Mouse Heart Transplantation Research Articles

Double Deficiency for RORγt and T-bet Drives Th2-Mediated Allograft Rejection in Mice

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

The protective effects of CD4+ CD25+ Treg cell on the allograft after infusion of dendritic cells with low expression of CD40 from donor in mouse heart transplantation

Objective To explore the effects of CD4+CD25+Treg cell on the allograft after infusion of dendritic cells (DCs) with low expression of CD40 from donor in mouse heart transplantation.Methods In vitro,mouse bone marrow-derived DCs were infected by CD40-RNAi lentiviral vector,and tolerogenic DCs (Tol-DCs) with low expression of CD40 were prepared.A heterotopic abdominal heart transplantation model was established in mice,and the other three groups that were control group,noninfected DC group and lentivirus infected DC group were designed correspondingly.Cardiac allograft survival time was recorded and pathological grading for acute rejection was assessed on the 7 d after heterotopic abdominal heart transplantation.Concentrations of CD4+CD25+Treg cells in peripheral blood were analyzed before and after transplantation by flow cytometry.Results After 48 h infection of DCs by CD40-RNAi lentiviral vector in vitro,the expression of CD40 mRNA was down-regulated significantly,whose inhibition rate was 80.9％.The expression of CD40 was decreased from 74.37％ ±4.08％ to 40.07％ ± 4.03％ (P＜0.05) after 48 h infection.Compared with the control group and the noninfected DC group,the cardiac allograft survival time was significantly prolonged in the CD40-RNAi lentivirus infected DC group,which was (14 ± 4) d(P＜0.01) ; concentrations of CD4+CD25+Treg cells in peripheral blood were increased both on the 3 d and the 7 d after transplantation (P＜0.05) ; the pathological grading for acute rejection was decreased on the 7 d after transplantation (P＜0.05).Conclusion The CD4+CD25+Treg cell in peripheral blood was protective to cardiac allograft in prolonging its survival time in mouse heart transplantation. Key words: Regulatory T cell; Costimulatory pathway; Dendritic cell; CD40; Heart transplantation

Immunosuppressive Effects of DTCM-G, a Novel Inhibitor of the mTOR Downstream Signaling Pathway

A newly developed compound, 3-[(dodecylthiocarbonyl)methyl]-glutarimide (DTCM-G), has been shown to inhibit nuclear translocation of c-Fos/c-Jun in a murine macrophage cell line. Herein, we studied the immunosuppressive properties and potency of DTCM-G. Using purified mouse T cells, the in vitro effects of DTCM-G on activation, cytokine production, proliferation, and cell cycle progression were assessed, and a possible molecular target of DTCM-G was investigated. In a BALB/c (H-2(d)) to C57BL/6 (H-2(d)) mouse heart transplantation model, transplant recipients were administered DTCM-G, a calcineurin inhibitor (tacrolimus), and a nuclear factor-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). Treatment drugs were administered daily for 14 days after transplantation. Alloimmune responses were assessed in addition to graft survival time. After anti-CD3+anti-CD28 monoclonal antibody stimulation, DTCM-G significantly suppressed proliferation, interferon-γ production, and cell cycle progression of activated T cells but not CD25 expression or interleukin-2 production. These effects were accompanied by inhibition of 70-kDa S6 protein kinase phosphorylation, a downstream kinase of the mammalian target of rapamycin. The addition of tacrolimus and DHMEQ to DTCM-G resulted in a robust inhibition of T-cell proliferation. In vivo combination therapy of DTCM-G plus either tacrolimus or DHMEQ significantly suppressed alloreactive interferon-γ-producing precursors and markedly prolonged cardiac allograft survival. Furthermore, combination of all three agents markedly inhibited alloimmune responses and permitted long-term cardiac allograft survival. DTCM-G inhibits T cells by suppressing the downstream signal of mammalian target of rapamycin. DTCM-G in combination with tacrolimus and DHMEQ induces a strong immunosuppressive effect in vivo.

