Kelly Hamby

NK Cells Mediate Costimulation Blockade-Resistant Rejection of Allogeneic Stem Cells During Nonmyeloablative Transplantation

Although T‐cell CD28/CD40 costimulation blockade represents a powerful mechanism to promote immune tolerance during murine allotransplantation, it has not yet been successfully translated to clinical transplantation. We determined the impact of natural killer (NK) cells on costimulation blockade‐resistant rejection of donor bone marrow. We found that NK cells represent a potent barrier to engraftment: host NK depletion led to increased donor stem cell survival, increased mixed hematopoietic chimerism and to engraftment of low doses of donor marrow (1 × 108/kg) that were otherwise rejected. To understand the mechanisms of NK alloreactivity, we employed an in vivo NK‐specific cytotoxicity assay. We found that an increased proportion of target cells were killed between days 2 and 8 after cell transfer, and that NK killing of parental targets was inducible: NK cells preprimed with allotargets were more efficient at their elimination upon reexposure. Finally, both transplant and in vivo NK‐killing models were used to determine the contribution of LFA‐1 to NK alloreactivity. Blockade of LFA‐1 led to decreased NK‐mediated killing, and increased alloengraftment. These results identify NK alloreactivity as an integral component to costimulation blockade‐resistant rejection, and suggest that its inhibition may represent an important target in the clinical translation of tolerance‐induction transplantation.

CD40 Blockade Combines with CTLA4Ig and Sirolimus To Produce Mixed Chimerism in an MHC-defined Rhesus Macaque Transplant Model

In murine models, T‐cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti‐CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, nondepleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well‐established primate bone marrow chimerism‐induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and nondepletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.

Infusion of stably immature monocyte-derived dendritic cells plus CTLA4Ig modulates alloimmune reactivity in rhesus macaques.

Background. Immature dendritic cells (DC) can promote long-term transplant survival in rodents. We assessed the impact of stably immature, donor-derived DC on alloimmune reactivity in rhesus macaques. Methods. CD14+ monocytes isolated from leukapheresis products of Macacca mulatta were cultured in granulocyte-macrophage colony stimulating factor plus interleukin (IL)-4±vitamin (vit) D3, and IL-10. Major histocompatibility complex class II and cosignaling molecule expression was determined on CD11c+ cells by flow cytometry. T-cell allostimulatory capacity of the DC, including DC exposed to proinflammatory cytokines, was determined in mixed leukocyte reaction. To test their influence in vivo, purified DC were infused intravenously into allogeneic recipients, either alone or followed by CTLA4Ig, 24 hr later. Proliferative responses of recipient CFSE-labeled T cells to donor or third party DC, cytokine production by stimulated T cells, and circulating alloantibody levels were determined by flow cytometry, up to 100 days postinfusion. Results. VitD3/IL-10-conditioned, monocyte-derived DC were resistant to maturation and failed to induce allogeneic T cell proliferation in vitro. After their infusion, an increase in anti-donor and anti–third party T-cell reactivity was observed, that subsequently subsided to fall significantly below pretreatment levels (by day 56) only in animals also given CTLA4Ig. No increase in circulating immunoglobulin (Ig) M or IgG anti-donor alloantibody titers compared with pretreatment values was detected. With DC+CTLA4Ig infusion, alloreactive IL-10-producing T cells were prevalent in the circulation after day 28. Conclusions. Maturation-resistant rhesus DC infusion is well-tolerated. DC+CTLA4Ig infusion modulates allogeneic T-cell responses and results in hyporesponsiveness to donor and third party alloantigens.

GVHD after haploidentical transplantation: a novel, MHC-defined rhesus macaque model identifies CD28− CD8+ T cells as a reservoir of breakthrough T-cell proliferation during costimulation blockade and sirolimus-based immunosuppression

We have developed a major histocompatibility complex-defined primate model of graft-versus-host disease (GVHD) and have determined the effect that CD28/CD40-directed costimulation blockade and sirolimus have on this disease. Severe GVHD developed after haploidentical transplantation without prophylaxis, characterized by rapid clinical decline and widespread T-cell infiltration and organ damage. Mechanistic analysis showed activation and possible counter-regulation, with rapid T-cell expansion and accumulation of CD8(+) and CD4(+) granzyme B(+) effector cells and FoxP3(pos)/CD27(high)/CD25(pos)/CD127(low) CD4(+) T cells. CD8(+) cells down-regulated CD127 and BCl-2 and up-regulated Ki-67, consistent with a highly activated, proliferative profile. A cytokine storm also occurred, with GVHD-specific secretion of interleukin-1 receptor antagonist (IL-1Ra), IL-18, and CCL4. Costimulation Blockade and Sirolimus (CoBS) resulted in striking protection against GVHD. At the 30-day primary endpoint, CoBS-treated recipients showed 100% survival compared with no survival in untreated recipients. CoBS treatment resulted in survival, increasing from 11.6 to 62 days (P < .01) with blunting of T-cell expansion and activation. Some CoBS-treated animals did eventually develop GVHD, with both clinical and histopathologic evidence of smoldering disease. The reservoir of CoBS-resistant breakthrough immune activation included secretion of interferon-γ, IL-2, monocyte chemotactic protein-1, and IL-12/IL-23 and proliferation of cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin-resistant CD28(-) CD8(+) T cells, suggesting adjuvant treatments targeting this subpopulation will be needed for full disease control.

