Makoto Kurachi

Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice

Tumor growth is associated with aberrant myelopoiesis, including the accumulation of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) that have the potential to promote tumor growth. However, the identity, growth, and migration of tumor-associated MDSCs remain undefined. We demonstrate herein that MDSCs at tumor site were composed primarily of bone marrow-derived CD11b(+)Gr-1(hi)Ly-6C(int) neutrophils and CD11b(+)Gr-1(int/dull)Ly-6C(hi) macrophages. Unexpectedly, in vivo bromodeoxyuridine (BrdU) labeling and parabiosis experiments revealed that tumor-infiltrating macrophages were replenished more rapidly than neutrophils. CCR2 deficiency caused striking conversion of infiltrating cellular dominance from macrophages to neutrophils in the tumor with the excessive production of CXCR2 ligands and granulocyte-colony stimulating factor in the tumor without affecting tumor growth. Overall, our data established the identity and dynamics of MDSCs in a tumor-bearing host mediated by chemokines and elucidated unexpected effects of the paucity of macrophages on tumor development.

Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade

Epigenetic profiling suggests that exhausted T cells are a distinct cell linage. The epigenetics of exhaustion During cancer or chronic infection, T cells become dysfunctional, eventually acquiring an “exhausted” phenotype. Immunotherapies aim to reverse this state. Using a mouse model of chronic infection, two studies now show that the epigenetic profile of exhausted T cells differs substantially from those of effector and memory T cells, suggesting that exhausted T cells are a distinct lineage (see the Perspective by Turner and Russ). Sen et al. defined specific functional modules of enhancers that are also conserved in exhausted human T cells. Pauken et al. examined the epigenetic profile of exhausted T cells after immunotherapy. Although there was transcriptional rewiring, the cells never acquired a memory T cell phenotype. Thus, epigenetic regulation may limit the success of immunotherapies. Science, this issue p. 1104, p. 1165; see also p. 1160 Blocking Programmed Death–1 (PD-1) can reinvigorate exhausted CD8 T cells (TEX) and improve control of chronic infections and cancer. However, whether blocking PD-1 can reprogram TEX into durable memory T cells (TMEM) is unclear. We found that reinvigoration of TEX in mice by PD-L1 blockade caused minimal memory development. After blockade, reinvigorated TEX became reexhausted if antigen concentration remained high and failed to become TMEM upon antigen clearance. TEX acquired an epigenetic profile distinct from that of effector T cells (TEFF) and TMEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that TEX are a distinct lineage of CD8 T cells. Nevertheless, PD-1 pathway blockade resulted in transcriptional rewiring and reengagement of effector circuitry in the TEX epigenetic landscape. These data indicate that epigenetic fate inflexibility may limit current immunotherapies.

The epigenetic landscape of T cell exhaustion

Epigenetic profiling suggests that exhausted T cells are a distinct cell linage. The epigenetics of exhaustion During cancer or chronic infection, T cells become dysfunctional, eventually acquiring an “exhausted” phenotype. Immunotherapies aim to reverse this state. Using a mouse model of chronic infection, two studies now show that the epigenetic profile of exhausted T cells differs substantially from those of effector and memory T cells, suggesting that exhausted T cells are a distinct lineage (see the Perspective by Turner and Russ). Sen et al. defined specific functional modules of enhancers that are also conserved in exhausted human T cells. Pauken et al. examined the epigenetic profile of exhausted T cells after immunotherapy. Although there was transcriptional rewiring, the cells never acquired a memory T cell phenotype. Thus, epigenetic regulation may limit the success of immunotherapies. Science, this issue p. 1104, p. 1165; see also p. 1160 Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells.

Bone marrow graft-versus-host disease: early destruction of hematopoietic niche after MHC-mismatched hematopoietic stem cell transplantation

Disrupted hematopoiesis and delayed immune reconstitution are life-threatening complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although graft-versus-host disease (GVHD) is a major risk factor for the bone marrow (BM) insufficiency, how GVHD impairs BM hematopoiesis has been largely unknown. We hypothesized that BM stromal niche could be a target of GVHD. In major histocompatibility complex (MHC)-mismatched murine models of GVHD, we have demonstrated the early destruction of osteoblasts that especially affected B-cell lineages. The defective B lymphopoiesis was due to the impaired ability of BM stroma and osteoblasts to support the hematopoiesis, as evidenced by the failure of GVHD-affected BM to reconstitute the hematopoietic cells. The administration of anti-CD4 monoclonal antibody (mAb) ameliorated these effects and improved B lymphopoiesis while preserving graft-versus-tumor effects. Genetic ablation of Fas-Fas ligand signaling also partially restored B lymphopoiesis. Our present study provided evidence of BM GVHD, with the identification of osteoblasts as the main target for GVHD in BM. Moreover, our data showed the potential for mAb therapies to enhance immune reconstitution in vivo for patients undergoing allo-HSCT.