Rapid, Simplified Technique for Heterotopic Cervical Heart Transplantation in Mice

Background: Methods for mouse vascularized heart transplantation using non-suture-cuff techniques have been described, but their delicate technique have prevented their widespread use. Here, we describe a simplified transplantation method with the cuff technique. Methods: By ligating the vessels and tissues including parts of the lungs and trachea en bloc, except ascending aorta, superior and inferior vena cava, and main pulmonary artery, it was possible to reduce the time of harvesting the donor heart. In heart transplantation, using 10-0 Ethilon, it became to be easy to pass the common Carotid Artery (CA) through the cuff. And using the ascending aorta of the donor heart, it also became to be easy to cover the end of the CA and fix it to the arterial cuff. Results: These modifications reduced the operating time. It typically takes 5 min to harvest the donor, 10 min to prepare the cervical vessels, and 15 min for anastomosis. All procedures, from donor harvest through skin closure in the recipient mice, can be completed within 40 min, and the cold ischemic time is less than 20 min. Conclusions: This method can be used to investigate mechanisms of MHC-disparate heart transplantation.

What Can Animal Models Teach Us About the Potential Safety and Efficacy of Regulatory Macrophage Therapy in Patients?

Cell-based immunosuppressive therapy is very much in its infancy, but with the inception of The ONE Study, a coordinated effort to bring such treatments to clinical trial is being made; therefore, we must ask, how great are the benefits of regulatory cell therapy in transplantation expected to be? Adoptive transfer of transplant tolerance with isolated populations of regulatory cell types is reliably achieved in homologous animal models, but whether such observations can be extrapolated to patients hinges on proving the equivalence between the animal and human cells in question. Our work centers on human regulatory macrophages (M reg) as a means of attenuating responses against alloantigen. The mouse proves a convenient model to test the combined effect of M reg treatment and conventional immunosuppression, but is only clinically relevant if mouse and human M regs are functional counterparts. M regs from both species arise from equivalent populations of monocytes under directly analogous culture conditions and share many morphological features and express unique markers, distinguishing them from other macrophages. Mouse and human M regs profoundly inhibit allogeneic T cell proliferation in co-culture, through deletion and phagocytosis of activated T cells, and a direct suppressive activity. Yet, differences between mouse and human M regs exist, and weighing these differences against the likenesses is necessarily subjective, so gene expression profiling by microarray was adopted as an unbiased approach to evaluating their true similarity. In co-clustering analyses, M regs from the two species grouped together when the entire set of orthologous genes was considered, so identifying human M regs as the closest counterpart of mouse M regs.[Figure]In a mouse heart transplant model, infusion of donor-strain M regs prior to transplantation significantly prolonged allograft survival in unconditioned, non-immunosuppressed, fully-allogeneic recipients, whereas recipient and third party-derived M regs were not effective. Co-treatment with M regs and rapamycin for 10-days post-transplant enhanced the effect of M regs compared to M reg or rapamycin treatment alone. After injection, M regs from CD45.2+ B6 mice or CD45.2+ BALB/c mice were detectable in the lung, blood, liver and spleen of CD45.1+ B6 recipients for up to two weeks. Donor-derived human M regs were administered to two renal transplant recipients 7-days prior to transplantation. Both patients were minimised to low-dose tacrolimus monotherapy within 24 weeks of transplantation and subsequently maintained excellent graft function. To assess their fate after infusion, M regs were labelled with Indium111-oxine for tracking in serial whole-body SPECT studies, which showed most M regs remained viable and trafficked from the pulmonary vasculature via the blood to liver, spleen and bone marrow.

New Approach to Selective Targeting of Dendritic Cells by Sirna, Resulting in Long Term Graft Survival in Mouse Heart Transplantation Model

New Approach to Selective Targeting of Dendritic Cells by Sirna, Resulting in Long Term Graft Survival in Mouse Heart Transplantation Model

Immunomodulatory Effect of Nuclear Factor-κB Inhibition by Dehydroxymethylepoxyquinomicin in Combination With Donor-Specific Blood Transfusion

