Swetha Ramakrishnan

Gene therapy of murine solid tumors with T cells transduced with a retroviral vascular endothelial growth factor-immunotoxin target gene

Solid tumor growth can be inhibited by targeting its neovasculature with vascular endothelial growth factor (VEGF)-toxin fusion proteins (FPs), but these agents have been limited by their inability to localize at the tumor site. In this study, we devised a gene therapy approach intended to deliver VEGF-toxin directly to tumor. Antigen-specific cytotoxic T lymphocytes (CTLs) served as vehicles to deliver a retroviral VEGF-toxin fusion protein to its specific leukemia cell target in vivo. A retroviral vector was constructed for gene therapy with VEGF positioned downstream of its 27-amino acid leader sequence, which promoted secretion of a catalytic immunotoxin containing either truncated diphtheria toxin or Pseudomonas exotoxin A. VEGF was chosen on the basis of the expression of VEGF receptor on endothelial cells in the tumor neovasculature. The VEGF FP was first expressed and secreted by mammalian NIH 3T3 cells. Intracellular expression of both VEGF and toxin was verified by immunofluorescence. In vitro, supernatants collected from transfected cells specifically inhibited the growth of VEGF receptor-expressing human umbilical vein endothelial cells (HUVECs), but not a control cell line. In vivo findings correlated with in vitro findings. A retroviral vector containing the target gene and a nerve growth factor receptor (NGFR) reporter gene was used to transiently transduce T15, a CD8(+) CTL line that specifically recognizes C1498, a lethal C57BL/6 myeloid tumor. Transduced T15 cells injected intravenously significantly inhibited the growth of subcutaneous tumor, whereas nontransduced controls did not. Together, these data indicate that gene therapy of T cells with retrovirus containing a VEGF-immunotoxin target gene may be a valid means of inhibiting a broad range of solid tumors dependent on angiogenesis.

Transcriptome analysis of GVHD reveals aurora kinase A as a targetable pathway for disease prevention

Transcriptomic profiling of primate T cells during acute graft-versus-host disease reveals signaling pathways that when inhibited, ameliorate disease. Dawn of new graft-versus-host disease therapies Hematopoietic stem cell transplant (HCT) is a common therapy for patients with damaged bone marrow or immunodeficiencies. However, HCT has its own risks: In cases where the donor is not a perfect match to the recipient, immune cells derived from the graft can attack their new home. Furlan et al. examined the gene expression profile of nonhuman primate T cells during acute graft-versus-host disease (GVHD). The transcriptomics signatures specific for alloreactive T cells identified pathways altered during acute GVHD that could serve as therapeutic targets. The authors then examined one target in particular, aurora kinase A, and demonstrated that pharmacologic inhibition could improve survival in a mouse model of GVHD. Graft-versus-host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of nonhuman primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3+ T cells from five cohorts: allogeneic transplant recipients receiving (i) no immunoprophylaxis (No Rx), (ii) sirolimus monotherapy (Siro), (iii) tacrolimus-methotrexate (Tac-Mtx), as well as (iv) autologous transplant recipients (Auto) and (v) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for alloreactive T cells and determine the impact of both mTOR (mechanistic target of rapamycin) and calcineurin inhibition on GVHD. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function, and cytokine synthesis. Within these pathways, we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD and suggest that AURKA should be considered a target for preventing GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD.

Evidence for kidney rejection after combined bone marrow and renal transplantation despite ongoing whole-blood chimerism in rhesus macaques.

Although there is evidence linking hematopoietic chimerism induction and solid organ transplant tolerance, the mechanistic requirements for chimerism‐induced tolerance are not clearly elucidated. To address this, we used an MHC‐defined primate model to determine the impact of impermanent, T cell‐poor, mixed‐chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow and renal transplant (“BMT/renal”) and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40‐directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole‐blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p = 0.46), with histopathology documenting T cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28–/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28‐negative Tem and costimulation blockade‐resistant rejection. These results suggest that in some settings, transient T cell‐poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.

CD8-predominant T-cell CNS infiltration accompanies GVHD in primates and is improved with immunoprophylaxis.

To the editor: A recently published article in Blood showed that, in mice, the central nervous system (CNS) is a target of graft-versus-host-disease (GVHD).[1][1] However, diagnosing CNS GVHD in the clinic is challenging,[2][2][⇓][3]-[4][4] and until now, evidence in a large-animal model has been

Inhibition of αvβ6 Promotes Acute Renal Allograft Rejection in Nonhuman Primates

The integrin αvβ6 activates latent transforming growth factor‐β (TGF‐β) within the kidney and may be a target for the prevention of chronic allograft fibrosis after kidney transplantation. However, TGF‐β also has known immunosuppressive properties that are exploited by calcineurin inhibitors (CNIs); thus, the net benefit of αvβ6 inhibition remains undetermined. To assess the acute impact of interference with αvβ6 on acute rejection, we tested a humanized αvβ6‐specific monoclonal antibody (STX‐100) in a randomized, double‐blinded, placebo‐controlled nonhuman primate renal transplantation study to evaluate whether αvβ6 blockade alters the risk of acute rejection during CNI‐based immunosuppression. Rhesus monkeys underwent renal allotransplantation under standard CNI‐based maintenance immunosuppression; 10 biopsy‐confirmed rejection‐free animals were randomized to receive weekly STX‐100 or placebo. Animals treated with STX‐100 experienced significantly decreased rejection‐free survival compared to placebo animals (p = 0.049). Immunohistochemical analysis confirmed αvβ6 ligand presence, and αvβ6 staining intensity was lower in STX‐100‐treated animals (p = 0.055), indicating an apparent blockade effect of STX‐100. LAP, LTBP‐1 and TGF‐β were all decreased in animals that rejected on STX‐100 compared to those that rejected on standard immunosuppression alone, suggesting a relevant effect of αvβ6 blockade on local TGF‐β. These data caution against the use of αvβ6 blockade to achieve TGF‐β inhibition in kidney transplantation.

A single-center analysis of abdominal imaging in the evaluation of kidney transplant recipients

Many transplantation programs utilize noninvasive abdominal and pelvic imaging in the pre‐operative evaluation of recipient candidates. Practice patterns vary, and consensus guidelines addressing the risks and benefits of computed tomography (CT) and magnetic resonance imaging (MRI) in the pre‐transplant evaluation process do not currently exist. In this single‐center study, we examined the frequency, clinical significance, and associated costs of CT and MRI findings during the pre‐transplant evaluation of renal transplant recipients. A retrospective chart review of 3041 adult patients who underwent a CT/CTA or MRI/MRA of the abdomen and pelvis for pre‐transplant evaluation between 2005 and 2010 was performed. Pre‐transplant imaging with MRI offered a more sensitive evaluation in comparison with CT, with the notable exception of abnormalities in which calcium was detected. Patients imaged with CT had a significantly greater proportion of subsequent clinical actions arising from imaging findings. The total financial cost of MRI was greater than that of CT. No cases of nephrogenic systemic fibrosis (NSF) in patients who received MultiHance gadolinium contrast were reported. In conclusion, the risks, benefits, and costs of CT/CTA and MRI/MRA must be carefully considered to optimize the pre‐operative evaluation of renal transplant recipients.