The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8 + T cells

The transcription factor BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and follicular helper T cells (TFH cells). Here we identified a fundamental role for BATF in regulating the differentiation of effector of CD8+ T cells. BATF-deficient CD8+ T cells showed profound defects in effector population expansion and underwent proliferative and metabolic catastrophe early after encountering antigen. BATF, together with the transcription factors IRF4 and Jun proteins, bound to and promoted early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors while paradoxically repressing genes encoding effector molecules (IFN-γ and granzyme B). Thus, BATF amplifies T cell antigen receptor (TCR)-dependent expression of transcription factors and augments the propagation of inflammatory signals but restrains the expression of genes encoding effector molecules. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved.

Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration

CXCR3 regulates CD8+ T cell recruitment to sites of inflammation, thus dictating CD8+ T cell contraction and subsequent effector/memory cell fate.

CCR7 mediates the migration of Foxp3+ regulatory T cells to the paracortical areas of peripheral lymph nodes through high endothelial venules.

Thymus‐derived forkhead box p3+ naturally occurring regulatory T cells (nTreg) are thought to circulate throughout the body to maintain peripheral immunological self‐tolerance through interactions with dendritic cells (DCs), resulting in regulation of conventional T cells. However, the chemokine receptors, which are putatively involved in the in vivo migration of nTreg, have not been fully established. Here, we demonstrated that lymph node nTreg preferentially migrated to the paracortical area of lymph nodes after adoptive transfer, where they were observed to make contact frequently with CD8α+ DCs and CD8α− CD11b− DCs. This migration of nTreg to the paracortical areas was impaired severely when cells were prepared from CCR7‐deficient mice. However, to some extent, CCR7‐independent migration of nTreg in such CCR7‐deficient mice was also observed, but this occurred mainly in the medullary high endothelial venules. Taken together, these data provide the evidence that CCR7 mediates nTreg migration to the paracortical areas of lymph nodes under steady‐state conditions; however, CCR7‐independent migration also takes place in the medulla.

Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) comprises an emerging spectrum of inherited and noninherited disorders of the immune system characterized by the excessive production of cytokines, including interferon-γ and interleukins 2, 6, and 10 (IL-2, IL-6, and IL-10). The Janus kinases (JAKs) transduce signals initiated following engagement of specific receptors that bind a broad array of cytokines, including those overproduced in HLH. Based on the central role for cytokines in the pathogenesis of HLH, we sought to examine whether the inhibition of JAK function might lessen inflammation in murine models of the disease. Toward this end, we examined the effects of JAK inhibition using a model of primary (inherited) HLH in which perforin-deficient (Prf1(-∕-)) mice are infected with lymphocytic choriomeningitis virus (LCMV) and secondary (noninherited) HLH in which C57BL/6 mice receive repeated injections of CpG DNA. In both models, treatment with the JAK1/2 inhibitor ruxolitinib significantly lessened the clinical and laboratory manifestations of HLH, including weight loss, organomegaly, anemia, thrombocytopenia, hypercytokinemia, and tissue inflammation. Importantly, ruxolitinib treatment also significantly improved the survival of LCMV-infectedPrf1(-∕-)mice. Mechanistic studies revealed that in vivo exposure to ruxolitinib inhibited signal transducer and activation of transcription 1-dependent gene expression, limited CD8(+)T-cell expansion, and greatly reduced proinflammatory cytokine production, without effecting degranulation and cytotoxic function. Collectively, these findings highlight the JAKs as novel, druggable targets for mitigating the cytokine-driven hyperinflammation that occurs in HLH. These observations also support the incorporation of JAK inhibitors such as ruxolitinib into future clinical trials for patients with these life-threatening disorders.

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.

Adoptive cytotoxic T lymphocyte therapy triggers a counter‐regulatory immunosuppressive mechanism via recruitment of myeloid‐derived suppressor cells

Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T‐cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel‐1‐specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor‐bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN‐γ, exerted anti‐tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b+Gr1+ myeloid‐derived suppressor cells (MDSCs). Notably, CD11b+Gr1intLy6G−Ly6C+ monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen‐specific CD8+ T cells. The anti‐tumor activity of the adoptively‐transferred CTLs and the accumulation of MDSCs both depended on IFN‐γ production on recognition of tumor antigens by the former. In CCR2−/− mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter‐regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment‐induced recruitment of these MDSCs would improve the efficacy of immunotherapy.