Todd V. Brennan

Heparan sulfate, an endogenous TLR4 agonist, promotes acute GVHD after allogeneic stem cell transplantation

Graft-versus-host disease (GVHD) remains the most common cause of nonrelapse-related morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although T-cell depletion and intensive immunosuppression are effective in the control of GVHD, they are often associated with higher rates of infection and tumor recurrence. In this study, we showed that heparan sulfate (HS), an extracellular matrix component, can activate Toll-like receptor 4 on dendritic cells in vitro, leading to the enhancement of dendritic cell maturation and alloreactive T-cell responses. We further demonstrated in vivo that serum HS levels were acutely elevated at the onset of clinical GVHD in mice after allo-HSCT. Treatment with the serine protease inhibitor α1-antitrypsin decreased serum levels of HS, leading to a reduction in alloreactive T-cell responses and GVHD severity. Conversely, an HS mimetic that increased serum HS levels accelerated GVHD. In addition, in patients undergoing allo-HSCT for hematologic malignancies, serum HS levels were elevated and correlated with the severity of GVHD. These results identify a critical role for HS in promoting acute GVHD after allo-HSCT, and they suggest that modulation of HS release may have therapeutic potential for the control of clinical GVHD.

Renal transplant imaging using magnetic resonance angiography with a nonnephrotoxic contrast agent.

Background In renal allograft recipients presenting with graft dysfunction, it is critical to determine the patency of the transplant vasculature to guide clinical management. Conventional modalities such as Doppler ultrasound, contrast-enhanced computed tomography, magnetic resonance angiography (MRA), and noncontrast MRA are each of limited use because of technical factors and toxicity of standard contrast agents. The purpose of this study was to retrospectively review our institutional experience with renal transplant MRA using ferumoxytol (a nonnephrotoxic medication) as a contrast agent and evaluate its use in the assessment of allograft vascular patency in patients with graft dysfunction, either delayed or slow graft function within hours to days after kidney transplantation or acute kidney injury weeks to months after kidney transplantation. Methods Sixteen kidney transplant recipients were retrospectively identified who underwent ferumoxytol-enhanced MRA after a nondiagnostic ultrasound for kidney dysfunction after transplantation. Image evaluation was performed by two radiologists, and clinical follow-up data were collected. Results In 1 of 16 subjects, MRA with ferumoxytol demonstrated complete arterial occlusion of an allograft. In 2 of 16 subjects, MRA detected moderate to severe anastomotic stenoses, which were confirmed at catheter angiography and successfully treated, resulting in the improvement of graft function. In 13 of 16 subjects, MRA demonstrated normal graft vasculature, and an alternative cause of allograft dysfunction was ultimately confirmed. Conclusions Our study suggests that ferumoxytol-enhanced MRA may be a novel, safe method to accurately detect graft artery abnormalities in renal transplant recipients without the risk of nephrotoxicity, when transplant ultrasound is nondiagnostic.

A New T-Cell Receptor Transgenic Model of the CD4+ Direct Pathway : Level of Priming Determines Acute Versus Chronic Rejection

Background. T-cell receptor transgenic (TCR-tg) mouse models with direct CD4+ alloreactivity will help elucidate mechanisms of transplant rejection and tolerance in vivo. Although such models exist, they are limited by unusual strain combinations or are based on model antigens. Methods. A TCR-tg mouse with direct CD4 specificity in the widely used BALB/c donor → C57BL/6 host strain combination was created. This TCR-tg mouse, named 4C, was selected for reactivity against BALB/c dendritic cells in order to model early priming events after transplantation. The response of 4C T cells to skin and heart transplants were characterized. Results. The alloantigen is restricted by I-Ad and appears to be widely distributed in mouse tissues. 4C T cells are able to acutely reject skin but not heart allografts. Paradoxically, heart grafts elicited a stronger proliferation and effector function of TCR-tg T cells than skin grafts. 4C T cells caused cardiac allograft vasculopathy in the absence of other T cells and alloantibodies, suggesting a role for the direct pathway in chronic rejection. Augmentation of priming with an infusion of donor-derived dendritic cells resulted in acute heart allograft rejection by 4C T cells, demonstrating that the level of priming can play a role in determining acute versus chronic rejection by the CD4 direct pathway. Conclusions. Rejection of a graft by the direct CD4 pathway is determined by graft susceptibility to rejection, as well as the degree of T-cell priming caused by the graft. Grafts that are not acutely rejected can develop transplant vasculopathy mediated by the direct CD4+ T cells.

