Autologous stem cell transplantation (ASCT) with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma, and a subset of patients achieve durable progression-free ...survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears to be a potent inhibitor of myeloma-specific immunity and represents a promising new checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8+ T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical anti-TIGIT antibodies that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8+ T cell-dependent, antimyeloma efficacy. Analysis of bone marrow (BM) CD8+ T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after ASCT.
Donor T-cell–derived interleukin-17A (IL-17A) can mediate late immunopathology in graft-versus-host disease (GVHD), however protective roles remain unclear. Using multiple cytokine and cytokine ...receptor subunit knockout mice, we demonstrate that stem cell transplant recipients lacking the ability to generate or signal IL-17 develop intestinal hyper-acute GVHD. This protective effect is restricted to the molecular interaction of IL-17A and/or IL-17F with the IL-17 receptor A/C (IL-17RA/C). The protection from GVHD afforded by IL-17A required secretion from, and signaling in, both hematopoietic and nonhematopoietic host tissue. Given the intestinal-specificity of the disease in these animals, we cohoused wild-type (WT) with IL-17RA and IL-17RC–deficient mice, which dramatically enhanced the susceptibility of WT mice to acute GVHD. Furthermore, the gut microbiome of WT mice shifted toward that of the IL-17RA/C mice during cohousing prior to transplant, confirming that an IL-17–sensitive gut microbiota controls susceptibility to acute GVHD. Finally, induced IL-17A depletion peritransplant also enhanced acute GVHD, consistent with an additional protective role for this cytokine independent of effects on dysbiosis.
•Recipient-derived IL-17A is critical for the prevention of intestinal acute GVHD.•Increased susceptibility to acute GVHD can be transferred to WT mice via cohousing with IL-17RA or IL-17RC–deficient mice.
The oral and colonic microbiota are distinct in healthy individuals. However, this distinction is diminished in common diseases such as colon cancer and inflammatory bowel disease, suggesting a ...potential pathogenic role for oral bacteria when ectopically colonized in the gut. A key mechanism for the segregation of oral and colonic microbiota niches is thought to be microbiota-mediated colonization resistance whereby the commensal gut microbiota outcompete and eliminate the ingested oral bacteria.
We tested this theory by analyzing exact amplicon sequence variants generated from concurrent fecal and oral samples from healthy volunteers exposed to a brief course of a single antibiotic (cohort 1), acute leukemia patients (cohort 2), and stem cell transplant recipients (cohort 3). Cohorts 2 and 3 represent extreme clinical scenarios with respect to antibiotic pressure and severity of gut microbiota injury.
While mild antibiotic exposure in cohort 1 was not sufficient for colonization of any oral bacteria in the gut, even with extreme antibiotic pressure and severe gut microbiota disruptions in cohorts 2 and 3, only one oral species in each cohort colonized the gut.
Colonization resistance is dispensable for segregation of oral and colonic microbiota in humans. This finding implies that the presence of oral bacteria in the distal gut in diseases such as colon cancer and inflammatory bowel disease is not driven by impaired colonization resistance.
The primacy of the gastrointestinal (GI) tract in dictating the outcome of graft-versus-host disease (GVHD) is broadly accepted; however, the mechanisms controlling this effect are poorly understood. ...Here, we demonstrate that GVHD markedly enhances alloantigen presentation within the mesenteric lymph nodes (mLNs), mediated by donor CD103(+)CD11b(-) dendritic cells (DCs) that migrate from the colon under the influence of CCR7. Expansion and differentiation of donor T cells specifically within the mLNs is driven by profound levels of alloantigen, IL-12, and IL-6 promoted by Toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signals. Critically, alloantigen presentation in the mLNs imprints gut-homing integrin signatures on donor T cells, leading to their emigration into the GI tract where they mediate fulminant disease. These data identify a critical, anatomically distinct, donor DC subset that amplifies GVHD. We thus highlight multiple therapeutic targets and the ability of GVHD, once initiated by recipient antigen-presenting cells, to generate a profound, localized, and lethal feed-forward cascade of donor DC-mediated indirect alloantigen presentation and cytokine secretion within the GI tract.
Idiopathic pneumonia syndrome (IPS) is a relatively common, frequently fatal clinical entity, characterized by noninfectious acute lung inflammation following allogeneic stem cell transplantation ...(SCT), the mechanisms of which are unclear. In this study, we demonstrate that immune suppression with cyclosporin after SCT limits T-helper cell (Th) 1 differentiation and interferon-γ secretion by donor T cells, which is critical for inhibiting interleukin (IL)-6 generation from lung parenchyma during an alloimmune response. Thereafter, local IL-6 secretion induces donor alloantigen-specific Th17 cells to preferentially expand within the lung, and blockade of IL-17A or transplantation of grafts lacking the IL-17 receptor prevents disease. Studies using IL-6−/− recipients or IL-6 blockade demonstrate that IL-6 is the critical driver of donor Th17 differentiation within the lung. Importantly, IL-6 is also dysregulated in patients undergoing clinical SCT and is present at very high levels in the plasma of patients with IPS compared with SCT recipients without complications. Furthermore, at the time of diagnosis, plasma IL-6 levels were higher in a subset of IPS patients who were nonresponsive to steroids and anti-tumor necrosis factor therapy. In sum, pulmonary-derived IL-6 promotes IPS via the induction of Th17 differentiation, and strategies that target these cytokines represent logical therapeutic approaches for IPS.
