Establishment of operational tolerance to sustain antitumor immunotherapy.

BACKGROUND: Commonly used immunosuppressive drugs are efficacious in the prevention of transplanted solid organ rejection but, are complicated by an increased rate of malignancies. Treatment of the latter with cancer immunotherapy is frequently associated with increased graft loss from rejection. METHODS: B16 melanoma was inoculated subcutaneously (s.c.) in the ear of host B6 mice carrying a heterotopic allogenic murine heart. Intratumoral (i.t.) immunotherapy with anti-programmed death-1 (PD-1) and a toll-like receptor 9 (TLR9) agonist was conducted to treat cancer. Cyclosporine A (CsA) therapy or replaced by other immunosuppressive agents was conducted to preserve heart allograft tolerance, respectively. RESULTS: Here we show that long-term allograft-bearing mice receiving CsA therapy and challenged with host-type melanoma resist cancer immunotherapy and reject their grafts after CsA withdrawal. However, when CsA therapy is replaced by intermittent administration of the PI4KIIIß inhibitor UCB9608, effective antitumor immunity is induced while preserving graft tolerance. UCB9608 switch combined with i.t. immunotherapy resulted in donor-specific tolerance with preserved third-party responses. This operational tolerance may not be dependent on regulatory T cells (Tregs). CONCLUSIONS: Immune effects induced by UCB9608 switch following CsA therapy allow for antitumor immunity, opening up new approaches to establish donor-specific operational tolerance in solid organ transplantation with cancer.

Immunization with alloantibodies-covered melanoma cells induces regional antitumor effects that become systemic when combined with 5-FU treatment.

Alloantibodies, in particular immunoglobulin G (allo-IgG), confer a rejection advantage to tumors sharing the same major histocompatibility complex (MHC) in mice. However, when administrated intratumorally, this effect can only be achieved in combination with dendritic cells (DCs) activation. Here, we developed high titer allo-IgG by multiple rounds of immunization with allogenic B16 melanoma cells, which allows for the strong binding with B16 cells. We demonstrate that B16 cells incubated with these allo-IgG (referred to as allo-IgG-B16) become highly immunogenic, which release tumor antigens that are efficiently presented by classic DCs in lymph nodes (LNs). Injection of allo-IgG-B16 turns the tumor into an immune hot one and even elicits a systemic antitumor response when used together with 5-fluorouracil (5-FU). This systemic response is tumor-specific and relies on the critical site - LNs. Our findings provide a rationale for the use of allo-IgG in cancer treatment.

Intratumoral immunotherapy with anti-PD-1 and TLR9 agonist induces systemic antitumor immunity without accelerating rejection of cardiac allografts.

Immune checkpoint inhibitors, such as programmed cell death 1 (PD-1) blockades, have revolutionized the field of cancer immunotherapy. However, there is a growing concern whether PD-1 inhibitors can be administered safely to transplant recipients with advanced cancer, as the T cells activated by checkpoint inhibitors may become reactive not only toward tumor antigens but also toward donor alloantigen, thereby resulting in allograft rejection. Here, immunotherapy with anti-PD-1/toll like receptor 9 agonist was administered to C57BL/6 mice bearing a cardiac allograft that were receiving maintenance immunosuppression or a PI4KIIIß inhibitor-based tolerogenic regimen. Intratumoral (i.t.), but not systemic, immunotherapy promoted potent anti-tumor responses, but did not accelerate allograft rejection. This effect was associated with a pro-immunogenic effect induced by i.t. immunotherapy resulting in systemic cellular and humoral immune anti-tumor responses. Furthermore, when the tumor and cardiac allograft shared major histocompatibility complex (MHC) antigens, i.t. immunotherapy promoted immune responses directed against tumor and the cardiac allograft resulting in allograft rejection. The anti-tumor effect was compromised by maintenance immunosuppression with cyclosporin A, indicating that an optimal balance between enhanced anti-tumor immunity and decreased transplant immunoreactivity is critical. A clinically relevant approach could be to temporarily withdraw maintenance immunosuppression and/or replace it with a PI4KIIIß inhibitor-based tolerance-inducing regimen to allow for effective immunotherapy to take place.

