Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.
Orhan Efe, Rodrigo B. Gassen, Leela Morena, Yoshikazu Ganchiku, Ayman Al Jurdi, Isadora T. Lape, Pedro Ventura-Aguiar, Christian LeGuern, Joren C. Madsen, Zachary Shriver, Gregory J. Babcock, Thiago J. Borges, Leonardo V. Riella
Tissue-intrinsic mechanisms that regulate severity of systemic pathogenic immune-mediated diseases, such as acute graft-versus-host disease (GVHD), remain poorly understood. Following allogeneic hematopoietic stem cell transplantation, autophagy, a cellular stress protective response, is induced in host nonhematopoietic cells. To systematically address the role of autophagy in various host nonhematopoietic tissues, both specific classical target organs of acute GVHD (intestines, liver, and skin) and organs conventionally not known to be targets of GVHD (kidneys and heart), we generated mice with organ-specific knockout of autophagy related 5 (ATG5) to specifically and exclusively inhibit autophagy in the specific organs. When compared with wild-type recipients, animals that lacked ATG5 in the gastrointestinal tract or liver showed significantly greater tissue injury and mortality, while autophagy deficiency in the skin, kidneys, or heart did not affect mortality. Treatment with the systemic autophagy inducer sirolimus only partially mitigated GVHD mortality in intestine-specific autophagy-deficient hosts. Deficiency of autophagy increased MHC class I on the target intestinal epithelial cells, resulting in greater susceptibility to damage by alloreactive T cells. Thus, autophagy is a critical cell-intrinsic protective response that promotes tissue tolerance and regulates GVHD severity.
Katherine Oravecz-Wilson, Emma Lauder, Austin Taylor, Laure Maneix, Jeanine L. Van Nostrand, Yaping Sun, Lu Li, Dongchang Zhao, Chen Liu, Pavan Reddy
Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs) that are critical for orchestrating the anti-inflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxia response through hypoxia-inducible factor-1 alpha (HIF-1a) were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted HIF-1a conditional knockout mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, scRNA-seq analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxia response genes. These findings support the importance of a glycolysis/HIF-1a axis in promoting G-MDSC anti-inflammatory activity and biofilm persistence during PJI.
Christopher M. Horn, Prabhakar Arumugam, Zachary Van Roy, Cortney E. Heim, Rachel W. Fallet, Blake P. Bertrand, Dhananjay Shinde, Vinai C. Thomas, Svetlana G. Romanova, Tatiana K. Bronich, Curtis W. Hartman, Kevin L. Garvin, Tammy Kielian
Adoptive transfer of T cell receptor-engineered T cells (TCR-T) is a promising strategy for immunotherapy against solid tumors. However, the potential of CD4+ T cells in mediating tumor regression has been neglected. Nasopharyngeal cancer is consistently associated with EBV. Here, to evaluate the therapeutic potential of CD4 TCR-T in nasopharyngeal cancer, we screened for CD4 TCRs recognizing EBV nuclear antigen 1 (EBNA1) presented by HLA-DP5. Using mass spectrometry, we identified EBNA567-581, a peptide naturally processed and presented by HLA-DP5. We isolated TCR135, a CD4 TCR with high functional avidity, that can function in both CD4+ and CD8+ T cells and recognizes HLA-DP5-restricted EBNA1567-581. TCR135-transduced T cells functioned in two ways: directly killing HLA-DP5+EBNA1+ tumor cells after recognizing EBNA1 presented by tumor cells and indirectly killing HLA-DP5-negative tumor cells after recognizing EBNA1 presented by antigen-presenting cells. TCR135-transduced T cells preferentially infiltrated into the tumor microenvironment and significantly inhibited tumor growth in xenograft nasopharyngeal tumor models. Additionally, we found that 62% of nasopharyngeal cancer patients showed 50%-100% expression of HLA-DP on tumor cells, indicating that nasopharyngeal cancer is well-suited for CD4 TCR-T therapy. These findings suggest that TCR135 may provide a new strategy for EBV-related nasopharyngeal cancer immunotherapy in HLA-DP5+ patients.
Chenwei Wang, Jiewen Chen, Jingyao Li, Zhihong Xu, Lihong Huang, Qian Zhao, Lei Chen, Xiaolong Liang, Hai Hu, Gang Li, Chengjie Xiong, Bin Wu, Hua You, Danyi Du, Xiaoling Wang, Hongle Li, Zibing Wang, Lin Chen
Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell–produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.
Sunny L. Sneed, Brian B. Reese, Ana F.S. Laureano, Sneha Ratnapriya, Isabella Fraschilla, Kate L. Jeffrey, Greg P. Coffey, Pamela B. Conley, Robert M. Anthony
Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal graft-versus-host disease (GI GVHD). CST is intended to target the immune system, but the glucocorticoid receptor is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids directly target intestinal epithelium, potentially worsening immune-mediated GI damage. Corticosteroids administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage, but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with “steroid-refractory” GR-deficient T cells or Interferon-γ. These findings correlated with corticosteroid-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, Interleukin-22 administration enhanced STAT3 activity and overcame corticosteroid-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.