Nuclear factor-κB (NF-κB) is a key molecule in alloimmune responses, however, its role in tolerance induction is not clear. We have previously reported that dehydroxymethylepoxyquinomycin (DHMEQ), a novel NF-κB inhibitor, prolongs cardiac allograft survival. In this study, we evaluated the immunomodulatory effects of DHMEQ when combined with a donor-specific blood transfusion (DST), and assessed whether the treatment induces tolerance in a mouse heart transplantation model. DST (20×10 splenocytes) was given intravenously at day -7. DHMEQ (30 mg/kg/day) was administered intraperitoneally for 14 days after DST. Graft survival and histology were evaluated. The underlying mechanisms of immunomodulation by DST and DHMEQ treatments were investigated by assessing alloimmune responses after transplantation. In fully mismatched H2-to-H2 heart transplants, DST alone prolonged allograft median survival time to 15 days, whereas when DST was combined with DHMEQ treatment, the graft median survival time was prolonged to 39.5 days. When the donor-recipient strain combination was reversed, that is, H2-to-H2, heart transplants were accepted (>150 days survival) in more than 60% of recipients treated with a DST and DHMEQ, whereas control allografts were all rejected within 8 days. The combined therapy markedly inhibited immune responses by both the direct and indirect allorecognition pathways mainly attributed to promotion of activation-induced cell death and Treg generation. Our results demonstrate the distinctive ability of NF-κB inhibition in combination with donor alloantigen to promote transplantation tolerance through multiple cellular mechanisms.

Decrease in donor heart injury by recombinant clusterin protein in cold preservation with University of Wisconsin solution

Donor organ injury during cold preservation before transplantation negatively impacts graft survival. Clusterin (CLU) is a chaperonic protein, and its expression confers donor hearts resistance to cold ischemic injury. This study was conducted to evaluate if the supplement of recombinant CLU protein (rCLU) protects donor organs from injury during cold storage with University of Wisconsin (UW) solution. Human endothelial cell cultures were used as an invitro model. Heart transplantation in mice was used as an invivo model. Cell membrane disruption or death was indicated by the release of lactate dehydrogenase (LDH). Donor injury was determined by its functional recovery, and histologic and biochemical analyses. Supplement of rCLU to UW solution protected cultured human endothelial cells from cold-induced cell necrosis, as evidenced by a decrease in both release of LDH and the number of ethidium bromide-stained necrotic cells. The protective activity of rCLU was associated with enhanced membrane fluidity at cold temperature. During cold storage of heart organs in UW solution, supplemental rCLU significantly decreased LDH release from heart tissue. In a preclinical model of transplantation, heart grafts after cold preservation with rCLU-containing UW solution had better functional recovery and decreased perivascular inflammation, neutrophil infiltration, and cardiac cell death, including apoptosis and necrosis, that correlated with lower levels of serum creatine kinase and LDH in recipients. Our data suggest that supplement of CLU protein in a cold preservation solution may have potential in improving cold preservation of donor organs in transplantation.

IL-33 Expands Suppressive CD11b+ Gr-1int and Regulatory T Cells, including ST2L+ Foxp3+ Cells, and Mediates Regulatory T Cell-Dependent Promotion of Cardiac Allograft Survival

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.

Effect of CD4 + Memory T Cells on Rejection Response of Ectopic Heart Transplantation in Mice

Background CD4 + memory T cells mediate resistance of the body to infection by exotic pathogens. This study investigated the effects of alloreactive CD4 + memory T cells on acute graft rejection responses toward ectopic hearts in the abdominal cavities of mice. Methods BALB/C mice were used as recipients and C57BL/6 mice as donors. The animals in the CD4 + memory T-cell group were infused with CD4 + memory T cells, those in the other group were infused with nonsensitized CD4 + T cells, and those in the control group received no CD4 + T cells. Heart transplantation was performed at 3 weeks after the cell infusion with cyclosporine administered beginning 1 day before transplantation via intraperitoneal injection. Results The survival among the CD4 + memory T-cell group was significantly shorter than that of the nonsensitized CD4 + T-cell group or the control group ( P < .01; n = 10). There was no significant difference between the survival times of the nonsensitized CD4 + T-cell group and the control group ( P > .05; n = 10). On the 5th day after the transplantation of heart, the histologic grades of the nonsensitized CD4 + T-cell and the blank control group were lower than those of the CD4 + memory T-cell group. There was no significant difference in the histologic grades between the nonsensitized CD4 + T-cell and control groups. Conclusion The CD4 + memory T cells that mediate acute rejection of allografted hearts are insensitive to cyclosporine.