Donor Lymphocyte-Derived Natural Killer Cells Control MHC Class I-Negative Melanoma.

Natural killer (NK) cells provide a natural defense against MHC-I-negative tumors, such as melanoma. Donor lymphocyte infusion (DLI) containing NK cells, a form of adoptive immunotherapy used after allogenic bone marrow transplantation (allo-BMT), promotes antitumor immune responses but is often associated with life-threatening complications such as graft-versus-host disease (GvHD). Here, we showed that without prior allo-BMT, DLI provoked melanoma control associated with the infiltration and persistence of the transferred NK cells. This allograft acceptance did not correlate with an increase of GvHD; instead it correlated with the expansion and activation of tumor-infiltrating NK cells that expressed the cytotoxic molecules (e.g., IFNγ and granzyme B) and maturation signatures (e.g., CD11bhiCD27lo and KLRGhi/CD43hi). The development of beneficial tumor-infiltrating NK cells of DLI origin required host CD4+ T-cell help in part by producing IL2, as well as by limiting regulatory CD4+ T cells (Treg). IL2 blockade impaired the NK-dependent melanoma control, which could not be rescued by IL2 administration beyond CD4+ T-cell help. Our findings linked NK allograft acceptance-CD4+ T-cell help crosstalk to melanoma development without the need of allo-BMT. We thereby helped define that tumor-infiltrating NK cells of DLI origin may serve as effective therapeutic targets for controlling melanoma.

Improved Anti-Tumour Adaptive Immunity Can Overcome the Melanoma Immunosuppressive Tumour Microenvironment.

Clinical benefits obtained from checkpoint blockade regimens demonstrate the importance of overcoming the immunosuppressive tumour microenvironment (TME) in cancer immunotherapy. Intravenous (i.v.) injection of B16 melanoma cells (H-2Kb) leads to lethal disseminated pulmonary metastasis in Balb/c recipients (H-2Kd). This lack of immune control is related to low major histocompatibility complex (MHC) expression on B16 cells which is associated with delayed and decreased anti-tumour adaptive immune responses (e.g., alloantibody formation) as: (i) other tumour types with normal H-2Kb expression are rejected with concomitant antibody production; (ii) preincubation of B16 with IFN-gamma to upregulate H-2Kb expression resulted in improved antibody production and anti-tumour activity. The delayed/decreased anti-tumour adaptive immune responses induced by B16 inoculation is not able to interrupt progression of primary metastases, while it is able to effectively eliminate secondary inoculated subcutaneously (s.c.) B16 cells from progression. This is due to the presence of an immunosuppressive TME within the primary metastases characterized by increased regulatory T cells (Tregs) and an increased T helper cells (Th) 2/1 profile. These tumour-induced immunosuppressive T cell populations are counteracted by improved adaptive immunity via active and passive immunization, resulting in effective elimination of the TME, destruction of the metastatic tumour and a reversal of Th2/1 profile in a time-sensitive manner. Thus, we here demonstrate that the TME is not irreversible and adaptive immunity is able to eradicate established solid tumour and its immunosuppressive TME. This study will help design treatments to overcome the immunosuppressive effect of the TME and improve efficacy of cancer immunotherapy.

Solid Tumor-Induced Immune Regulation Alters the GvHD/GvT Paradigm after Allogenic Bone Marrow Transplantation.