Requirements for Prolongation of Allograft Survival with Regulatory T Cell Infusion in Lymphosufficient Hosts

BACKGROUND For the clinical applicability of regulatory T cells (Tregs) in transplantation, it is critical to determine if donor antigen specificity is required for their immunosuppressive function. We developed an allospecific CD4(+) T cell receptor transgenic (TCR-tg) mouse as a source for large numbers of Tregs with defined allospecificity and tested whether they are more effective than polyclonal Tregs at suppressing allograft rejection. MATERIALS AND METHODS CD4(+)CD25(+)CD62L(hi) T cells were sorted from the spleen and peripheral lymph nodes of wild-type (WT-Tregs) and TCR-tg (Allo-Tregs) mice, and expanded using IL-2 and anti-CD3/anti-CD28 conjugated magnetic beads. Tregs were tested for their ability to suppress the proliferation and cytokine production of alloreactive CD4(+)CD25(-) T cells in mixed leukocyte assays. Syngeneic WT hosts were adoptively transferred 5 × 10(6) Tregs and transplanted with allogeneic hearts. RESULTS Using anti-CD3/anti-CD28 conjugated beads, Tregs were expanded in vitro 100-fold and maintained their suppressor phenotype and function. Allo-Tregs were 6-8 times more potent on a cell-for-cell basis than WT-Tregs in suppressing allospecific proliferation in vitro. Allo-Tregs were unable to suppress in the absence of allo-antigen. Adoptive transfer of expanded Allo-Tregs into WT recipients prolonged the graft survival in a F1 heart transplant model compared with WT-Treg or no treatment [20.0 ± 4.4 d (n = 6) versus 10.4 ± 1.2 (n = 8) and 9.7 ± 1.6 d (n = 6)]. CONCLUSIONS Unlike polyclonal Tregs, allospecific Tregs are able to prolong allograft survival. However, large numbers of Allo-Tregs were unable to induce tolerance, suggesting that Treg therapy in immunocompetent recipients will require conditioning and/or additional immunomodulation for the induction of tolerance.

Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation

Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell-based antitumor therapies.

Posterior Reversible Encephalopathy Syndrome Independently Associated With Tacrolimus and Sirolimus After Multivisceral Transplantation

Posterior reversible encephalopathy syndrome (PRES) is a small vessel microangiopathy of the cerebral vasculature that occurs in 0.5–5% of solid organ transplant recipients, most commonly associated with tacrolimus (Tac). Clinical manifestations include hypertension and neurologic symptoms. We report an adult multivisceral transplant recipient who experienced recurrent PRES initially associated with Tac and subsequently with sirolimus. A 49‐year‐old woman with short bowel syndrome underwent multivisceral transplantation due to total parenteral nutrition–related liver disease. She was initially maintained on Tac, mycophenalate mofetil (MMF) and prednisone. Three months after transplantation, she developed renal dysfunction, leading to a reduction in Tac and the addition of sirolimus. Eight months after transplantation, she developed PRES. Tac was discontinued and PRES resolved. Sirolimus was increased to maintain trough levels of 12–15 ng/mL. Fourteen months after transplant, she experienced recurrent PRES which resolved after discontinuing sirolimus. Currently 3 years posttransplant, she is maintained on cyclosporine, MMF and prednisone with no PRES recurrence. In addition to calcineurin inhibitors, sirolimus may also be associated with PRES after solid organ transplantation. Ours is the first report of sirolimus‐associated PRES in the setting of multivisceral transplantation. Identifying a safe alternative immunosuppression regimen was challenging but ultimately successful.

