Michelle L. West

In vitro-differentiated TH17 cells mediate lethal acute graft-versus-host disease with severe cutaneous and pulmonary pathologic manifestations

The morbidity and mortality associated with graft-host-disease (GVHD) is a significant obstacle to the greater use of allogeneic stem cell transplantation. Donor T cells that predominantly differentiate into TH1/Tc1 T cells and generate pro-inflammatory cytokines such as interferon-gamma (IFN-gamma) mediate GVHD. Although numerous studies have described a pathogenic role for IFN-gamma, multiple reports have demonstrated that the lack of IFN-gamma paradoxically exacerbated GVHD lethality. This has led to speculation that another subset of T cells may significantly contribute to GVHD mortality. Several groups have demonstrated a new lineage of CD4+ T helper cell development distinct from TH1 or TH2 differentiation. This lineage is characterized by production of interleukin (IL)-17A, IL-17F, IL-22, and IL-21 and has been termed TH17 cells. Here, we demonstrate that a highly purified population of TH17 cells is capable of inducing lethal GVHD, hallmarked by extensive pathologic cutaneous and pulmonary lesions. Upon transfer, these cells migrate to and expand in GVHD target organs and secondary lymphoid tissues. Finally, we demonstrate differential roles for tumor necrosis factor-alpha (TNF-alpha) and IL-17A in the clinical manifestations of GVHD induced by TH17 cells. Our studies demonstrate that cells other than TH1/Tc1 can mediate acute GVHD.

Separation of graft-versus-host disease from graft-versus-leukemia responses by targeting CC-chemokine receptor 7 on donor T cells

CC-chemokine receptor 7 (CCR7) is expressed on the surface of naive T cells, and plays a critical role in their movement into secondary lymphoid tissue. Here, we show that murine T cells lacking CCR7 (CCR7(-/-)) generate attenuated graft-versus-host disease (GVHD) responses compared with wild-type (WT) cells, with the difference varying inversely with the degree of major histocompatibility complex (MHC) disparity between the donor and recipient. CCR7(-/-) T cells exhibited an impaired ability to traffic to recipient lymph nodes, with an increased capacity to home to the spleen. CCR7(-/-) T cells, however, demonstrated a reduced ability to undergo in vivo expansion in the spleen due to impaired interactions with splenic antigen-presenting cells. On a cellular level, CCR7(-/-) T cells were functionally competent, demonstrating a normal in vitro proliferative capacity and a preserved ability to produce inflammatory cytokines. Importantly, CCR7(-/-) T cells were capable of generating robust graft-versus-leukemia (GVL) responses in vivo, as well as complete donor T-cell reconstitution. CCR7(-/-) regulatory T cells were able to protect against lethal GVHD when administered before WT conventional T cells. Our data suggest that CCR7 inhibition in the early posttransplantation period may represent a feasible new therapeutic approach for acute GVHD attenuation without compromising GVL responses.

Attenuation of Acute Graft-versus-Host Disease in the Absence of the Transcription Factor RORγt

Graft-versus-host disease (GVHD) remains the most significant complication after allogeneic stem cell transplantation. Previously, acute GVHD had been considered to be mediated predominantly by Th1-polarized T cells. Recently, investigators have identified a second proinflammatory lineage of T cells termed Th17 that is critically dependent on the transcription factor retinoic acid-related orphan receptor (ROR)γt. In this study, we have evaluated the role of Th17 cells in murine acute GVHD by infusing donor T cells lacking RORC and as a consequence the isoform RORγt. Recipients given donor CD4+ and CD8+ T cells lacking RORC had significantly attenuated acute GVHD and markedly decreased tissue pathology in the colon, liver, and lung. Using a clinically relevant haploidentical murine transplantation model, we showed that RORC−/− CD4+ T cells alone diminished the severity and lethality of acute GVHD. This was not found when CD4+ T cells from RORC−/− mice were given to completely mismatched BALB/c mice, and it was correlated with absolute differences in the generation of TNF in the colon after transplant. Thus, CD4+ T cell expression of RORC is important in the pathogenesis of acute GVHD.

CC chemokine receptor 8 potentiates donor Treg survival and is critical for the prevention of murine graft-versus-host disease.

