Jinhua Piao

Autoimmune lymphoproliferative syndrome-like disease with somatic KRAS mutation

Autoimmune lymphoproliferative syndrome (ALPS) is classically defined as a disease with defective FAS-mediated apoptosis (type I-III). Germline NRAS mutation was recently identified in type IV ALPS. We report 2 cases with ALPS-like disease with somatic KRAS mutation. Both cases were characterized by prominent autoimmune cytopenia and lymphoadenopathy/splenomegaly. These patients did not satisfy the diagnostic criteria for ALPS or juvenile myelomonocytic leukemia and are probably defined as a new disease entity of RAS-associated ALPS-like disease (RALD).

GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25(-)CD4(+) effector (Teff) and CD25(+)CD4(+) regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4(+) T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4(+) T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists.

Enhancement of T-cell-mediated anti-tumour immunity via the ectopically expressed glucocorticoid-induced tumour necrosis factor receptor-related receptor ligand (GITRL) on tumours

Glucocorticoid‐induced tumour necrosis factor receptor‐related receptor (GITR) costimulates functions of both effector and regulatory T cells. The administration of agonistic anti‐GITR monoclonal antibodies efficiently enhances various T‐cell‐mediated immune responses; however, it is unknown to what extent the ligand of GITR (GITRL) contributes to T‐cell responses. We investigated the involvement of endogenously expressed GITRL on dendritic cells and ectopically expressed GITRL on tumours in T‐cell‐mediated immunity. Expression of GITRL on dendritic cells in secondary lymphoid organs was limited, and treatment with anti‐GITRL monoclonal antibodies did not substantially affect T‐cell‐mediated immunity to alloantigens, a specific protein antigen (ovalbumin), or tumour antigens. The introduction of GITRL promoted anti‐tumour immunity in four tumour models. Tumour‐associated GITRL greatly augmented the effector function of CD8+ T cells and enhanced the contribution of CD8+ T cells. These events reduced the crucial contribution of CD25+ CD4+ regulatory T cells, which were found to inhibit immunity against tumours lacking GITRL. Peritumoral injection of GITRL tumour vaccine efficiently inhibited the growth of established tumours. Our results suggest that the ectopic expression of GITRL in tumour cells enhances anti‐tumour immunity at peripheral tumour sites. Consequently, the combined use of a GITRL tumour vaccine with methods aimed at enhancing the activation of host antigen‐presenting cells in secondary lymphoid tissues may be a promising strategy for tumour immunotherapy.

The glucocorticoid-induced TNF receptor-related protein (GITR)-GITR ligand pathway acts as a mediator of cutaneous dendritic cell migration and promotes T cell-mediated acquired immunity.

Glucocorticoid-induced TNFR-related protein (GITR) has various roles in the activation of T cells and inflammation. In this study, we investigated the roles of the GITR-GITR ligand (GITRL) pathway in contact hypersensitivity (CH). Treatment with anti-GITRL mAb at sensitization inhibited CH responses. Depletion studies using an anti-CD25 or anti-PDCA-1 mAb revealed that regulatory T cells and plasmacytoid dendritic cells (DCs), known to express high levels of GITR and GITRL, respectively, were not apparently involved in GITRL-mediated CH responses. Treatment with/addition of anti-GITRL mAb in the experiments for hapten-specific T cell proliferation and IFN-γ production showed a minor contribution of the GITRL, which was weakly expressed on DCs in draining lymph nodes (dLNs). Interestingly, anti-GITRL mAb treatment inhibited the migration of cutaneous DCs to the dLNs. Epidermal keratinocytes (KCs) constitutively express GITR, whereas Langerhans cells (LCs) express higher levels of GITRL compared with DCs in dLNs. GITR ligation, by an anti-GITR mAb, in KCs promoted expression of multiple proinflammatory cytokines and blockade of GITRL-inhibited IL-1β and CCR7 expression in sensitized skin. These results suggest that the GITR-GITRL pathway promotes epidermal inflammatory cytokine production by KCs and LCs, resulting in migration of cutaneous DCs from the skin to the dLNs. This is the first report demonstrating the involvement of the GITR-GTRL pathway in interactions with KCs and LCs and the migration of DCs. Our findings provide important implications for understanding the molecular bases of KC-LC interactions and for developing new therapeutic strategies in skin disease.

