Bone marrow adipocytes is a new player in supporting myeloma cells proliferation and survival in myeloma microenvironment.

Multiple myeloma (MM) is a lethal B cell neoplasm characterized by clonal expansion of malignant plasma cells in the bone marrow and remains incurable due to disease relapse and drug resistance. Bone marrow adipocytes (BMAs) are emerging as playing active functions that can support myeloma cell growth and survival. The aim of this study is to investigate myeloma-mesenchymal stem cells (MSCs) interaction and the impact of such interactions on the pathogenesis of MM using in vitro co-culture assay. Here we provide evidence that MM cell up-regulated MSCs to express PPAR-γ and pushes MSCs differentiation toward adipocytes at the expense of osteoblasts in co-culture manner. The increased BMAs can effectively enhance MM cell to proliferation, migration, and chemoresistance via cell-cell contact and/or cytokines release regulated by PPAR-γ signal pathway. This effect was partially reversed in medium containing PPAR-γ antagonist G3335 and indicated that G3335 distorts the maturation of MSC-derived adipocytes and cytokines release by adipocytes through inhibition of PPAR-γ, a key transcriptional factor for the activation of adipogenesis, or cell to cell contact, or both. In meantime, we observed higher expression of adipocyte differentiation associated genes DLK1, DGAT1, FABP4, and FASN both in MSCs and MSC derived adipocytes, but the osteoblast differentiation-associated gene ALP was down regulated in MSCs. These finding mean that direct consequence of MM/MSC interaction that play a role in MM pathogenesis. Consistent with those in vitro results, our primary clinical observation also showed that bone marrow samples from MM patients had significantly higher bone adiposity in comparison with controls and the number of adipocytes decreased in those who were response to anti-MM therapy. Our finding suggested that BMAs may have an important contribution to MM progression, particularly in drugs resistant of MM cells, and plays an important contribution in MM bone disease and treatment failure, but more clinical studies are needed to confirm its role.

Intratumoral expression of interleukin 23 variants using oncolytic vaccinia virus elicit potent antitumor effects on multiple tumor models via tumor microenvironment modulation.

Background: Newly emerging cancer immunotherapy has led to significant progress in cancer treatment; however, its efficacy is limited in solid tumors since the majority of them are "cold" tumors. Oncolytic viruses, especially when properly armed, can directly target tumor cells and indirectly modulate the tumor microenvironment (TME), resulting in "hot" tumors. These viruses can be applied as a cancer immunotherapy approach either alone or in combination with other cancer immunotherapies. Cytokines are good candidates to arm oncolytic viruses. IL-23, an IL-12 cytokine family member, plays many roles in cancer immunity. Here, we used oncolytic vaccinia viruses to deliver IL-23 variants into the tumor bed and explored their activity in cancer treatment on multiple tumor models. Methods: Oncolytic vaccinia viruses expressing IL-23 variants were generated by homologue recombination. The characteristics of these viruses were in vitro evaluated by RT-qPCR, ELISA, flow cytometry and cytotoxicity assay. The antitumor effects of these viruses were evaluated on multiple tumor models in vivo and the mechanisms were investigated by RT-qPCR and flow cytometry. Results: IL-23 prolonged viral persistence, probably mediated by up-regulated IL-10. The sustainable IL-23 expression and viral oncolysis elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, TNF-α, Perforin, IL-2, Granzyme B and activated T cells in the TME, transforming the TME to be more conducive to antitumor immunity. This leads to a systemic antitumor effect which is dependent on CD8+ and CD4+ T cells and IFN-γ. Oncolytic vaccinia viruses could not deliver stable IL-23A to the tumor, attributed to the elevated tristetraprolin which can destabilize the IL-23A mRNA after the viral treatment; whereas vaccinia viruses could deliver membrane-bound IL-23 to elicit a potent antitumor effect which might avoid the possible toxicity normally associated with systemic cytokine exposure. Conclusion: Either secreted or membrane-bound IL-23-armed vaccinia virus can induce potent antitumor effects and IL-23 is a candidate cytokine to arm oncolytic viruses for cancer immunotherapy.

CSN8 is a key regulator in hypoxia-induced epithelial-mesenchymal transition and dormancy of colorectal cancer cells.