•IL-6 is dysregulated after experimental allogeneic SCT and promotes alloantigen-dependent Th17 expansion within the lung.•IL-6 is dysregulated in patients with IPS after clinical allogeneic SCT.
The stimulation of naive donor T cells by recipient alloantigen is central to the pathogenesis of graft-versus-host disease after bone marrow transplantation (BMT). Using mouse models of ...transplantation, we have observed that donor cells become "cross-dressed" in very high levels of recipient hematopoietic cell-derived MHC class I and II molecules following BMT. Recipient-type MHC is transiently present on donor dendritic cells (DCs) after BMT in the setting of myeloablative conditioning but is persistent after nonmyeloablative conditioning, in which recipient hematopoietic cells remain in high numbers. Despite the high level of recipient-derived alloantigen present on the surface of donor DCs, donor T cell proliferative responses are generated only in response to processed recipient alloantigen presented via the indirect pathway and not in response to cross-dressed MHC. Assays in which exogenous peptide is added to cross-dressed MHC in the presence of naive TCR transgenic T cells specific to the MHC class II-peptide combination confirm that cross-dressed APC cannot induce T cell proliferation in isolation. Despite failure to induce T cell proliferation, cross-dressing by donor DCs contributes to generation of the immunological synapse between DCs and CD4 T cells, and this is required for maximal responses induced by classical indirectly presented alloantigen. We conclude that the process of cross-dressing by donor DCs serves as an efficient alternative pathway for the acquisition of recipient alloantigen and that once acquired, this cross-dressed MHC can assist in immune synapse formation prior to the induction of full T cell proliferative responses by concurrent indirect Ag presentation.
Allogeneic hematopoietic stem cell transplantation remains the cornerstone of curative therapy for haematological malignancies but is limited by graft-versus-host disease (GVHD), both acute and ...chronic. Exciting new approaches have been developed to prevent GVHD in the last 5 years and some have already successfully translated into the clinic. These strategies have been born from new model systems that allow the dissection of alloantigen specific responses that have in turn informed new disease paradigms. In this review, we discuss these recent insights into the biology of GVHD that appear to be highly predictive for improved clinical outcomes.
BackgroundTigilanol Tiglate (TT) is a novel small molecule under development for local treatment of solid tumours via intratumoral (I.T.) injection. TT is a protein kinase C (PKC)/C1 domain activator ...that disrupts tumour vasculature and causes direct oncolysis of tumour cells. Together, these activities lead to haemorrhagic necrosis of injected tumours with enduring ablation of >70% of target tumours in both pre-clinical xenograft/syngeneic mouse models and cutaneous tumours presenting in the veterinary clinic.1–3 TT has completed a Phase I/IIa dose-escalation trial in humans (ACTRN12614000685617), with strong evidence of local anti-cancer efficacy and signs of abscopal effects in some patients.4 However, the underlying mechanism of action (MOA) of TT, together with its immunotherapeutic potential in oncology, is not fully understood.MethodsA combination of microscopy, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, LDH release assays and mixed lymphocyte reactions were used to probe the MOA of TT in vitro. TT-mediated damage associated molecular pattern (DAMP) release/externalization was assessed using luciferase (ATP), ELISA (HMGB1), flow cytometry and immunohistochemical (HMGB1, calreticulin) approaches. In vivo experimentation with TT utilized CT-26 and B16-F10 tumor bearing mice. Analysis of DAMP release and immune cell infiltration into TT treated human head and neck tumours (ACTRN12619001407189) was performed by immunohistochemistry.ResultsOur data reveal that therapeutic concentrations of TT induce the death of cancer and endothelial cell lines via a pathway involving caspase activation and cleavage of the pore forming protein gasdermin E. TT promotes this mechanism of cell death by interacting with ER membranes, causing an ER stress response that results in loss of mitochondrial membrane potential, ATP depletion, organelle swelling and oncosis/pyroptosis. Treatment of cells with TT also led to the release of damage associated molecular patterns (DAMPs), indicative of an immunogenic cell death (ICD) pathway that also resulted in the generation of tumour-specific T cells in CT-26 tumor bearing mice. Whilst the induction of ICD is largely PKC-independent in vitro, PKC/C1 domain signaling appears necessary for efficacious tumour ablation in vivo. Consistent with our pre-clinical data, immunohistochemical analysis of TT-treated head and neck tumors found that drug stimulated DAMP release/externalisation and the recruitment of immune cells, principally CD8+ T cells, into remnant tumour mass.ConclusionsThese data indicate that TT is an oncolytic small molecule with the potential to ablate target tumours and enhance immunotherapy combinations through promoting immune cell infiltration. TT is currently undergoing Phase II trials in head and neck cancer (NCT05234437) and soft tissue sarcoma (NCT05755113).ReferencesBoyle GM, D’Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, Maslovskaya L, Gordon VA, Reddell PW, Parsons PG. