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Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

Nationwide Children’s Hospital

Human cytomegalovirus (HCMV) donor positive (D+) serostatus with acute rejection is associated with renal allograft loss, but the impact of recipient positive (R+) serostatus is unclear. In an allogeneic renal transplant model, antiviral natural killer (NK) and CD8+ T cell memory responses in murine CMV (MCMV) D+/R+ transplants were compared to D-/R- and D+/R- transplants, with recipient infection varied by MCMV dose and strain. D+/R- transplants had high primary antiviral cytolytic (interferon-γ+) and cytotoxic (granzyme B+) NK responses, whereas NK memory responses were lower in D+/R+ recipients receiving a high primary MCMV dose. Despite MCMV immunity, D+/R+ recipients receiving a low MCMV dose showed primary-like high cytolytic and cytotoxic NK responses. D+/R+ transplants infected with different D/R strains had low cytolytic NK responses but high cytotoxic NK responses. NK memory also induced a novel TNF-α+ NK response among high-dose virus recipients. MCMV+ transplants had greater Th17 responses than MCMV-uninfected transplants and Th17 inhibition ameliorated graft injury. All MCMV+ recipients had similar CD8+ T cell responses. In sum, NK and Th17 responses, but not CD8+ T cells, varied according to conditions of primary recipient infection. This variability could contribute to variable graft outcomes in HCMV D+/R+ renal transplantation.

NK cell and Th17 responses are differentially induced in murine cytomegalovirus infected renal allografts and vary according to recipient virus dose and strain.

 To characterize early events in neotissue formation during the first 2 weeks after vascular scaffold implantation.  Biodegradable polymeric scaffolds were implanted as abdominal inferior vena cava interposition grafts in wild-type mice.  All scaffolds explanted at day 1 contained a platelet-rich mural thrombus. Within the first few days, the majority of cell infiltration appeared to be from myeloid cells at the peritoneal surface with modest infiltration along the lumen. Host reaction to the graft was distinct between the scaffold and mural thrombus; the scaffold stimulated an escalating foreign body reaction, whereas the thrombus was quickly remodeled into collagen-rich neotissue.  Mural thrombi remodel into neotissue that persistently occludes the lumen of vascular grafts.

Early natural history of neotissue formation in tissue-engineered vascular grafts in a murine model.

Wall stress is often lower in tissue-engineered constructs than in comparable native tissues due to the use of stiff polymeric materials having thicker walls. In this work, we sought to design a murine arterial graft having a more favorable local mechanical environment for the infiltrating cells; we used electrospinning to enclose a compliant inner core of poly(glycerol sebacate) with a stiffer sheath of poly(caprolactone) to reduce the potential for rupture. Two scaffolds were designed that differed in the thickness of the core as previous computational simulations found that circumferential wall stresses could be increased in the core toward native values by increasing the ratio of the core:sheath. Our modified electrospinning protocols reduced swelling of the core upon implantation and eliminated residual stresses in the sheath, both of which had contributed to the occlusion of implanted grafts during pilot studies. For both designs, a subset of implanted grafts occluded due to thrombosis or ruptured due to suspected point defects in the sheath. However, there were design-based differences in collagen content and mechanical behavior during early remodeling of the patent samples, with the thinner-core scaffolds having more collagen and a stiffer behavior after 12 weeks of implantation than the thicker-core scaffolds. By 24 weeks, the thicker-core scaffolds also became stiff, with similar amounts of collagen but increased smooth muscle cell and elastin content. These data suggest that increasing wall stress toward native values may provide a more favorable environment for normal arterial constituents to form despite the overall stiffness of the construct remaining elevated due to the absolute increase in load-bearing constituents.

Electrospun Tissue-Engineered Arterial Graft Thickness Affects Long-Term Composition and Mechanics.

Human cytomegalovirus (CMV) infection is associated with renal allograft dysfunction and loss, particularly in combination with acute rejection. Emerging literature suggests that non-HLA antibodies may contribute to antibody-mediated rejection, but pathogen-induced antibodies have not been investigated in this context. This study examines the presence of CMV-induced antibodies in murine CMV (MCMV)-infected renal allografts during acute rejection.

Murine Cytomegalovirus-induced Complement-fixing Antibodies Deposit in Murine Renal Allografts During Acute Rejection.

