant repertoires was uniformly low at 1–2 months after transplant but increased over the balance of the first posttransplant year. Blood TRB repertoires at ≥3 months after transplant were often dominated by clones present in the donor blood/marrow memory CD8⁺ compartment. Limited overlap was observed between the TRB repertoires of T cells infiltrating the skin or gastrointestinal tract versus the blood. Although public TRB sequences associated with herpesvirus- or alloantigen-specific CD8⁺ T cells were detected in some patients, posttransplant TRB and IGH repertoires were unique to each individual. These data define the immune dynamics occurring after PTCy and establish a benchmark against which immune recovery after other transplantation approaches can be compared.

Authors

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

Nonhematopoietic Nrf2 dominantly impedes adult progression of sickle cell anemia in mice

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Abstract

The prevention of organ damage and early death in young adults is a major clinical concern in sickle cell disease (SCD). However, mechanisms that control adult progression of SCD during the transition from adolescence are poorly defined with no cognate prophylaxis. Here, we demonstrate in a longitudinal cohort of homozygous SCD (SS) mice a link between intravascular hemolysis, vascular inflammation, lung injury, and early death. Prophylactic Nrf2 activation in young SS mice stabilized intravascular hemolysis, reversed vascular inflammation, and attenuated lung edema in adulthood. Enhanced Nrf2 activation in endothelial cells in vitro concurred with the dramatic effect on vascular inflammation in the mice. BM chimeric SS mice lacking Nrf2 expression in nonhematopoietic tissues were created to dissect the role of nonerythroid Nrf2 in SCD progression. The SS chimeras developed severe intravascular hemolysis despite having erythroid Nrf2. In addition, they developed premature vascular inflammation and pulmonary edema and died younger than donor littermates with intact nonhematopoietic Nrf2. Our results reveal a dominant protective role for nonhematopoietic Nrf2 against tissue damage in both erythroid and nonerythroid tissues in SCD. Furthermore, we show that prophylactic augmentation of Nrf2-coordinated cytoprotection effectively impedes onset of the severe adult phenotype of SCD in mice.

Authors

Samit Ghosh, Chibueze A. Ihunnah, Rimi Hazra, Aisha L. Walker, Jason M. Hansen, David R. Archer, Amma T. Owusu-Ansah, Solomon F. Ofori-Acquah

Functional significance of the discordance between transcriptional profile and left ventricular structure/function during reverse remodeling

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Abstract

To elucidate the mechanisms for reverse LV remodeling, we generated a conditional (doxycycline [dox] off) transgenic mouse tetracycline transactivating factor–TRAF2 (tTA-TRAF2) that develops a dilated heart failure (HF) phenotype upon expression of a proinflammatory transgene, TNF receptor–associated factor 2 (TRAF2), and complete normalization of LV structure and function when the transgene is suppressed. tTA-TRAF2 mice developed a significant increase in LV dimension with decreased contractile function, which was completely normalized in the tTA-TRAF2 mice fed dox for 4 weeks (tTA-TRAF2dox4W). Normalization of LV structure and function was accompanied by partial normalization (~60%) of gene expression associated with incident HF. Similar findings were observed in patients with dilated cardiomyopathy who underwent reverse LV remodeling following mechanical circulatory support. Persistence of the HF gene program was associated with an exaggerated hypertrophic response and increased mortality in tTA-TRAF2dox4W mice following transaortic constriction (TAC). These effects were no longer observed following TAC in tTA-TRAF2dox8W, wherein there was a more complete (88%) reversal of the incident HF genes. These results demonstrate that reverse LV remodeling is associated with improvements in cardiac myocyte biology; however, the persistence of the abnormal HF gene program may be maladaptive following perturbations in hemodynamic loading conditions.

Authors

Veli K. Topkara, Kari T. Chambers, Kai-Chien Yang, Huei-Ping Tzeng, Sarah Evans, Carla Weinheimer, Attila Kovacs, Jeffrey Robbins, Philip Barger, Douglas L. Mann

Vaccinia vaccine–based immunotherapy arrests and reverses established pulmonary fibrosis

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal disease without any cure. Both human disease and animal models demonstrate dysregulated wound healing and unregulated fibrogenesis in a background of low-grade chronic T lymphocyte infiltration. Tissue-resident memory T cells (Trm) are emerging as important regulators of the immune microenvironment in response to pathogens, and we hypothesized that they might play a role in regulating the unremitting inflammation that promotes lung fibrosis. Herein, we demonstrate that lung-directed immunotherapy, in the form of i.n. vaccination, induces an antifibrotic T cell response capable of arresting and reversing lung fibrosis. In mice with established lung fibrosis, lung-specific T cell responses were able to reverse established pathology — as measured by decreased lung collagen, fibrocytes, and histologic injury — and improve physiologic function. Mechanistically, we demonstrate that this effect is mediated by vaccine-induced lung Trm. These data not only have implications for the development of immunotherapeutic regimens to treat IPF, but also suggest a role for targeting tissue-resident memory T cells to treat other tissue-specific inflammatory/autoimmune disorders.

Authors

Samuel L. Collins, Yee Chan-Li, MinHee Oh, Christine L. Vigeland, Nathachit Limjunyawong, Wayne Mitzner, Jonathan D. Powell, Maureen R. Horton

Chronic lymphocytic leukemia cells diversify and differentiate in vivo via a nonclassical Th1-dependent, Bcl-6–deficient process

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Abstract

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 IGHV-D-J mutations and occurs via an activation-induced deaminase-dependent pathway that upregulates IRF4 and Blimp-1 without appreciable levels of the expected Bcl-6. These processes were induced in IGHV-unmutated and IGHV-mutated clones by Th1-polarized T-bet⁺ T cells, not classical T follicular helper (Tfh) cells. Thus, the block in B cell maturation, defects in T cell action, and absence of antigen-receptor diversification, which are often cardinal characteristics of CLL, are not inherent but imposed by external signals and the microenvironment. Although these activities are not dominant features in human CLL, each occurs in tissue proliferation centers where the mechanisms responsible for clonal evolution operate. Thus, in this setting, CLL B cell diversification and differentiation develop by a nonclassical germinal center–like reaction that might reflect the cell of origin of this leukemia.