Hypoxic stress plays a pivotal role in cancer progression; however, how hypoxia drives tumors to become more aggressive or metastatic and adaptive to adverse environmental stress is still poorly understood. In this study, we revealed that CSN8 might be a key regulatory switch controlling hypoxia-induced malignant tumor progression. We demonstrated that the expression of CSN8 increased significantly in colorectal cancerous tissues, which was correlated with lymph node metastasis and predicted poor patient survival. CSN8 overexpression induces the epithelial-mesenchymal transition (EMT) process in colorectal cancer cells, increasing migration and invasion. CSN8 overexpression arrested cell proliferation, upregulated key dormancy marker (NR2F1, DEC2, p27) and hypoxia response genes (HIF-1α, GLUT1), and dramatically enhanced survival under hypoxia, serum deprivation, or chemo-drug 5-fluorouracil treatment conditions. In particular, silenced CSN8 blocks the EMT and dormancy processes induced by the hypoxia of 1% O2 in vitro and undermines the adaptive capacity of colorectal cancer cells in vivo. The further study showed that CSN8 regulated EMT and dormancy partly by activating the HIF-1α signaling pathway, which increased HIF-1α mRNA expression by activating NF-κB and stabilized the HIF-1α protein via HIF-1α de-ubiquitination. Taken together, CSN8 endows primary colorectal cancer cells with highly aggressive/metastatic and adaptive capacities through regulating both EMT and dormancy induced by hypoxia. CSN8 could serve as a novel prognostic biomarker for colorectal cancer and would be an ideal target of disseminated dormant cell elimination and tumor metastasis, recurrence, and chemoresistance prevention.

Oncolytic vaccinia virus delivering tethered IL-12 enhances antitumor effects with improved safety.

Immune checkpoint blockade is arguably the most effective current cancer therapy approach; however, its efficacy is limited to patients with "hot" tumors, warranting an effective approach to transform "cold" tumors. Oncolytic viruses (especially properly armed ones) have positive effects on almost every aspect of the cancer-immunity cycle and can change the cancer-immune set point of a tumor. Here, we tested whether oncolytic vaccinia virus delivering tethered interleukin 12 (IL-12) could turn a "cold" tumor into a "hot" tumor while avoiding IL-12's systemic toxicity. Our data demonstrated that tethered IL-12 could be maintained in the tumor without treatment-induced toxic side effects. Moreover, the treatment facilitated tumor infiltration of more activated CD4+ and CD8+ T cells and less Tregs, granulocytic myeloid-derivedsuppressor cells, and exhausted CD8+ T cells, with increased interferon γ and decreased transforming growth factor ß, cyclooxygenase-2, and vascular endothelial growth factor expression, leading to transformed, immunogenic tumors and improved survival. Combined with programmed cell death 1 blockade, vaccinia virus expressing tethered IL-12 cured all mice with late-stage colon cancer, suggesting immediate translatability to the clinic.

Nuclear-localized costimulatory molecule 4-1BBL promotes colon cancer cell proliferation and migration by regulating nuclear Gsk3ß, and is linked to the poor outcomes associated with colon cancer.

Anti-tumor immune response and the prognosis of tumor are the results of competition between stimulatory and inhibitory checkpoints. Except for upregulating inhibitory checkpoints, lowering some immune accelerating molecules to convert an immunostimulatory microenvironment into an immunodormant one through "decelerating the accelerator" might be another effective immune escape pattern. 4-1BBL is a classical transmembrane costimulatory molecule involving in antitumor immune responses. In contrast, we demonstrated that 4-1BBL is predominantly localized in the nuclei of cancer cells in colon cancer specimens and is positively correlated with tumor size, lymph node metastasis, and a lower survival ratio. Furthermore, the nuclear localization of 4-1BBL was also ascertained in vitro. 4-1BBL knockout (KO) arrests the proliferation and impaired the migration and invasion ability of colon cancer cells in vitro and retarded tumor growth in vivo. 4-1BBL KO increased the accumulation of Gsk3ß in the nuclei of colon cancer cells and consequently decreased the expression of Wnt pathway target genes and thus alter tumor biological behavior. We hypothesized that unlike membrane-expressed 4-1BBL, which stimulates the 4-1BB signaling of antitumor cytotoxic T cells, the nuclear-localized 4-1BBL could facilitate the malignant behavior of colon cancer cells by circumventing antitumor signaling and driving some key oncotropic signal pathway in the nucleus. Nuclear-localized 4-1BBL might be an indicator of colon cancer malignancy and serve as a promising target of immunotherapy.