Early immune activation of donor organs after brain death in a mouse heart transplantation model

Early immune activation of donor organs after brain death in a mouse heart transplantation model

Administration of anti-interleukin-6 monoclonal antibody prolongs cardiac allograft survival

To investigate the effects of anti-IL-6 monoclonal antibody (anti-IL-6 mAb) on acute allograft rejection and the potential mechanisms in a mouse heart transplantation model. Heterotopical heart graft model was performed. The anti-IL-6 mAb was administered to recipient mice after cardiac grafting. Results were compared with administration of anti-IL-17 mAb or anti-IL-6 mAb+anti-IL-17 mAb (the 'double' treatment). The cardiac allograft survival was monitored by daily palpation in combination with histological evaluation. Quantitative polymerase chain reaction assay, mixed lymphocyte reaction, and flow cytometric analysis were employed to determine the mRNA expression of pro-inflammatory cytokines, allogeneic T-cell proliferation, and the proportion of CD4(+) CD25(+) Foxp3(+) regulatory T cells in graft-infiltrating lymphocytes and splenocytes of recipients, respectively. The results showed that the cardiac allograft survival in anti-IL-6 mAb-treated mice was prolonged significantly when compared with that of the untreated or anti-IL-17 mAb-treated mice. Meanwhile, the 'double-treated' did not prolong graft survival significantly when compared with those treated with anti-IL-6 mAb. The increase of graft survival induced by anti-IL-6 mAb was associated with reduced transcript levels for IFN-γ and IL-17, accompanied by a dramatic reduction of T-cell proliferation capacity to alloantigen stimuli and a higher proportion of Treg cells. Thus, anti-IL-6 mAb may be protective against acute rejection after cardiac transplantation through suppressing the activation of effector T cells and promoting the induction of Treg cells.

Clinical Summaries

Clinical Summaries

ICOS-Ig combined with CsA induces long term survival of cardiac allografts in mouse

Objective To study the synergistic effect of ICOS-Ig combined with cyclosporine (CsA) on mouse heart transplantation and explore its therapeutic potential. Methods ICOS-Ig fusion protein was generated by fusing the extracellular portion of human ICOS and Fc portion of human IgG. To investigate the effect of ICOS-Ig on T-cell proliferation in vitro, ICOS-Ig or IgG was added to the primary MLR cultures (BALB/c spleen T cells as responder cells and irradiated C57BL/6 spleen cells as stimulator cells). The cells responsiveness rates were detected by 3H-TdR methods. Then the T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 (donor) or C3H (third party) spleen cells as stimulator cells. To study the effect of ICOS-Ig on T-cell proliferation in vivo, CFSE-labeled C57BL/6 spleen cells were transferred to irradiated BALB/c mice. Mice were then treated with IgG, ICOS-Ig or CsA. Seventy two hours after transfer, the spleen cells of the mice were harvested for the detection of CD4 +CFSE + and CD8 +CFSE + by FACS. C57BL/6 mouse underwent transplantation of the hearts of BALB/c mouse and were then randomly divided into five equal groups: no treatment group, control IgG treated group (250 μg i.p. d2, 4, 6), ICOS-Ig treated group (250 μg i.p. d2, 4, 6), CsA treated group (10 mg/kg i.p. d0-6), ICOS-Ig combined with CsA group. The cardiac allograft survival was monitored by daily palpation. Results In primary MLR, ICOS-Ig inhibited T-cell proliferation, (inhibition ratio 58±8.2% in 50 μg/ml). In secondary MLR, ICOS-Ig specifically inhibited donor spleen cells, which suggested ICOS-Ig could induce donor-specific hyporesponsiveness. In the CFSE dye assay, CD4 +CFSE + and CD8 +CFSE + in ICOS-Ig and CsA group was stronger than those in control group, which showed ICOS-Ig and CsA could inhibit the proliferation of allo-reactive T cells in vivo. In mouse heart transplantation model, survival was significantly prolonged in animals treated with ICOS-Ig or CsA as compared with controls. Moreover, ICOS-Ig combined with CsA group had even longer engraftment (>100 d) than ICOS-Ig or CsA used alone. In histological examination, it was found that there were congestions and edemas in no treatment and IgG treated recipients, together with a lot of inflammatory cells infiltrated. Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealed milder histological changes. It was revealed in mechanical analysis that splenic T cells from recipients also exhibited depressed mixed leukocyte reactions (MLR) and cytotoxic lymphocyte reactions (CTL). Conclusion These data suggest that ICOS-Ig combined with CsA induces a long-term survival of mouse cardiac allografts, whereas monotherapy is less effective in this regard. Thus, ICOS-Ig combined with CsA treatment may be a novel regimen to combat allograft rejection.