Growth of solid tumors is often associated with the development of an immunosuppressive tumor microenvironment (TME). It has been suggested that the influence of the TME may extend beyond the local tumor and results in systemic immunosuppression. Here, we utilize two murine cancer models to explore the influence of solid tumors on the occurrence of alloreactivity-driven GvHD and graft-versus-solid tumor (GvT) effects following MHC-mismatched allogeneic bone marrow transplantation (allo-BMT). Melanoma- or colon carcinoma-bearing C57BL/6 mice did not develop GvHD after BMT even when the bone marrow inoculum was supplemented with donor-type splenocytes. This protection against GvHD required the presence of tumors because its resection prior to allo-BMT promptly resulted in development of GvHD. In addition, tumor-bearing mice given T-cell-depleted allo-BMT (allo-TCD-BMT) failed to develop GvHD and also showed significantly stronger GvT effects than mice given allo-BMT. The GvT effects in allo-TCD-BMT recipients were associated with profound changes in tumor-infiltrating cells compared with that in allo-BMT recipients, with significantly reduced donor-derived regulatory T cells (Treg), increased cytotoxic effector (IFNγhi) CD8 T cells, and increased M1 macrophages (iNOShi, arginaselo, and IL10lo); the use of macrophage-depleted bone marrow abrogated the GvT effects. Collectively, these results indicate that the presence of M1 macrophages may disrupt the generation of donor-type Treg cells so that the immunomodulatory effect of the TME can affect systemic immunity. SIGNIFICANCE: These findings show that cells such as T cells or macrophages in the bone marrow inoculum may interfere with the systemic and local immune reactivity against tumors.

Activation of ATF4 mediates unwanted Mcl-1 accumulation by proteasome inhibition.

Myeloid cell leukemia-1 (Mcl-1) protein is an anti-apoptotic Bcl-2 family protein that plays essential roles in multiple myeloma (MM) survival and drug resistance. In MM, it has been demonstrated that proteasome inhibition can trigger the accumulation of Mcl-1, which has been shown to confer MM cell resistance to bortezomib-induced lethality. However, the mechanisms involved in this unwanted Mcl-1 accumulation are still unclear. The aim of the present study was to determine whether the unwanted Mcl-1 accumulation could be induced by the unfolded protein response (UPR) and to elucidate the role of the endoplasmic reticulum stress response in regulating Mcl-1 expression. Using quantitative RT-PCR and Western blot, we found that the translation of activating transcription factor-4 (ATF4), an important effector of the UPR, was also greatly enhanced by proteasome inhibition. ChIP analysis further revealed that bortezomib stimulated binding of ATF4 to a regulatory site (at position -332 to -324) at the promoter of the Mcl-1 gene. Knocking down ATF4 was paralleled by down-regulation of Mcl-1 induction by bortezomib and significantly increased bortezomib-induced apoptosis. These data identify the UPR and, more specifically, its ATF4 branch as an important mechanism mediating up-regulation of Mcl-1 by proteasome inhibition.

Inhibition of N-myc downstream-regulated gene 2 in prostatic carcinoma.

To study the expression of N-myc Downstream Regulated Gene-2 (NDRG2) in prostatic carcinoma (PCA) tissue and in different PCA cell lines, and to investigate its clinical and pathological implications, 144 PCA and benign prostatic hyperplasia (BPH) tissue sections were analyzed retrospectively with immunohistochemistry (S-P method). The expression levels of NDRG2 and c-Myc in prostate cell lines were detected through Western blot. The effects of adenovirus-mediated NDRG2 on PC3 cells and PC3 nude mouse xenografts was observed through cell growth curves, tumor growth curves, flow cytometry (FCM), transmission electron microscopy (TEM) and TUNEL staining. The NDRG2 gene was highly expressed in BPH tissues, but not in carcinomatous ones (χ(2)=25.98, p < 0.001). Furthermore, positive expression of NDRG2 was negatively correlated with the Gleason score (r = -0.445, p< 0.001) and the c-myc level (r = -0.311, p < 0.001). However, positive expression of NDRG2 was not correlated with pTNM tumor stages or the serum concentration of prostate-specific antigen (PSA) (p > 0.05). The expression of the NDRG2 genes was low in the three PCA cell lines. PC3 cells infected by pAD-cmv-NDRG2 showed inhibition of proliferation both in vitro and vivo. To sum up, NDRG2 may be involved in the carcinogenesis and progression of PCA. Moreover, adenovirus-mediated NDRG2 can suppress the proliferation of PC3 cells significantly both in vitro and in vivo. These results indicate that NDRG2 may become a new target gene for PCA diagnosis and therapy.

Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation.

HAb18G/CD147, a glycoprotein of the immunoglobulin super-family (IgSF), is a T cell activation-associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4(+) and CD8(+) T cells was up-regulated. In vitro cross-linking of T cells with an anti-HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co-stimulation inhibited T cell proliferation by down-regulating the expression of CD25 and interleukin-2 (IL-2), decreased production of IL-4 but not interferon-γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti-HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody-antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N-terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co-stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.

CD167 acts as a novel costimulatory receptor in T-cell activation.

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. CD167 is a tyrosine kinase receptor for native type I collagen, its physiologic functions include matrix homeostasis and cell growth, adhesion, branching, and migration, but the specific role of CD167 in T cells has not yet been characterized. In this study, we found that CD167 expression on T cells was up-regulated after activation. Cooperation of CD167 engagement with suboptimal TCR/CD3 signals induced T-cell proliferation, enhanced expression of activation markers such as CD25 and CD69, elevated intracellular calcium mobilization and tyrosine phosphorylation, and introduced a bias toward a TH1/Tc1 immune response. Cooperation of CD167 engagement also enhanced mixed lymphocyte responses to alloantigens. Moreover, CD167 rapidly localized to the aggregated lipid rafts upon T-cell activation, this provided a molecular base for the signaling machinery of CD167. Together these findings, we demonstrate for the first time that CD167 could serve as a novel costimulatory receptor for T-cell activation.

Expression pattern of epithelial cell adhesion molecule on normal and malignant colon tissues.

AIM: To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance. METHODS: cDNA encoding Ep-CAM extracellular domain was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from excised malignant colon tissues and inserted into a glutathione S-transferase (GST)-tagged vector. Ep-CAM-GST fusion protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with glutathione-sepharose. The Ep-CAM-GST fusion protein was mixed with Freund's adjuvant and Balb/c mice were immunized with it. Sp2/0 myeloma cells were fused with the spleen cells of the immunized mice. After having selected by indirect ELISA, the anti-Ep-CAM monoclonal antibodies (MAbs) were generated and the corresponding ascites were obtained. Finally, the human colon carcinoma tissue array prepared from seventy individual patients was stained with the anti-Ep-CAM MAbs. RESULTS: The isolated Ep-CAM cDNA sequence was identical to the data in GenBank. The expressed fusion protein was almost soluble and had a molecular weight (MW) of 53 ku. Four MAbs against Ep-CAM were obtained and designated as FMU-Ep1, FMU-Ep2, FMU-Ep3 and FMU-Ep4 respectively. Among them, FMU-Ep4 could recognize the natural Ep-CAM on Colo205 and SW480 cells, and all of them could be used for immunohistochemical staining of tissue sections. It was found that Ep-CAM was distributed differently in normal and various malignant colon tissues, including squamous cell carcinoma, signet-ring cell carcinoma and adenocarcinoma. In normal colon gland epithelia, Ep-CAM antigen was mainly distributed on the basolateral membrane and in the region between the basolateral membrane and the cytoplastic part near the nuclei, whereas the expression pattern of colon malignancies was mainly on the whole surface of epithelia and the expression was much higher than the normal colon tissues. The staining pattern of tissue array showed in adenocarcinoma and papillary adenocarcinoma, and the expression of Ep-CAM was increased from grade I to grade III. CONCLUSION: MAbs against Ep-CAM might be useful for research on the structure and function of Ep-CAM and may have diagnostic and therapeutic value to various colon carcinomas.