Process for immune defect and chromosomal translocation during early thymocyte development lacking ATM.

Immune defect in ataxia telangiectasia patients has been attributed to either the failure of V(D)J recombination or class-switch recombination, and the chromosomal translocation in their lymphoma often involves the TCR gene. The ATM-deficient mouse exhibits fewer CD4 and CD8 single-positive T cells because of a failure to develop from the CD4(+)CD8(+) double-positive phase to the single-positive phase. Although the occurrence of chromosome 14 translocations involving TCR-δ gene in ATM-deficient lymphomas suggests that these are early events in T-cell development, a thorough analysis focusing on early T-cell development has never been performed. Here we demonstrate that ATM-deficient mouse thymocytes are perturbed in passing through the β- or γδ-selection checkpoint, leading in part to the developmental failure of T cells. Detailed karyotype analysis using the in vitro thymocyte development system revealed that RAG-mediated TCR-α/δ locus breaks occur and are left unrepaired during the troublesome β- or γδ-selection checkpoints. By getting through these selection checkpoints, some of the clones with random or nonrandom chromosomal translocations involving TCR-α/δ locus are selected and accumulate. Thus, our study visualized the first step of multistep evolutions toward lymphomagenesis in ATM-deficient thymocytes associated with T-lymphopenia and immunodeficiency.

Haploinsufficiency of TNFAIP3 (A20) by germline mutation is involved in autoimmune lymphoproliferative syndrome

Background Autoimmune diseases in children are rare and can be difficult to diagnose. Autoimmune lymphoproliferative syndrome (ALPS) is a well‐characterized pediatric autoimmune disease caused by mutations in genes associated with the FAS‐dependent apoptosis pathway. In addition, various genetic alterations are associated with the ALPS‐like phenotype. Objective The aim of the present study was to elucidate the genetic cause of the ALPS‐like phenotype. Methods Candidate genes associated with the ALPS‐like phenotype were screened by using whole‐exome sequencing. The functional effect of the identified mutations was examined by analyzing the activity of related signaling pathways. Results A de novo heterozygous frameshift mutation of TNF‐&agr;–induced protein 3 (TNFAIP3, A20), a negative regulator of the nuclear factor &kgr;B pathway, was identified in one of the patients exhibiting the ALPS‐like phenotype. Increased activity of the nuclear factor &kgr;B pathway was associated with haploinsufficiency of TNFAIP3 (A20). Conclusion Haploinsufficiency of TNFAIP3 (A20) by a germline heterozygous mutation leads to the ALPS phenotype.

Enhancement of effector CD8+ T‐cell function by tumour‐associated B7‐H3 and modulation of its counter‐receptor triggering receptor expressed on myeloid cell‐like transcript 2 at tumour sites

B7‐H3 is a B7‐family co‐stimulatory molecule and is broadly expressed on various tissues and immune cells. Transduction of B7‐H3 into some tumours enhances anti‐tumour responses. We have recently found that a triggering receptor expressed on myeloid cell‐like transcript 2 (TLT‐2) is a receptor for B7‐H3. Here, we examined the roles of tumour‐associated B7‐H3 and the involvement of TLT‐2 in anti‐tumour immunity. Ovalbumin (OVA)257–264‐specific OT‐I CD8+ T cells exhibited higher cytotoxicity against B7‐H3‐transduced OVA‐expressing tumour cells (B7‐H3/E.G7) in vitro and selectively eliminated B7‐H3/E.G7 cells in vivo. The presence of B7‐H3 on target cells efficiently augmented CD8+ T‐cell‐mediated cytotoxicity against alloantigen or OVA, whereas the presence of B7‐H3 in the priming phase did not affect the induced cytotoxicity. B7‐H3 transduction into five tumour cell lines efficiently reduced their tumorigenicity and regressed growth. Treatment with either anti‐B7‐H3 or anti‐TLT‐2 monoclonal antibody accelerated growth of a tumour that expressed endogenous B7‐H3, suggesting a co‐stimulatory role of the B7‐H3–TLT‐2 pathway. The TLT‐2 was preferentially expressed on CD8+ T cells in regional lymph nodes, but was down‐regulated in tumour‐infiltrating CD8+ T cells. Transduction of TLT‐2 into OT‐I CD8+ T cells enhanced antigen‐specific cytotoxicity against both parental and B7‐H3‐transduced tumour cells. Our results suggest that tumour‐associated B7‐H3 directly augments CD8+ T‐cell effector function, possibly by ligation of TLT‐2 on tumour‐infiltrating CD8+ T cells at the local tumour site.