Arsenic Trioxide Attenuated the Rejection of Major Histocompatibility Complex Fully-Mismatched Cardiac Allografts in Mice

We investigated the effects of arsenic trioxide (As 2O 3) on allogeneic immune response using a mouse heart transplantation model. Mice were randomly divided into 4 groups of 6 animals each. The control group received phosphate-buffered saline (PBS); the As 2O 3-treated group, intraperitoneal (IP) injection of As 2O 3 (1 mg/kg) from days −3 to 10 after heart transplantation. The cyclosporine (CsA)-treated group was given a subtherapeutic dose of CsA (10 mg/kg) IP, and the As 2O 3 plus CsA-treated group, a combined protocol of As 2O 3 and CsA. Six days after transplantation, cardiac allografts were harvested for immunohistology and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The survival of the allografts was significantly improved among the As 2O 3-treated group compared with the control group (17.2 ± 1.9 vs 8.0 ± 0.9 days; P < .05). A marked prolongation (28.6 ± 6.0 days) of graft survival was achieved by the combined protocol compared with the CsA-treated group (9.6 ± 3.0 days; P < .05) or the As 2O 3-treated group. Allografts of As 2O 3-treated and As 2O 3 plus CsA-treated mice showed a changing pattern of Th1/Th2 cytokine mRNA expression. Allograft rejection was apparently alleviated by low-dose As 2O 3, and particularly when combined with a subtherapeutic CsA dose.

STAT-1 decoy oligodeoxynucleotide inhibition of acute rejection in mouse heart transplants.

During acute rejection of cardiac transplants endothelial cell–leukocyte interaction fuelled by co-stimulatory molecules like CD40/CD154 may ultimately lead to graft loss. One key player in up-regulating the expression of such pro-inflammatory gene products is the interferon-γ-dependent transcription factor STAT-1. Hence down-regulating interferon-γ-stimulated pro-inflammatory gene expression in the graft endothelial cells by employing a decoy oligodeoxynucleotide (dODN) neutralising STAT-1 may protect the graft. To verify this hypothesis, heterotopic mouse heart transplantation was performed in the allogeneic B10.A(2R) to C57BL/6 and syngeneic C57BL/6 to C57BL/6 strain combination without immunosuppression. Graft vessels were pre-treated with STAT-1 dODN, mutant control ODN (10 μM each) or vehicle (Ringer solution). Cellular rejection (vascular and interstitial component) was graded histologically and CD40, ICAM-1, VCAM-1, MCP-1, E-selectin and RANTES expression in the graft monitored by real time PCR 24 h and 9 days post-transplantation. Nine days after transplantation both rejection scores were significantly diminished by 85 and 70%, respectively, in STAT-1 dODN-treated allografts as compared to mutant control ODN-treated allografts. According to immunohistochemistry analysis, this was accompanied by a reduced infiltration of monocyte/macrophages and T cells into the graft myocardium. In addition, pro-inflammatory gene expression was strongly impaired by more than 80% in STAT-1 dODN-treated allografts 24 h post-transplantation but not in mutant control ODN or vehicle-treated allografts. This inhibitory effect on pro-inflammatory gene expression was no longer detectable 9 days post-transplantation. Single periprocedural treatment with a STAT-1 dODN thus effectively reduces cellular rejection in mouse heart allografts. This effect is associated both with an early decline in pro-inflammatory gene expression and a later drop in mononuclear cell infiltration.