Modifying the cancer-immune set point using vaccinia virus expressing re-designed interleukin-2.

The complex immune tumour microenvironment requires an equally complex immunotherapy approach, especially when the cancer-immune set point is non-inflamed. Oncolytic viruses expressing immune activating cytokines might optimally modify the immune microenvironment and improve the antitumour effects. In this study, we have explored a variety of IL-2 constructs expressed by a tumour-selective oncolytic vaccinia virus, designed to maintain IL-2 in the tumour microenvironment to reduce systemic toxicity. An IL-2 construct combining a glycosylphosphatidylinositol (GPI) anchor with a rigid peptide linker leads to functional IL-2 expression on the tumour cell surface and in the tumour microenvironment. This virus construct effectively modifies the cancer-immune set point and treats a variety of murine tumour models with no toxic side effects. In combination with PD-1/PD-L1 blockade this virus cures most of the mice with a high tumour burden. This combination represents a treatment for cancers which are to date unresponsive to immunotherapy.

Impact of cell fusion in myeloma marrow microenvironment on tumor progression.

BACKGROUND: Mesenchymal stem cells (MSCs) represent a subset of non-hematopoietic adult stem cells, which can also fuse with other cells spontaneously in bone marrow and capable of adopting the phenotype of other cells. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of bone marrow mesenchymal stem cells(BM-MSCs) and MM cells demonstrate that the fused cells can exhibit stemness and cancer cell-like characteristics. RESULTS: We successfully produced a hybrid cells that acquired larger size and multinucleation, in which partial chromatin condensation, a visible nucleolus, and one or more round or oval nucleus. Experiments results showed that the stemness markers highly expressed in these fused cells and there were much more chromosomes in fused cells than those in parental cells as well as exhibited increased resistance to drug treatment. CONCLUSIONS: Our results suggest that cell fusion between BM-MSCs and MM cells could contribute it genomic heterogeneity and play a role on disease progression. METHODS: We fused human BM-MSCs with MM cells lines RPMI 8226 or XG1 in vitro by polyethylene glycol (PEG), and the hybrid cells were sorted by sedimentation assays. The growth, migration, cell cycle, chromosome and drug sensitive of hybrids were assessed by cell counting, cell colony formation, transwell assays, cytogenetic assay and flow cytometry (FCM). The proteins and genes related to stemness and cytokines were tested by western blot and/or real-time quantitative RT-PCR.

Dietary quercetin ameliorates experimental colitis in mouse by remodeling the function of colonic macrophages via a heme oxygenase-1-dependent pathway.

Inflammatory bowel disease (IBD) results from a chronic intestinal inflammation and tissue destruction via an aberrant immune-driven inflammatory response towards an altered gut microbiota. Dietary intervention is becoming an attractive avenue for the therapy of colitis because diet is a key determinant of the mucosal immune response. Quercetin (QCN) is the most common in nature and the major representative of dietary antioxidant flavonoids, which has been demonstrated to influence the progression of colitis. However, the underlying mechanism of QCN on intestinal immunomodulation remains unclear. Here, our study demonstrated dietary QCN could ameliorate experimental colitis in part by modulating the anti-inflammatory effects and bactericidal capacity of macrophages via Heme oxygenase-1 (Hmox1, HO-1) dependent pathway. It suggested that QCN might restore the proper intestinal host-microbe relationship to ameliorate the colitis via rebalancing the pro-inflammatory, anti-inflammatory and bactericidal function of enteric macrophages. Hence, modulating the function of intestinal macrophages with dietary administration of QCN to restore the immunological hemostasis and rebalance the enteric commensal flora is a potential and promising strategy for IBD therapy.

Impact of connexin 43 coupling on survival and migration of multiple myeloma cells.