Possible involvement of soluble B7-H4 in T cell-mediated inflammatory immune responses.

B7-H4, a newly identified B7 family molecule, is reported to regulate T cell activation. However, the expression and function of B7-H4 remain controversial. Here, we demonstrated that B7-H4 expression in immune cells was undetectable at both the transcription and cell-surface protein levels. B7-H4 transfectants augmented anti-CD3 mAb-induced re-directed cytotoxicity and this was inhibited by anti-B7-H4 mAb. In a hapten-induced contact hypersensitivity model, treatment with anti-B7-H4 mAb at sensitization, but not at challenge, efficiently suppressed the ear swelling and CD8(+) T cell activation assessed by CD25 expression and IFN-gamma production. We found that cells expressing B7-H4 secreted soluble B7-H4 and the serum B7-H4 level increased with disease progression in lupus-prone and collagen-induced arthritis autoimmune mice and after the antigen challenge in allergic inflammatory diseases. Our results suggest a different action of B7-H4 in T cell-mediated inflammatory responses and the possible involvement of soluble B7-H4 in inflammatory immune responses.

Artemis-dependent DNA double-strand break formation at stalled replication forks.

Stalled replication forks undergo DNA double‐strand breaks (DSBs) under certain conditions. However, the precise mechanism underlying DSB induction and the cellular response to persistent replication fork stalling are not fully understood. Here we show that, in response to hydroxyurea exposure, DSBs are generated in an Artemis nuclease‐dependent manner following prolonged stalling with subsequent activation of the ataxia–telangiectasia mutated (ATM) signaling pathway. The kinase activity of the catalytic subunit of the DNA‐dependent protein kinase, a prerequisite for stimulation of the endonuclease activity of Artemis, is also required for DSB generation and subsequent ATM activation. Our findings indicate a novel function of Artemis as a molecular switch that converts stalled replication forks harboring single‐stranded gap DNA lesions into DSBs, thereby activating the ATM signaling pathway following prolonged replication fork stalling.

ATM-dependent DNA damage-response pathway as a determinant in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase, and subsequently progresses to an accelerated or blastic phase. Although several genes are known to be involved in the progression to blastic phase, molecular mechanisms for the evolution toward blast crisis have not been fully identified. Oncogenic stimuli enforce cell proliferation, which requires DNA replication. Unscheduled DNA replication enforced by oncogenic stimuli leads to double strand breaks on DNA. We found the DNA damage-response pathway is activated in bone marrow of chronic-phase CML patients possibly due to an enforced proliferation signal by BCR-ABL expression. Since ataxia telangiectasia mutated (ATM) is a central player of the DNA damage-response pathway, we studied whether loss of this pathway accelerates blast crisis. We crossed Atm-knockout mice with BCR-ABL transgenic mice to test this hypothesis. Interestingly, the loss of one of the Atm alleles was shown to be enough for the acceleration of the blast crisis, which is supported by the finding of increased genomic instability as assayed by breakage-fusion-bridge (BFB) cycle formation. In light of these findings, the DNA damage-response pathway plays a vital role for determination of susceptibility to blast crisis in CML.