Assessment of graft function in rodent models of heart transplantation

Heterotopic heart transplantation in rats and mice is the most commonly used model to study allograft immune response and to test immunosuppressive drugs and tolerance induction protocols. Standardization of both the surgical procedure and the evaluation of graft function is essential for data interpretation. The most popular way to monitor graft function has been the palpation method. However, there are some proposal for more objective assessment methods like electrocardiogram and echocardiogram. Although, complementary tests might add some relevant information when assessing minor effects of immunosuppressive therapy, palpation by an experienced investigator is very predictive and so far the simplest method to determine heart allograft function. Minor complications during the surgical procedure and unreliable assessment can have a major impact on the interpretation of experiment results. Here, the author reviews the literature and presents some suggestions that help eliminating biases on the assessment of heart allograft function.

Altered Distribution of H60 Minor H Antigen-Specific CD8 T Cells and Attenuated Chronic Vasculopathy in Minor Histocompatibility Antigen Mismatched Heart Transplantation in Cxcr3−/− Mouse Recipients

Chemokine-chemokine receptor interactions and the subsequent recruitment of T lymphocytes to the graft are believed to be among the initial events in the development of acute and chronic rejection of heart transplants. We sought to determine the role of chemokine receptor Cxcr3 on the development of acute and chronic rejection in a multiple minor Ag mismatched mouse heart transplant model. The frequencies and kinetics of immunodominant H60 (LTFNYRNL) miHA-specific CD8 T cells in wild-type or Cxcr3-/- C57BL/6 recipients were monitored using MHC class I tetramer after BALB/b donor hearts were transplanted. Acceptance of grafts, severity of rejection, and infiltration of T cells were not altered in Cxcr3-/- recipients. However, graft survival was moderately prolonged in Cxcr3-/- recipient mice undergoing acute rejection. Analyses of splenocytes, PBLs, and graft-infiltrating cells revealed increased alloreactive T cells (H60-specific CD8 T cells) in the peripheral blood and spleen but not in the graft. Adoptively transferred Cxcr3-/- CD8 T cells in the BALB/b heart-bearing B6 scid mice showed retention of alloreactive CD8 T cells in the blood but less infiltration into the graft. Cxcr3-/- recipients with long-term graft survival also showed a marked decrease of CD8+ T cell infiltration and reduced neo-intimal hyperplasia. These data indicate that Cxcr3 plays a critical role in the trafficking as well as activation of alloreactive T cells. This role is most eminent in a transplant model when a less complex inflammatory milieu is involved such as a well-matched graft and chronic rejection.

Cardiac proteasome dysfunction during cold ischemic storage and reperfusion in a murine heart transplantation model

Recent observations suggest that the ubiquitin–proteasome system (UPS) contributes to the pathophysiology of myocardial ischemia–reperfusion injury. Since its regulation during cold ischemia–reperfusion is unknown, we evaluated the cardiac UPS in a model of heart transplantation in mice. Cardiac ubiquitylation rates and ubiquitin–protein conjugates increased after 3 h of cold ischemia (CI) and normalized post-transplant. 20S proteasome content and proteasome peptidase activities were unchanged after CI. 4 h/24 h post-transplant 20S proteasome concentrations decreased and chymotryptic-like but not tryptic-like proteasome peptidase activity was inactivated. Epoxomicin sensitivity of the proteasome increased 5.7-fold during CI and normalized 4 h/24 h post-transplant. This was accompanied by the disappearance of a 13.5 kDa-ubiquitin-conjugate during CI that could be attenuated by addition of epoxomicin to the preservation fluid. We conclude that substrate specificity of the proteasome changes during cold ischemia and that proteasome inhibition preserves the physiological ubiquitin–protein conjugate pool during organ preservation. Reduced proteasome activity during reperfusion is caused by a decrease in proteasome content and enzyme inhibition.

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis. When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.