Authors

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

Synergism of FAK and tyrosine kinase inhibition in Ph⁺ B-ALL

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Abstract

BCR-ABL1⁺ B progenitor acute lymphoblastic leukemia (Ph⁺ B-ALL) is an aggressive disease that frequently responds poorly to currently available therapies. Alterations in IKZF1, which encodes the lymphoid transcription factor Ikaros, are present in over 80% of Ph⁺ ALL and are associated with a stem cell–like phenotype, aberrant adhesion molecule expression and signaling, leukemic cell adhesion to the bone marrow stem cell niche, and poor outcome. Here, we show that FAK1 is upregulated in Ph⁺ B-ALL with further overexpression in IKZF1-altered cells and that the FAK inhibitor VS-4718 potently inhibits aberrant FAK signaling and leukemic cell adhesion, potentiating responsiveness to tyrosine kinase inhibitors, inducing cure in vivo. Thus, targeting FAK with VS-4718 is an attractive approach to overcome the deleterious effects of FAK overexpression in Ph⁺ B-ALL, particularly in abrogating the adhesive phenotype induced by Ikaros alterations, and warrants evaluation in clinical trials for Ph⁺ B-ALL, regardless of IKZF1 status.

Authors

Michelle L. Churchman, Kathryn Evans, Jennifer Richmond, Alissa Robbins, Luke Jones, Irina M. Shapiro, Jonathan A. Pachter, David T. Weaver, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock, Charles G. Mullighan

Interleukin-1 signaling contributes to acute islet compensation

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Abstract

IL-1β is a well-established inducer of both insulin resistance and impaired pancreatic islet function. Despite this, findings examining IL-1 receptor deficiency or antagonism in in vivo animal models, as well as in clinical studies of type 2 diabetic (T2D) patients, have led to conflicting results, suggesting that the actions of IL-1β on glycemic control may be pleiotropic in nature. In the present work, we find that the ability of IL-1β to amplify glucose-stimulated insulin secretion from human islets correlates with donor BMI. Islets from obese donors are sensitized to the insulinotropic effects of this cytokine, whereas the stimulatory effects of IL-1β are lost in islets from obese T2D patients, suggesting a role for IL-1 signaling in islet compensation. Indeed, mice deficient in IL-1 receptor type I become glucose intolerant more rapidly than their WT littermates and have impaired secretory responses during the acute stages of inflammatory and metabolic stress induced by LPS and high-fat diet, respectively. IL-1β directly enhances β cell insulin secretion by increasing granule docking and soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex formation at the plasma membrane. Together, our study highlights the importance of IL-1β signaling in islet compensation to metabolic and inflammatory stress.

Authors

Catherine Hajmrle, Nancy Smith, Aliya F. Spigelman, Xiaoqing Dai, Laura Senior, Austin Bautista, Mourad Ferdaoussi, Patrick E. MacDonald

NFAT restricts osteochondroma formation from entheseal progenitors

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Abstract

Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in Aggrecan-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth.

Authors

Xianpeng Ge, Kelly Tsang, Lizhi He, Roberto A. Garcia, Joerg Ermann, Fumitaka Mizoguchi, Minjie Zhang, Bin Zhou, Bin Zhou, Antonios O. Aliprantis

Eosinophilic esophagitis–linked calpain 14 is an IL-13–induced protease that mediates esophageal epithelial barrier impairment

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Abstract

We recently identified a genome-wide genetic association of eosinophilic esophagitis (EoE) at 2p23 spanning the calpain 14 (CAPN14) gene, yet the causal mechanism has not been elucidated. We now show that recombinant CAPN14 cleaves a calpain-specific substrate and is inhibited by 4 classical calpain inhibitors: MDL-28170, acetyl-calpastatin, E-64, and PD151746. CAPN14 is specifically induced (>100-fold) in esophageal epithelium after IL-13 treatment. Epithelial cells overexpressing CAPN14 display impaired epithelial architecture, characterized by acantholysis, epidermal clefting, and epidermolysis. CAPN14 overexpression impairs epithelial barrier function, as demonstrated by decreased transepithelial resistance (2.1-fold) and increased FITC-dextran flux (2.6-fold). Epithelium with gene-silenced CAPN14 demonstrates increased dilated intercellular spaces (5.5-fold) and less organized basal cell layering (1.5-fold) following IL-13 treatment. Finally, CAPN14 overexpression results in loss of desmoglein 1 (DSG1) expression, whereas the IL-13–induced loss of DSG1 is normalized by CAPN14 gene silencing. Importantly, these findings were specific to CAPN14, as they were not observed with modulation of CAPN1 expression. These results, along with the potent induction of CAPN14 by IL-13 and genetic linkage of EoE to the CAPN14 gene locus, demonstrate a molecular and cellular pathway that contributes to T helper type 2 responses in mucosal epithelium.

Authors

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

Innate immune reconstitution with suppression of HIV-1

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Abstract

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 TNFA promoter locus in monocytes that are associated with viremia, suggesting a molecular mechanism for the observed changes in innate immune function following initiation of ART. These data indicate that suppression of HIV-1 viremia is associated with changes in innate cellular function that may in part determine the restoration of protective immune responses.

Authors

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