INTRODUCTION: Gap junctions (GJs) represent the best known intercellular communication (IC) system and are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules to pass from cell to cell. In this study, we constructed an amino terminus of human Cx43 (Cx43NT-GFP), verified the overexpression of Cx43-NT in HUVEC cells and explored the impact of gap junctions (GJs) on multiple myeloma (MM). MATERIAL AND METHODS: The levels of phosphorylated Cx43(s368) and the change of MAPK pathway associated molecules (ERK1/2, JNK, p38, NFκB) were also investigated in our cell models. Cx43 mRNA and proteins were detected in both MM cell lines and mesenchymal stem cells (MSCs). Dye transfer assays demonstrated that gap junction intercellular communication (GJIC) occurring via Cx43 situated between MM and MSCs or MM and HUVECCx43NT is functional. RESULTS: Our results present evidence for a channel-dependent modulator action of connexin 43 on the migratory activity of MM cells toward MSCs or HUVECCx43-N was higher than those of spontaneous migration (p < 0.05) and protection them from apoptosis in the presence of dexamethasone via cytokines secretion. In the meantime, the migration of MM cells involves an augmented response of p38 and JNK signaling pathway of carboxyl tail of the protein. CONCLUSIONS: Our data suggest that GJIC between MM and MSCs is one of the essential factors in tumor cell proliferation and drug sensitivity, and is implicated in MM pathogenesis.

Immune checkpoint molecule PD-1 acts as a novel biomarker for the pathological process of gestational diabetes mellitus.

AIM: Accumulating evidence suggested that challenge of the maternal-fetal interaction during pregnancy might cause the impairment of immunological hemostasis and lead to gestational diabetes mellitus (GDM) pathological process. Immune checkpoint molecule PD-1 is one of the critical molecule balancing immune response and immunological tolerance. METHODS: PD-1 expressions on T-cell subsets of GDM patients and control groups were measured via flow cytometric analysis and followed up. RESULTS: Downregulation of PD-1 acted as an indicator for GDM occurrence in the third trimester of pregnancy. With the recovery of GDM, PD-1 expression restored to normal level. CONCLUSION: PD-1 expression on T-cell subsets is a novel biomarker for the occurrence and recovery of GDM.

Directional delivery of RSPO1 by mesenchymal stem cells ameliorates radiation-induced intestinal injury.

Radiation-induced intestinal injury (RIII) commonly occurs in patients who received radiotherapy for pelvic or abdominal cancer, or who suffered from whole-body irradiation during a nuclear accident. RIII can lead to intestinal disorders and even death given its integrity damage that results from intestinal stem cell (ISC) loss. Recovery from RIII relies on the intensity of supportive treatment, which can attenuate lethal infection and give surviving stem cells an opportunity to regenerate. It has been reported that RSPO1 is a cytokine with potent and specific proliferative effects on intestinal crypt cells. MSCs have multiple RIII-healing effects, including anti-inflammatory and anti-irradiation injury properties, due to its negative immune regulation and its homing ability to the damaged intestinal epithelia. To combine the comprehensive anti-injury potential of MSCs, and the potent ability of RSPO1 as a mitogenic factor for ISCs, we constructed RSPO1-modified C3H10 T1/2 cells and expected that RSPO1, the ISC-proliferative cytokine, could be delivered to the site of injury in a targeted manner. In this study, we transferred C3H10/RSPO1 intravenously via the retro-orbital sinus into mice suffering from abdominal irradiation at lethal dosages. Our findings demonstrated that C3H10/RSPO1 cells are able to directionally migrate to the injury site; enhance ISC survival, proliferation, and differentiation; and effectively repair the radiation-damaged intestinal epithelial cells. This study suggests that the directional delivery of RSPO1 by MSCs is a promising strategy to ameliorate, and even cure, RIII.

Impact of Global and Gene-Specific DNA Methylation in de Novo or Relapsed Acute Myeloid Leukemia Patients Treated with Decitabine.

In this investigation, global DNA methylation patterns and the specific methylation status of 5 genes were studied in DNA from peripheral blood (PB) and impact on progression free survival (PFS) and overall-survival (OS) in patients with de novo or relapsed acute myeloid leukemia (AML) treated with decitabine-based regimens waas assessed. DNA was isolated from PB samples at the time of -1, 1, and 7 days of chemotherapy. Global methylation was determined by ELISA, and the CpG island DNA methylation profile of 5 genes using a DNA methylation PCR system. Our data demonstrated that patients with a high level of 5-mC had a poor prognosis after demethylation therapy and those who have low levels of 5-mC in PB achieved higher CR and better SO, but there was no significant correlation found between the 5-mC levels and other clinical features before treatment except the disease status. Higher methylation status of Sox2 and Oct4 genes was associated with differential response to demethylation therapy. A relatively low methylation percentage in one or both of these two genes was also associated with longer OS after decitabine based chemotherapy. We also suggest that global DNA and Oct-4/Sox2 methylation might impact on the pathogenesis of leukemia and play an important role in the initiation and progression. Moreover, dynamic analysis of 5-mC and Oct-4/Sox2 in peripheral blood nucleated cells of leukemia patients may provide clues to important molecular diagnostic and prognostic targets.

