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Principal Investigator: Dan Jane-Wit
Organization: VA CONNECTICUT HEALTHCARE SYSTEM
Fiscal Year: 2024
Funding agency: Veterans Affairs
Ischemia reperfusion injury (IRI) is a pathological process affecting solid organ allografts and occurs
when a transplanted organ subjected to prolonged disruption of blood flow undergoes tissue damage following
surgical implantation. IRI frequently occurs among deceased donors and predisposes to chronic antibody-
mediated rejection (CABMR), decreased graft survival, and worsened patient outcomes. This condition has no
effective medical treatments and is especially relevant to veteran transplant recipients who undergo higher
rates of deceased donor transplants and are thus at risk for developing IRI and its related complications.
In recognition of the significant lifetime health burden and costs incurred by IRI to veteran patients and
to the VA Healthcare System, respectively, recent federal mandates have established mechanisms to
systemically improve care for veteran recipients of solid organ transplants, in particular renal transplants. This
mandate will increase the number of transplanting VA centers, institute systems-based changes to improve
pre- and post-operative access to care, and promote basic research to improve allograft survival.
In this context, we submit this transplant immunology application to explore a role for Hedgehog (Hh)
signaling in CABMR, an IRI-associated complication in solid organ transplantation. Complement (C') are
immune proteins involved in host defense that are pathologically activated on endothelial cells (ECs) during
IRI, a process we have found selectively expands T peripheral helper (TPH cells), a recently discovered CD4+ T
cell subset specialized in provision of B cell help. Hh signaling is a widely studied pathway regulating wound
healing by eliciting vascular cell proliferation. We reasoned that Hh ligands are released by C'-injured ECs
during IRI as a signal for autologous wound repair, but concurrently these same ligands could expand
alloimmune TPH cells that go on to promote alloantibody responses and CABMR-related pathologies.
To test this notion, we used humanized models in vitro and in vivo, and we used prospectively collected
patient samples to increase the clinical relevance of our findings. Our data showed that IRI-treated ECs
produced Hh ligands in a C'-dependent manner that selectively activated T peripheral helper (TPH) cells, a
newly described T cell subset specialized in provision of B cell help. Mechanistically, Hh induced ZFYVE21, a
novel Rab5 effector we discovered, to elicit Akt-mediated NRLP3 inflammasomes in TPH cells. This resulted in
IL-18 release and IL-18-mediated expansion of IL-18R1+TPH cells that promoted CABMR-like pathologies
including vascular inflammation, alloAb production, and vasculopathy. These exciting preliminary data address
an important and prevalent clinical problem that is a focus area of recent federal initiatives at the VA.
From these data, we hypothesize that Hedgehog ligands released by C'-injured EC during IRI activate
CD4+ TPH cells to elicit CABMR-associated pathologies. We will explore this hypothesis in 2 non-
interdependent Aims. In Aim 1, we will employ biochemical and functional assays to identify mechanisms(s) by
which Hh agonism activates a ZFYVE21-Akt-Casp1 axis to elicit inflammasome-dependent expansion of TPH
cells. We propose cutting edge approaches including phosphoproteomic analyses to facilitate mechanistic
discovery. In Aim 2, we will test the relevance of ZFYVE21-Akt-Casp1 signaling in vivo using 2 humanized
mouse models of CABMR, and we will test the clinical relevance of lead molecule(s) using prospectively
collected patient specimens. Our studies explore Hedgehog signaling as a new pathway that may be feasibly
modulated in clinical settings with recently FDA-approved agents to putatively block IRI-associated
complications like CABMR in solid organ transplantation. As such, findings from these studies may be relevant
for improving allograft survival in veteran patients who are at risk for IRI and its associated complications.