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLOS ONE 2014;9:e108887.Cullen JK, Boyle GM, Yap PY, Elmlinger S, Simmons JL, Broit N, Johns J, Ferguson B, Maslovskaya LA, Savchenko AI, Mirzayans PM, Porzelle A, Bernhardt PV, Gordon VA, Reddell PW, Pagani A, Appendino G, Parsons PG, Williams CM. Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes. Sci. Rep. 2021;11:207.De Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, Kozak W, Pittenger ST, Pruitt JB, Riehl J, White J, Wiest ML, Johannes CM, Morton J, Jones PD, Schmidt PF, Gordon VA, Reddell PW. Randomized controlled clinical study evaluating the efficacy and safety of intratumoral treatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J. Vet. Intern. Med. 2021;35:415–429.Panizza BJ, de Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I dose-escalation study to determine the safety, tolerability, preliminary efficacy and pharmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EBioMedicine. 2019;50:433–441.Ethics ApprovalThe study obtained ethics approval from the following commitees and boards: Metro South Human Research Ethics Committee, 199 Ipswich Road, Woolloongabba, QLD, 4102, Australia. Ethics approval number: HREC/2019/QMS/54004. Bellberry Limited, 123 Glen Osmond Road, Eastwood, SA 5063, Australia. Ethics approval number: 2019–10-846 (REGIS 2019/ETH13063). Tata Memorial Hospital - Institutional Review Board, IRB Office, Dr. E. Borges Marg, Parel, Mumbai - 400 012, India. Ethics approval number: IEC/1219/3370/001. Tata Medical Center - Institutional Review Board, 14 Major Arterial Road (EW), New Town, Rajarhat, Kolkata - 700 160, India. Ethics approval number: 2019/PHARMA/57/IRB39.
Although the effects of type II-IFN (IFN-γ) on GVHD and leukemia relapse are well studied, the effects of type I-interferon (type I-IFN, IFN-α/β) remain unclear. We investigated this using type I-IFN ...receptor-deficient mice and exogenous IFN-α administration in established models of GVHD and GVL. Type I-IFN signaling in host tissue prevented severe colon-targeted GVHD in CD4-dependent models of GVHD directed toward either major histocompatibility antigens or multiple minor histocompatibility antigens. This protection was the result of suppression of donor CD4+ T-cell proliferation and differentiation. Studies in chimeric recipients demonstrated this was due to type I-IFN signaling in hematopoietic tissue. Consistent with this finding, administration of IFN-α during conditioning inhibited donor CD4+ proliferation and differentiation. In contrast, CD8-dependent GVHD and GVL effects were enhanced when type I-IFN signaling was intact in the host or donor, respectively. This finding reflected the ability of type I-IFN to both sensitize host target tissue/leukemia to cell-mediated cytotoxicity and augment donor CTL function. These data confirm that type I-IFN plays an important role in defining the balance of GVHD and GVL responses and suggests that administration of the cytokine after BM transplantation could be studied prospectively in patients at high risk of relapse.
Donor T cells play pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects following bone marrow transplantation (BMT). DNAX accessory molecule 1 (DNAM-1) is a ...costimulatory and adhesion molecule, expressed mainly by natural killer cells and CD8+ T cells at steady state to promote adhesion to ligand-expressing targets and enhance cytolysis. We have analyzed the role of this pathway in GVHD and GVL. The absence of DNAM-1 on the donor graft attenuated GVHD in major histocompatibility complex (MHC)-mismatched and MHC-matched BMT following conditioning with lethal and sublethal irradiation. In contrast, DNAM-1 was not critical for GVL effects against ligand (CD155) expressing and nonexpressing leukemia. The effects on GVHD following myeloablative conditioning were independent of CD8+ T cells and dependent on CD4+ T cells, and specifically donor FoxP3+ regulatory T cells (Treg). The absence of DNAM-1 promoted the expansion and suppressive function of Treg after BMT. These findings provide support for therapeutic DNAM-1 inhibition to promote tolerance in relevant inflammatory-based diseases characterized by T-cell activation.
•The DNAM-1 adhesion and costimulatory pathway promotes GVHD via effects on regulatory T cells.•Effective GVL can still occur in the absence of DNAM-1, making the pathway an attractive therapeutic target.
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