Viktor Arnhold, Winston Y. Chang, Suze A. Jansen, Govindarajan Thangavelu, Marco Calafiore, Paola Vinci, Ya-Yuan Fu, Takahiro Ito, Shuichiro Takashima, Anastasiya Egorova, Jason Kuttiyara, Adam Perlstein, Marliek van Hoesel, Chen Liu, Bruce R. Blazar, Caroline A. Lindemans, Alan M. Hanash
Radiotherapy (RT) is considered immunogenic, but clinical data demonstrating RT-induced T-cell priming are scarce. Here, we show in a mouse tumor model representative of human lymphocyte-depleted cancer that RT enhances spontaneous priming of thymus-derived (FOXP3+ Helios+) regulatory T-cells (Tregs) by the tumor. These Tregs acquire an effector phenotype, populate the tumor and impede tumor control by a simultaneous, RT-induced CD8+ cytotoxic T-cell (CTL) response. Combination of RT with CTLA-4 or PD-1 blockade, which enables CD28 costimulation, further increased this Treg response and failed to improve tumor control. We discovered that upon RT, CD28-ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector (e)Treg response, enriched the tumor-draining lymph node for PD-L1+CD80+ migratory, conventional dendritic cells (cDCs) and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1, enhanced intra-tumoral CTL accumulation and the combination significantly increased RT-induced tumor regression and overall survival. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since they drive Treg responses in this context. However, combining RT with CD86 blockade may promote control of such tumors by enabling a CTL response.
Elselien Frijlink, Douwe M.T. Bosma, Julia Busselaar, Thomas W. Battaglia, Mo D. Staal, Inge Verbrugge, Jannie Borst
Neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). To assess the PMN phenotypic and functional diversification during inflammatory ulceration to CRC transition we analyzed the transcriptomic landscape of blood and tissue PMNs. Transcriptional programs effectively separated PMNs based on their localization to peripheral blood, inflamed colon, and tumors. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and mechanistic studies to be highly induced in TANs, acting to critically regulate endothelial cell chemotaxis and branching. TCGA dataset and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in UC patients. Pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. Our findings, demonstrate a niche-directed PMN functional specialization, and identify TAN contributions to tumor vascularization, delineating a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.
Triet M. Bui, Lenore K. Yalom, Edward Ning, Jessica M. Urbanczyk, Xingsheng Ren, Caroline J. Herrnreiter, Jackson A. DiSario, Brian Wray, Matthew J. Schipma, Yuri S. Velichko, David P. Sullivan, Kouki Abe, Shannon M. Lauberth, Guang-Yu Yang, Parambir S. Dulai, Stephen B. Hanauer, Ronen Sumagin
The measles, mumps and rubella (MMR) vaccine protects against all-cause mortality in children, but the immunological mechanisms mediating these effects are poorly known. We systematically investigated whether MMR can induce long-term functional changes in innate immune cells, a process termed trained immunity, that could at least partially mediate this heterologous protection. In a randomized placebo-controlled trial, 39 healthy adults received either the MMR vaccine or a placebo. By using single-cell RNA-sequencing, we found that MMR caused transcriptomic changes in CD14-positive monocytes and NK cells, but most profoundly in γδ T cells. Monocyte function was not altered by MMR vaccination. In contrast, the function of γδ T cells was markedly enhanced by MMR vaccination, with higher production of TNF and IFNγ, as well as upregulation of cellular metabolic pathways. In conclusion, we describe a trained immunity program characterized by modulation of γδ T cell function induced by MMR vaccination.
Rutger J. Röring, Priya A. Debisarun, Javier Botey-Bataller, Tsz Kin Suen, Ozlem Bulut, Gizem Kilic, Valerie A.C.M. Koeken, Andrei Sarlea, Harsh Bahrar, Helga Dijkstra, Heidi Lemmers, Katharina L. Gössling, Nadine Rüchel, Philipp N. Ostermann, Lisa Müller, Heiner Schaal, Ortwin Adams, Arndt Borkhardt, Yavuz Ariyurek, Emile J. de Meijer, Susan L. Kloet, Jaap ten Oever, Katarzyna Placek, Yang Li, Mihai G. Netea
Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel Cell Polyomavirus, driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the non-responding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class-I haplotypes. We observed a broad T-cell recognition of T-Ags, including identification of 20 novel T-Ag-derived epitopes. Broadening of the T-Ag recognition profile and increased T-cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.
Ulla Kring Hansen, Candice D. Church, Ana Micaela Carnaz Simões, Marcus Svensson Frej, Amalie Kai Bentzen, Siri A. Tvingsholm, Jürgen C. Becker, Steven P. Fling, Nirasha Ramchurren, Suzanne L. Topalian, Paul T. Nghiem, Sine Reker Hadrup