A Mouse Model for Polyomavirus‐Associated Nephropathy of Kidney Transplants

Polyomavirus‐associated nephropathy is an important cause of dysfunction and failure of renal transplants. BK virus is an ubiquitous human polyoma virus that persistently infects the kidney. This otherwise silent infection can reactivate in immunosuppressed individuals, resulting in renal complications. Because polyoma viruses are highly species‐specific, we developed a mouse polyoma virus‐renal transplant model in order to investigate the pathogenesis of polyomavirus‐associated nephropathy. Using this model, we found that polyoma virus preferentially replicates in the allogeneic kidney grafts, accelerating graft failure; thus, this animal model is able to mimic the polyomavirus‐associated nephropathy seen in human renal transplant patients. Acute polyoma virus infection of mouse allograft recipients augmented the alloreactive CD8+ T‐cell response, while maintaining the anti‐viral CD8+ T‐cell response. In addition to the known virus‐induced cytopathology, these findings demonstrate a potential role for an enhanced anti‐donor T‐cell response in the pathogenesis of polyomavirus‐associated nephropathy.

NK Cells Rapidly Reject Allogeneic Bone Marrow in the Spleen Through a Perforin- and Ly49D-Dependent, but NKG2D-Independent Mechanism

We have used a sensitive and specific in vivo killing assay to monitor the kinetics, anatomic location and mechanisms controlling NK‐mediated rejection of Balb/c bone marrow by C57BL/6 natural killer (NK) cells. We find that NK killing of fully allogeneic bone marrow is a rapid, highly efficient process, leading to substantial rejection of transplanted marrow within 6 h of transplant and elimination of 85% of the transplanted cells within 2 days. NK‐mediated rejection occurred predominantly in the spleen, with sparing of rejection in the bone marrow and lymph nodes. Rejection was dependent on Perforin gene function, but was independent of interferon‐gamma. Finally, rejection of Balb/c bone marrow by B6 NK cells required signaling through the Ly49D receptor, but occurred despite blockade of NKG2D, which distinguishes these results from previous studies using semiallogeneic transplant pairs. These results identify NK cells as highly active mediators of bone marrow rejection, and suggest that inhibiting NK function early during transplantation may increase the efficiency of engraftment and allow successful engraftment of limiting doses of donor bone marrow.

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.

Expanded nonhuman primate Tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses

We have established two complementary strategies for purifying naturally occurring regulatory T cells (Tregs) from rhesus macaques in quantities that would be sufficient for use as an in vivo cellular therapeutic. The first strategy identified Tregs based on their being CD4+/CD25bright. The second incorporated CD127, and purified Tregs based on their expression of CD4 and CD25 and their low expression of CD127. Using these purification strategies, we were able to purify as many as 1×106 Tregs from 120 cc of peripheral blood. Cultures of these cells with anti‐CD3, anti‐CD28 and IL‐2 over 21 days yielded as much as a 450‐fold expansion, ultimately producing as many as 4.7×108 Tregs. Expanded Treg cultures potently inhibited alloimmune proliferation as measured by a carboxyfluorescein succinimidyl ester‐ mixed lymphocyte reaction (CFSE‐MLR) assay even at a 1:100 ratio with responder T cells. Furthermore, both responder‐specific and third‐party Tregs downregulated alloproliferation similarly. Both freshly isolated and cultured Tregs had gene expression signatures distinguishable from concurrently isolated bulk CD4+ T‐cell populations, as measured by singleplex reverse transcriptase‐polymerase chain reaction (RT‐PCR) and gene array. Moreover, an overlapping yet distinct gene expression signature seen in freshly isolated compared to expanded Tregs identifies a subset of Treg genes likely to be functionally significant.

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.

The Knife's Edge of Tolerance: Inducing Stable Multilineage Mixed Chimerism But With A Significant Risk of CMV Reactivation and Disease in Rhesus Macaques

Although stable mixed‐hematopoietic chimerism induces robust immune tolerance to solid organ allografts in mice, the translation of this strategy to large animal models and to patients has been challenging. We have previously shown that in MHC‐matched nonhuman primates (NHPs), a busulfan plus combined belatacept and anti‐CD154‐based regimen could induce long‐lived myeloid chimerism, but without T cell chimerism. In that setting, donor chimerism was eventually rejected, and tolerance to skin allografts was not achieved. Here, we describe an adaptation of this strategy, with the addition of low‐dose total body irradiation to our conditioning regimen. This strategy has successfully induced multilineage hematopoietic chimerism in MHC‐matched transplants that was stable for as long as 24 months posttransplant, the entire length of analysis. High‐level T cell chimerism was achieved and associated with significant donor‐specific prolongation of skin graft acceptance. However, we also observed significant infectious toxicities, prominently including cytomegalovirus (CMV) reactivation and end‐organ disease in the setting of functional defects in anti‐CMV T cell immunity. These results underscore the significant benefits that multilineage chimerism‐induction approaches may represent to transplant patients as well as the inherent risks, and they emphasize the precision with which a clinically successful regimen will need to be formulated and then validated in NHP models.