Optimizing living donor kidney graft function by donor-recipient pair selection.

Background. With the rising prevalence of living donor kidney transplantations (LDKT), we increasingly encounter transplant candidates who present with multiple potential living donors. For a given candidate, it can be unclear which donor offers the best opportunity for optimal posttransplant graft function. This study was undertaken to determine the relative contributions of individual donor demographic factors on graft function following LDKT. Methods. All LDKT donor-recipient pairs between January 1, 1999 and December 31, 2002 entered into the Scientific Registry of Transplant Recipients (SRTR) were reviewed. Suboptimal one year graft function was defined as a serum creatinine (Cr) greater than 1.5 mg/dL. Results. Of 20,528 adult LDKTs performed, 8,603 donor-recipient pairs had complete donor, recipient, and one year graft function data. Over one third (36%) of all LDKTs had suboptimal one year graft function. Logistic regression identified simple recipient and donor characteristics associated with suboptimal one year graft function. Four recipient factors (age, gender, race, and size), three donor factors (age, gender, and size) and recipient-donor relatedness were used to derive an equation that predicts the risk of suboptimal one year graft function posed by each potential living donor for a given transplant candidate. Conclusions. In the setting of multiple potential living kidney donors, this quantitative tool may facilitate the choice of the optimal donor.

Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.

Renal outcomes of simultaneous liver-kidney transplantation compared to liver transplant alone for candidates with renal dysfunction.

It is unclear whether a concomitant kidney transplant grants survival benefit to liver transplant (LT) candidates with renal dysfunction (RD). We retrospectively studied LT candidates without RD (n = 714) and LT candidates with RD who underwent either liver transplant alone (RD‐LTA; n = 103) or simultaneous liver–kidney transplant (RD‐SLKT; n = 68). RD was defined as renal replacement therapy (RRT) requirement or modification of diet in renal disease (MDRD)–glomerular filtration rate (GFR) <25 mL/min/1.73 m2. RD‐LTAs had worse one‐yr post‐transplant survival compared to RD‐SLKTs (79.6% vs. 91.2%, p = 0.05). However, RD‐LTA recipients more often had hepatitis C (60.2% vs. 41.2%, p = 0.004) and more severe liver disease (MELD 37.9 ± 8.1 vs. 32.7 ± 9.1, p = 0.0001). Twenty RD‐LTA recipients died in the first post‐transplant year. Evaluation of the cause and timing of death relative to native renal recovery revealed that only four RD‐LTA recipients might have derived survival benefit from RD‐SLKT. Overall, 87% of RD‐LTA patients recovered renal function within one month of transplant. One yr after RD‐LTA or RD‐SLKT, serum creatinine (1.5 ± 1.2 mg/dL vs. 1.4 ± 0.5 mg/dL, p = 0.63) and prevalence of stage 4 or 5 chronic kidney disease (CKD; 5.9% vs. 6.8%, p = 0.11) were comparable. Our series provides little evidence that RD‐SLKT would have yielded substantial short‐term survival benefit to RD‐LTA recipients.

Use of Vascularized Posterior Rectus Sheath Allograft in Pediatric Multivisceral Transplantation - Report of Two Cases

Restoring abdominal wall cover and contour in children undergoing bowel and multivisceral transplantation is often challenging due to discrepancy in size between donor and recipient, poor musculature related to birth defects and loss of abdominal wall integrity from multiple surgeries. A recent innovation is the use of vascularized posterior rectus sheath to enable closure of abdomen. We describe the application of this technique in two pediatric multivisceral transplant recipients—one to buttress a lax abdominal wall in a 22‐month‐old child with megacystis microcolon intestinal hypoperistalsis syndrome and another to accommodate transplanted viscera in a 10‐month child with short bowel secondary to gastoschisis and loss of domain. This is the first successful report of this procedure with long‐term survival. The procedure has potential application to facilitate difficult abdominal closure in both adults and pediatric liver and multivisceral transplantation.