Terms: <19S Gamma Globulin><Access to Care><Address><Affect><Alloantibodies><Allografting><Antigen-Presenting Cells><Area><Assay><Autologous><B blood cells><B cell><B cells><B-Cells><B-Lymphocytes><B-cell><Basic Research><Basic Science><Bioassay><Biochemical><Biological Assay><Blood Vessels><Blood flow><Body Tissues><C 5b-9><C5b-9><CD4 Cells><CD4 Positive T Lymphocytes><CD4 T cells><CD4 helper T cell><CD4 lymphocyte><CD4+ T-Lymphocyte><CD4-Positive Lymphocytes><Caring><Cell Body><Cell Communication and Signaling><Cell Function><Cell Growth in Number><Cell Multiplication><Cell Physiology><Cell Process><Cell Proliferation><Cell Signaling><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular Proliferation><Cellular injury><Chronic><Clinical><Complement><Complement Complex C5b-9><Complement Membrane Attack Complex><Complement Proteins><Complication><Cytolytic Terminal Complement Complex><Data><Diagnostic><Donor person><Endothelial Cells><Erinaceidae><FDA approved><Graft Survival><Grafting Procedure><Health><Health Care Systems><Health Services Accessibility><Healthcare Systems><Hedgehog (Hh) signal transduction pathway><Hedgehogs><Helper Cells><Helper T-Cells><Helper T-Lymphocytes><Helper-Inducer T-Cells><Helper-Inducer T-Lymphocyte><Host Defense><Human><Hypoxia><Hypoxic><IFN-Gamma-Inducing Factor Gene><IFN-gamma-Inducing Factor><IGIF><IGIF Gene><IL-1 Gamma><IL-1 Gamma Gene><IL-18><IL-18 Gene><IL-1g><IL-1g Gene><IL18><IL18 Protein><IL18 gene><IL1F4><IL1F4 Gene><IgM><Immune><Immunes><Immunoglobulin M><In Vitro><Inducer Cells><Inducer T-Lymphocytes><Inflammasome><Innate Immunity><Interferon-Gamma-Inducing Factor Gene><Interferon-gamma-Inducing Factor><Interleukin 18 (Interferon-Gamma-Inducing Factor)><Interleukin 18 (Interferon-Gamma-Inducing Factor) Gene><Interleukin 18 Proprotein><Interleukin 18 Proprotein Gene><Interleukin-1 Gamma><Interleukin-1 Gamma Gene><Interleukin-18><Interleukin-18 Precursor><Interleukin-18 Precursor Gene><Intracellular Communication and Signaling><Ischemia-Reperfusion Injury><Isoantibodies><Kidney Grafting><Kidney Transplantation><Kidney Transplants><Lead><Lesion><Ligands><Link><MGC12320><MGC12320 Gene><Mediating><Medical><Membrane Attack Complex><Modeling><Modern Man><Molecular><Native Immunity><Natural Immunity><Non-Specific Immunity><Nonspecific Immunity><Operative Procedures><Operative Surgical Procedures><Organ Transplantation><Organ Transplants><Outcome><Oxygen Deficiency><Pathologic><Pathologic Processes><Pathological Processes><Pathology><Pathway interactions><Patient outcome><Patient-Centered Outcomes><Patient-Focused Outcomes><Patients><Pb element><Peripheral><Phosphorylation><Population><Post-Operative><Postoperative><Postoperative Period><Process><Production><Property><Protein Phosphorylation><Proteins><Renal Grafting><Renal Transplantation><Renal Transplants><Reperfusion Damage><Reperfusion Injury><Research Specimen><Respondent><Risk><Rodent><Rodentia><Rodents Mammals><Role><Sampling><Signal Transduction><Signal Transduction Systems><Signaling><Site><Solid><Specimen><Subcellular Process><Surgical><Surgical Interventions><Surgical Procedure><System><T-Cell Subsets><T-Cells><T-Lymphocyte><T-Lymphocyte Subsets><T4 Cells><T4 Lymphocytes><Terminal Complement Complex><Testing><Tissues><Transplant Recipients><Transplant immunology><Transplantation><Transplantation Immunology><Vascular Diseases><Vascular Disorder><Veterans><Work><Wound Repair><access to health services><access to services><access to treatment><accessibility to health services><accessory cell><alloimmunity><antibody rejection><antibody-mediated rejection><availability of services><biological signal transduction><blood vessel disorder><care access><cell damage><cell injury><cellular damage><clinical relevance><clinically relevant><complementation><cost><damage to cells><delayed graft function><donor antibodies><donor-specific antibody><effective therapy><effective treatment><genetic approach><genetic strategy><health service access><health services availability><heavy metal Pb><heavy metal lead><hedgehog signaling><hedgehog signaling pathway><hh signaling pathway><humanized mice><humanized mouse><implantation><improved><in vitro Model><in vivo><injured><injury to cells><isoimmunity><kidney tx><mouse model><murine model><novel><organ allograft><organ graft><organ xenograft><pathway><patient oriented outcomes><pharmacologic><phospho-proteomics><phosphoproteomics><prospective><response><service availability><smoothened signaling pathway><social role><surgery><thymus derived lymphocyte><transplant><transplant donor><transplant patient><treatment access><vascular><vascular dysfunction><vascular inflammation><vasculopathy><wound healing><wound recovery><wound resolution>