CCR5 KO kidney transplant (KTx) recipients are extraordinarily high alloantibody producers and develop pathology that mimics human antibody-mediated rejection (AMR). C57BL/6 and CCR5 KO mice (H-2 ) were transplanted with A/J kidneys (H-2 ); select cohorts received adoptive cell therapy (ACT) with alloprimed CXCR5 CD8 T cells (or control cells) on day 5 after KTx. ACT efficacy was evaluated by measuring posttransplant alloantibody, pathology, and allograft survival. Recipients were assessed for the quantity of CXCR5 CD8 T cells and CD8-mediated cytotoxicity to alloprimed IgG B cells. Alloantibody titer in CCR5 KO recipients was four-fold higher than in C57BL/6 recipients. The proportion of alloprimed CXCR5 CD8 T cells 7 days after KTx in peripheral blood, lymph node, and spleen was substantially lower in CCR5 KO compared to C57BL/6 recipients. In vivo cytotoxicity towards alloprimed IgG B cells was also reduced six-fold in CCR5 KO recipients. ACT with alloprimed CXCR5 CD8 T cells (but not alloprimed CXCR5 CD8 or third-party primed CXCR5 CD8 T cells) substantially reduced alloantibody titer, ameliorated AMR pathology, and prolonged allograft survival. These results indicate that a deficiency in quantity and function of alloprimed CXCR5 CD8 T cells contributes to high alloantibody and AMR in CCR5 KO recipient mice, which can be rescued with ACT.

Antibody-suppressor CXCR5 CD8 T cellular therapy ameliorates antibody-mediated rejection following kidney transplant in CCR5 KO mice.

Human cytomegalovirus (HCMV) infection is associated with renal allograft failure. Allograft damage in animal models is accelerated by CMV-induced T helper 17 (Th17) cell infiltrates. However, the mechanisms whereby CMV promotes Th17 cell-mediated pathological organ inflammation are uncharacterized. Here we demonstrate that murine CMV (MCMV)-induced intragraft Th17 cells have a Th1/17 phenotype co-expressing IFN-γ and/or TNF-α, but only a minority of these cells are MCMV specific. Instead, MCMV promotes intragraft expression of CCL20 and CXCL10, which are associated with recruitment of CCR6 CXCR3 Th17 cells. MCMV also enhances Th17 cell infiltrates after ischemia-reperfusion injury, independent of allogeneic responses. Pharmacologic inhibition of the Th17 cell signature cytokine, IL-17A, ameliorates MCMV-associated allograft damage without increasing intragraft viral loads or reducing MCMV-specific Th1 cell infiltrates. Clinically, HCMV DNAemia is associated with higher serum IL-17A among renal transplant patients with acute rejection, linking HCMV reactivation with Th17 cell cytokine expression. In summary, CMV promotes allograft damage via cytokine-mediated Th1/17 cell recruitment, which may be pharmacologically targeted to mitigate graft injury while preserving antiviral T cell immunity.

Murine cytomegalovirus promotes renal allograft inflammation via Th1/17 cells and IL-17A.

Our understanding of tissue-resident memory T (T) cell biology has been largely developed from acute infection models in which antigen is cleared and sterilizing immunity is achieved. Less is known about T cells in the context of chronic antigen persistence and inflammation. We investigated factors that underlie T maintenance in a kidney transplantation model in which T cells drive rejection. In contrast to acute infection, we found that T cells declined markedly in the absence of cognate antigen, antigen presentation, or antigen sensing by the T cells. Depletion of graft-infiltrating dendritic cells or interruption of antigen presentation after T cells were established was sufficient to disrupt T maintenance and reduce allograft pathology. Likewise, removal of IL-15 transpresentation or of the IL-15 receptor on T cells during T maintenance led to a decline in T cells, and IL-15 receptor blockade prevented chronic rejection. Therefore, antigen and IL-15 presented by dendritic cells play nonredundant key roles in CD8 T cell maintenance in settings of antigen persistence and inflammation. These findings provide insights that could lead to improved treatment of chronic transplant rejection and autoimmunity.

Qiang Zeng

Department of Surgery

Nationwide Children’s Hospital

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Qiang Zeng

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Surgery

Nationwide Children’s Hospital

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Department of Surgery