Aneesah Garrett

CMV reactivation drives posttransplant T-cell reconstitution and results in defects in the underlying TCRβ repertoire

Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme B(high)/CD28(low)/CD57(high)/CD8(+) effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31(+)/CD4(+) putative thymic emigrants. T-cell receptor β (TCRβ) deep sequencing revealed a striking contraction of CD8(+) Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T-cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in the underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that, in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T-cell repertoire. This trial was registered at www.clinicaltrials.gov as #NCT01012492.

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.

Immune parameter analysis of children with sickle cell disease on hydroxycarbamide or chronic transfusion therapy

Sickle cell disease (SCD) is increasingly appreciated as an inflammatory condition associated with alterations in immune phenotype and function. In this cross‐sectional study we performed a multiparameter analysis of 18 immune markers in 114 paediatric SCD patients divided by treatment group [those receiving hydroxycrabamide (HC, previously termed hydroxyurea), chronic transfusion (CT), or no disease‐modifying therapy] and 29 age‐matched African American healthy controls. We found global elevation of most immune cell counts in SCD patients receiving no disease‐modifying therapy at steady state. Despite the decrease in percentage of haemoglobin S associated with CT therapy, the abnormal cellular immune phenotype persisted in patients on CT. In contrast, in both univariate and multivariate analysis, treatment with HC was associated with normalization of the vast majority of leucocyte populations. This study provides additional support for HC treatment in SCD, as it appears that HC decreases the abnormally elevated immune cell counts in patients with SCD.

Viewing transplantation immunology through today's lens: new models, new imaging, and new insights.

The last several decades have brought significant immunologic advances in the fields of inflammation, infection, and transplantation tolerance. Heretofore, our understanding of how complex immune interactions occur has been limited to static in situ tissue analysis and in vitro dynamic studies using isolated cells devoid of stromal elements typically present in vivo. Recent advances in molecular, flow cytometry, and intravital imaging have provided new insight into the dynamic interactions occurring among a variety of cells within the bone marrow (BM) and immune systems, ranging from undifferentiated hematopoietic progenitors to fully committed effector memory cells, which will likely have direct clinical and translational implications. In this review we highlight how the application of these cutting-edge technologies will sculpt the landscape of the next generation of immunologic advances.

CD28 blockade controls T cell activation to prevent graft-versus-host disease in primates

Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab′, in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-&ggr; response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.