[Sensitivity to bortezomib of RPIM8226 cells after co-cultured with down-regulated Cav-1 expression HUVECs].

OBJECTIVE: To investigate the sensitivity to bortezomib of RPMI8226 cells after co-cultured with down-regulated Caveolin (Cav)-1 expression of HUVECs by transfection with Cav-1 shRNA (HUVECs(Cav-1 low)). METHODS: Exposure to bortezomib with or without 50 nmol/L dexamethasone at different concentration, the proliferation of RPMI8226 was analyzed by MTT assay when it was cultured alone or co-cultured with HUVECs(Cav-1 low). Cav-1 expression was detected by using of Western blot and cell cycle, apoptosis and the level of reactive oxygen species (ROS) were analyzed by flow cytometry. RESULTS: Cav-1 expression was notably down-regulated in HUVECs(Cav-1 low) (0.2199±0.0288 vs 1.3195±0.2393) (P<0.01). The IC(50) of bortezomib for RPMI8226 cultured alone, co-cultured with HUVECs orHUVECCav- 1 low were 20 nmol/L, 50 nmol/L and 65 nmol/L, respectively. The percentages of G0/G1 phase in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) were 28.49%, 30.41%, and 36.15% respectively. The protection of RPMI 8226 against apoptosis by HUVECs was demonstrated that the apoptosis/death rates were 66.8%, 10.7% and 8.6% in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) after exposure to 20 nmol/L bortezomib for 24 h. RPMI8226 could induce the oxidative stress of HUVECs before and after co-culture. The ROS level was raised from 15.0% to 35.2% in RPMI8226, from 80.4% to 91.0% in HUVECs, and from 84.6% to 96.8% in HUVECs(Cav-1 low). CONCLUSION: The down-regulated Cav-1 expression of HUVECs could promote proliferation and induce apoptosis of RMPI8226 cells, lead to G0/G1 phase arrest, and reduce the sensitivity to bortezomib.

An anti-human CD13 monoclonal antibody that suppresses the suppressive function of Treg cells.

CD13 (CD13/aminopeptidase N, APN, or CD13/APN) is a widely expressed type II membrane-bound metalloprotease. It is often overexpressed on cancer cells and expressed on CD4(+)CD25(hi) Treg cell subpopulation with higher suppressive ability. It has been determined to be a promising target in cancer diagnosis and therapy. In this study, a functional anti-human CD13 monoclonal antibody, MAb 9E4, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the CD13 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 9E4 suppresses the suppressive function of Treg cells. This functional anti-human CD13 MAb provides a valuable tool for further study targeting the CD13 positive Treg cells.

Blockade of PD-1/PD-L1 immune checkpoint during DC vaccination induces potent protective immunity against breast cancer in hu-SCID mice.

Immune checkpoints, such as the PD-1/PD-L1 pathway, negatively interfere in the efficiency of dendritic cell (DC) vaccination in cancer immunotherapy. In this study, we demonstrated that the blockade of PD-L1 signaling could promote DC maturation, proliferation, and IL-12 secretion, augment DC primed T cell response and reverse tumor cell dampened T cell impairment. Blockade of PD-L1 signaling during DC vaccination showed better therapeutic effects than classic DC vaccination by preventing tumor growth and prolonging survival times in a breast tumor-bearing hu-SCID model. Taken together, suppressing immune checkpoints during DC vaccination might be a more efficient strategy for cancer therapy.

[Eukaryotic expression and biological activity of recombinant human IL-17B protein].

AIM: To construct pCEP4/hIL-17B recombinant expression vector and express it stably in eukaryotic cells and investigate the biological activity in vitro. METHODS: The CDS region of human IL-17B gene was cloned by RT-PCR. After identification by sequencing, the hIL-17B gene was inserted into expression vector of pCEP4 to construct the recombinant vector pCEP4/hIL-17B, then transfected into 293T cells. The transgenic 293T cell line stably expressing rhIL-17B protein was selected in the presence of Hygromycin B. After FCS-free cultivation and sub-cloning, The IL-17B/mFc gene and protein expression was confirmed by RT-PCR, ELISA and Western blot analysis. To investigate the ability of combination with IL-17B receptor on human leukemic monocytic cell line, THP-1, by Flow cytometrical analysis (FACS) and of stimulation to secret cytokines in vitro. RESULTS: The recombinant pCEP4/hIL-17B and its transgenic 293T cells stably expressing rhIL-17B protein were obtained successfully. FACS analysis showed its high affinity with its receptor and it can stimulated THP-1 cell line to excrete IL-1ß and TNF-α in vitro and consistently caused a dose-dependent influx of neutrophil into the peritoneal cavity by intraperitoneal injection in vivo. CONCLUSION: The obtainment of transgenic 293T cell line stably expressing rhIL-17B protein paved the way for further study on biological functions of hIL-17B.