The infusion of donor regulatory T cells (Tregs) has been used to prevent acute graft-versus-host disease (GVHD) in mice and has shown promise in phase 1 clinical trials. Previous work suggested that early Treg migration into lymphoid tissue was important for GVHD prevention. However, it is unclear how and where Tregs function longitudinally to affect GVHD. To better understand their mechanism of action, we studied 2 Treg-associated chemokine receptors in murine stem cell transplant models. CC chemokine receptor (CCR) 4 was dispensable for donor Treg function in the transplant setting. Donor Tregs lacking CCR8 (CCR8(-/-)), however, were severely impaired in their ability to prevent lethal GVHD because of increased cell death. By itself, CCR8 stimulation was unable to rescue Tregs from apoptosis. Instead, CCR8 potentiated Treg survival by promoting critical interactions with dendritic cells. In vivo, donor bone marrow-derived CD11c(+) antigen-presenting cells (APCs) were important for promoting donor Treg maintenance after transplant. In contrast, host CD11c(+) APCs appeared to be dispensable for early activation and expansion of donor Tregs. Collectively, our data indicate that a sustained donor Treg presence is critical for their beneficial properties, and that their survival depends on CCR8 and donor but not host CD11c(+) APCs.

Intravital imaging of donor allogeneic effector and regulatory T cells with host dendritic cells during GVHD

Graft-versus-host disease (GVHD) is a systemic inflammatory response due to the recognition of major histocompatibility complex disparity between donor and recipient after hematopoietic stem cell transplantation (HSCT). T-cell activation is critical to the induction of GVHD, and data from our group and others have shown that regulatory T cells (Tregs) prevent GVHD when given at the time of HSCT. Using multiphoton laser scanning microscopy, we examined the single cell dynamics of donor T cells and dendritic cells (DCs) with or without Tregs postallogeneic transplantation. We found that donor conventional T cells (Tcons) spent very little time screening host DCs. Tcons formed stable contacts with DCs very early after transplantation and only increased velocity in the lymph node at 20 hours after transplant. We also observed that Tregs reduced the interaction time between Tcons and DCs, which was dependent on the generation of interleukin 10 by Tregs. Imaging using inducible Tregs showed similar disruption of Tcon-DC contact. Additionally, we found that donor Tregs induce host DC death and down-regulate surface proteins required for donor T-cell activation. These data indicate that Tregs use multiple mechanisms that affect host DC numbers and function to mitigate acute GVHD.

L-selectin is dispensable for T regulatory cell function postallogeneic bone marrow transplantation.

In murine models, the adoptive transfer of CD4+/CD25+ regulatory T cells (Tregs) inhibited graft‐versus‐host disease (GvHD). Previous work has indicated a critical role for the adhesion molecule L‐selectin (CD62L) in the function of Tregs in preventing GvHD. Here we examined the capacity of naive wild‐type (WT), CD62L−/− and ex vivo expanded CD62LLo Tregs to inhibit acute GvHD. Surprisingly, we found that CD62L−/− Tregs were potent suppressors of GvHD, whereas CD62LLo Tregs were unable to inhibit disease despite being functionally competent to suppress allo T cell responses in vitro. Concomitant with improved outcomes, WT and CD62L−/− Tregs significantly reduced liver pathology and systemic pro‐inflammatory cytokine production, although CD62L−/− Tregs were less effective in reducing lung pathology. While accumulation of CD62L−/− Tregs in GvHD target organs was equivalent to WT Tregs, CD62L−/− Tregs did not migrate as well as WT Tregs to peripheral lymph nodes (PLNs) over the first 2 weeks posttransplantation. This work demonstrated that CD62L was dispensable for Treg‐mediated protection from GvHD.

The immunosuppressive tumor environment is the major impediment to successful therapeutic vaccination in Neu transgenic mice.

We earlier showed that therapeutic vaccination of FVB/N mice with alphaviral replicon particles expressing rat neuET-VRP induced regression of established neu-expressing tumors. In this study, we evaluated the efficacy of neuET-VRPs in a tolerant mouse model using mice with transgenic expression of neu. Using the same approach that induced regression of 70 mm2 tumors in FVB/N mice, we were unable to inhibit tumor growth in tolerant neu-N mice, despite showing neu-specific B-cell and T-cell responses post vaccination. As neu-N mice have a limited T-cell repertoire specific to neu, we hypothesized that the absence of these T cells led to differences in the vaccine response. However, transfer of neu-specific T cells from vaccinated FVB/N mice was not effective in inducing tumor regression, as these cells did not proliferate in the tumor-draining lymph node. Vaccination given with low-dose cyclophosphamide to deplete regulatory T cells delayed tumor growth but did not result in tumor regression. Finally, we showed that T cells given with vaccination were effective in inhibiting tumor growth, if administered with approaches to deplete myeloid-derived suppressor cells. Our data show that both central deletion of lymphocytes and peripheral immunosuppressive mechanisms are present in neu-N mice. However, the major impediment to successful vaccination is the peripheral tumor-induced immune suppression.

C-C chemokine receptor 5 on pulmonary mesenchymal cells promotes experimental metastasis via the induction of erythroid differentiation regulator 1.

C-C Chemokine receptor 5 knockout (Ccr5−/−) mice develop fewer experimental pulmonary metastases than wild-type (WT) mice. This phenomenon was explored by applying gene expression profiling to the lungs of mice with these metastases. Consequently, erythroid differentiation regulator 1 (Erdr1) was identified as upregulated in the WT mice. Though commonly associated with bone marrow stroma, Erdr1 was differentially expressed in WT pulmonary mesenchymal cells (PMC) and murine embryonic fibroblasts (MEF). Moreover, the Ccr5 ligand Ccl4 increased its expression by 3.36 ± 0.14–fold. Ccr5 signaling was dependent on the mitogen-activated protein kinase kinase (Map2k) but not the phosphoinositide 3-kinase (Pi3k) pathway because treatment with U0126 inhibited upregulation of Erdr1, but treatment with LY294002 increased the expression by 3.44 ± 0.92–fold (P < 0.05). The effect Erdr1 on B16-F10 melanoma metastasis was verified by the adoptive transfer of WT MEFs into Ccr5−/− mice. In this model, MEFs that had been transduced with Erdr1 short hairpin RNA (shRNA) lowered metastasis by 33% compared with control transduced MEFs. The relevance of ERDR1 on human disease was assessed by coculturing chronic lymphocytic leukemia (CLL) cells with M2-10B4 stromal cells that had been transfected with shRNA or control plasmids. After 96 hours of coculture, the cell counts were higher with control cell lines than with Erdr1 knockdown lines [odds ratio (OR), 1.88 ± 0.27, 2.52 ± 0.66, respectively]. This increase was associated with a decrease in apoptotic cells (OR, 0.69 ± 0.18, 0.58 ± 0.12, respectively). Implications: Therefore, ERDR1 is a stromal-derived factor that promotes cancer cell survival in vitro and in an experimental metastasis model. Mol Cancer Res; 12(2); 274–82. ©2013 AACR.

Altered T-cell entry and egress in the absence of Coronin 1A attenuates murine acute graft versus host disease.

Acute graft‐versus‐host disease (aGvHD) is a major limitation to the use of allogeneic stem cell transplantation for the treatment of patients with relapsed malignant disease. Previous work using animals lacking secondary lymphoid tissue (SLT) suggested that activation of donor T cells in SLT is critically important for the pathogenesis of aGvHD. However, these studies did not determine if impaired migration into, and more importantly, out of SLT, would ameliorate aGvHD. Here, we show that T cells from mice lacking Coronin 1A (Coro 1A−/−), an actin‐associated protein shown to be important for thymocyte egress, do not mediate acute GvHD. The attenuation of aGvHD was associated with decreased expression of the critical trafficking proteins C‐C chemokines receptor type 7 (CCR7) and sphingosine 1 phosphate receptor on donor T cells. This was mediated in part by impaired activation of the canonical NF‐κB pathway in the absence of Coro 1A. As a result of these alterations, donor T cells from Coro 1A−/− mice were not able to initially traffic to SLT or exit SLT after BM transplantation. However, this alteration did not abrogate the graft‐versus‐leukemia response. Our data suggest that blocking T‐cell migration into and out of SLT is a valid approach to prevent aGvHD.