Anti-human FLT3 monoclonal antibody that inhibits proliferation of monocytic leukemia cell line SHI-1.

FLT3, a transmembrane molecule, was found on hematopoietic stem/progenitor cells and leukemia cells and determined to be a promising target in leukemia diagnosis and therapy. In this study a functional anti-human FLT3, monoclonal antibody (MAb) 10G6, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the FLT3 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 10G6 inhibited the proliferation and migration ability and induced the apoptosis of SHI-1 cells that derived from a human monocytic leukemia. This functional anti-human FLT3 MAb provides a valuable tool for further study targeting the FLT3 on leukemia cells.

Functional expression of chimeric Fab of an anti-CD40L mAb: Vector design and culture condition optimization.

CD40L is an important costimulatory molecule in the induction of the humoral and cell-mediated immune responses. 4F1, a specific murine antagonistic monoclonal antibody against human CD40L molecule, is a promising candidate biomedicine for autoimmune diseases, transplantation rejection and anti-angiogenesis therapy of cancer. To avoid the mAb induced thromboembolism as a consequence of platelet surface FcγR activation, we attempted to construct a chimeric Fab of 4F1 to minimize its side effects for potential clinical use. A chimeric version of anti-CD40L Fab was generated by transferring mouse variable regions into a human framework. Our study indicated that 4F1 could be simply and rapidly converted to chimeric Fab which could be expressed in bacteria and purified in reasonable quantities. This chimeric antibody maintained its bioreactivity to human CD40L.

T-bet and eomesodermin are required for T cell-mediated antitumor immune responses.

Cell-mediated adaptive immunity is very important in tumor immune surveillance and tumor vaccination. However, the genetic program underlying an effective adaptive antitumor immunity is elusive. T-bet and Eomesodermin (Eomes) have been suggested to be master regulators of Th1 cells and CD8(+) T cells. However, whether they are important for T cell-mediated antitumor immunity is controversial. In this paper, we show that the combined germline deletion of T-bet and T cell-specific deletion of Eomes resulted in profound defects in adaptive antitumor immune responses. T-bet and Eomes drive Tc1 differentiation by preventing alternative CD8(+) T cell differentiation to Tc17 or Tc2 cells. Surprisingly, T-bet and Eomes are not critical for the generation of systemic CTL activities against cancer cells. Instead, T-bet and Eomes are crucial for tumor infiltration by CD8(+) T cells. This study defines T-bet and Eomes as critical regulators of T cell-mediated immune responses against tumor.

Gadd45b and Gadd45g are important for anti-tumor immune responses.

An effective Th1 type cell-mediated immune response against cancer cells is critical in limiting cancer progression. Gadd45b, a signaling molecule highly up-regulated during Th1 type responses, is studied for its role in limiting tumor growth. Mouse B16 melanoma cells implanted into Gadd45b(-/-) mice grew faster than those in WT or Gadd45b(+/-) littermate controls. The defect of Gadd45b(-/-) mice in tumor immunosurveillance was attributed to the reduced expression of IFN-gamma, granzyme B, and CCR5 in Gadd45b(-/-) CD8(+) T cells at the tumor site. Activation of p38 MAP kinase, but not ERK or JNK, by either TCR-stimuli or IL-12 and IL-18 is diminished in Gadd45b(-/-) CD8(+) T cells, resulting in reduced production of IFN-gamma. In addition, mRNA of T-bet and Eomes were reduced in Gadd45b(-/-) CD8(+) T cells, supporting a critical role of Gadd45b in shaping the Th1 fate. More importantly, the tumor vaccination, which is effective in WT mice, failed in Gadd45b/Gadd45g doubly deficient mice. Collectively, these data demonstrate that members of the Gadd45 gene family are important for anti-tumor immune responses.