Systemic antitumor effect by regional hyperthermia combined with low-dose chemotherapy and immunologic correlates in an adolescent patient with rhabdomyosarcoma - a case report.

Introduction: An abscopal effect is a clinical observation whereby a local treatment is associated with regression of metastatic cancer at a site distant from the primary location of treatment. Here, we describe the clinical systemic effect induced by regional hyperthermia combined with low-dose chemotherapy and provide immunologic correlates.Case presentation: A 15-year-old patient had been diagnosed with alveolar rhabdomyosarcoma (ARMS). All previous treatment options failed in the patient including haploidentical stem cell transplantation and donor lymphocyte infusion. The patient presented with local and metastatic disease, and upon admission, underwent regional hyperthermia combined with low-dose chemotherapy. Immediately following therapy severe skin reactions were observed. Skin biopsies revealed an intraepithelial lymphocytic infiltration dominated by CD3+/CD8+ T cells with a regular network of dendritic cells. Clinical images compared before and during sequential treatment cycles showed complete metabolic response of the local tumor for more than 10 months of therapy. In addition, metastases completely regressed although they were not direct targets of regional hyperthermia. The systemic effect was associated with enhanced frequency of NK cells and T cells expressing the lectin-like natural-killer group 2 D activating receptor (NKG2D), an increase of the CD56bright subset of NK cells, as well as an increase of effector/memory and effector CD8+ and CD4+ T cells in the blood while the percentage of CD25+FOXP3+ regulatory T cells declined.Conclusions: Regional hyperthermia combined with low-dose chemotherapy had the potential to create a systemic effect which was associated with activation of NK cells and T cells.

Induction of porcine-specific regulatory T cells with high specificity and expression of IL-10 and TGF-ß1 using baboon-derived tolerogenic dendritic cells.

BACKGROUND: Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo-organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen-specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non-human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno-specific Treg would benefit research on immune tolerance in xenotransplantation using this model system. METHOD: Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti-inflammatory cytokines. After loading with porcine-specific (PS) in vitro-transcribed RNA (ivtRNA), tolDC were used to induce CD4+ T cells to become porcine-specific Treg (PSTreg) in cocultures supplemented with IL-2 and rapamycin for 10 days. Anti-inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine-specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity. RESULTS: TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL-10 and TGF-ß1, whereas IL-12p40 and IFN-γ were not expressed. PSTreg were successfully generated in cocultures of CD4+ T cells and PS ivtRNA-loaded tolDC. They exhibited a CD3+  CD4+  CD25+  CD127low/-  CD45RAlow  Foxp3+ phenotype and were characterized by high expression of IL-10 and TGF-ß1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL-10 and TGF-ß1 cytokine upon interaction with PSTeff and suppressing IFN-γ expression on PSTeff. CONCLUSION: In this study, a fast 3-day method to generate baboon-derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine-antigen specificity and expression of IL-10 and TGF-ß1. Porcine-specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.

A novel and effective method to generate human porcine-specific regulatory T cells with high expression of IL-10, TGF-ß1 and IL-35.

Organ transplantation remains the most effective treatment for patients with late stage organ failure. Transgenic pigs provide an alternative organ donor source to the limited availability of human organs. However, cellular rejection still remains to be the obstacle for xenotransplantation. Superior to other methods, antigen-specific regulatory T cells (Treg) alleviate cellular rejection with fewer side effects. Here we demonstrate the use of a fast method to provide tolerogenic dendritic cells (tolDC) that can be used to generate effective porcine-specific Treg cells (PSTreg). TolDC were produced within three days from human monocytes in medium supplemented with anti-inflammatory cytokines. Treg were generated from naïve CD4+ T cells and induced to become PSTreg by cocultivation with porcine-antigen-loaded tolDC. Results showed that PSTreg exhibited the expected phenotype, CD4+CD25+CD127low/- Foxp3+, and a more activated phenotype. The specificity of PSTreg was demonstrated by suppression of effector T cell (Teff) activation markers of different stages and inhibition of Teff cell proliferation. TolDC and PSTreg exhibited high expression of IL-10 and TGF-ß1 at both protein and RNA levels, and PSTreg also highly expressed IL-35 at RNA levels. Upon restimulation, PSTreg retained the activated phenotype and specificity. Taken together, the newly developed procedure allows efficient generation of highly suppressive PSTreg.

Transcript signature predicts tissue NK cell content and defines renal cell carcinoma subgroups independent of TNM staging.

Clear cell renal cell carcinoma (ccRCC) is an aggressive and difficult to manage cancer. Immunotherapy has the potential to induce long-lasting regression in a small group of patients. However, severe side effects limit broad application which highlights the need for a marker to distinguish responder from nonresponder. TNMG staging, referring to tumor size, lymph node involvement, presence of metastasis, and grade of tumor differentiation, represents an important prognostic system but is not useful for predicting responders to immunotherapy. NK cells are potent antitumor effector cells, and a role as prognostic marker in some solid tumors has been suggested. As NK cells are responsive to various immune modifiers, they may be important mediators of patient response to immunotherapies, in particular those including IL-2. We report that the NK cell percentage within RCC-infiltrating lymphocytes, as determined by flow cytometry, allows ccRCC subgrouping in NK(high)/NK(low) tissues independent of TNMG classification. Quantitative reverse transcriptase polymerase chain reaction using whole-tissue RNA identified four markers (NKp46, perforin, CX(3)CL1, and CX(3)CR1) whose transcript levels reproduced the NK(high)/NK(low) tissue distinction identified by flow cytometry with high selectivity and specificity. Combined in a multiplex profile and analyzed using neural network, the accuracy of predicting the NK(high)/NK(low) groups was 87.8%, surpassing that of each single marker. The tissue transcript signature, based on a robust high-throughput methodology, is easily amenable to archive material and clinical translation. This now allows the analysis of large patient cohorts to substantiate a role of NK cells in cancer progression or response to immunotherapy.

Human renal cell carcinoma induces a dendritic cell subset that uses T-cell crosstalk for tumor-permissive milieu alterations.

Tissue dendritic cells (DCs) may influence the progression of renal cell carcinoma (RCC) by regulating the functional capacity of antitumor effector cells. DCs and their interaction with T cells were analyzed in human RCC and control kidney tissues. The frequency of CD209(+) DCs in RCCs was found to be associated with an unfavorable T(H)1 cell balance in the tissue and advanced tumor stages. The CD209(+) DCs in RCC were unusual because most of them co-expressed macrophage markers (CD14, CD163). The phenotype of these enriched-in-renal-carcinoma DCs (ercDCs) could be reiterated in vitro by carcinoma-secreted factors (CXCL8/IL-8, IL-6, and vascular endothelial growth factor). ErcDCs resembled conventional DCs in costimulatory molecule expression and antigen cross-presentation. They did not suppress cognate cytotoxic T-lymphocyte function and did not cause CD3ζ down-regulation, FOXP3 induction, or T-cell apoptosis in situ or in vitro; thus, they are different from classic myeloid-derived suppressor cells. ErcDCs secreted high levels of metalloproteinase 9 and used T-cell crosstalk to increase tumor-promoting tumor necrosis factor α and reduce chemokines relevant for T(H)1-polarized lymphocyte recruitment. This modulation of the tumor environment exerted by ercDCs suggests an immunologic mechanism by which tumor control can fail without involving cytotoxic T-lymphocyte inhibition. Pharmacologic targeting of the deviated DC differentiation could improve the efficacy of immunotherapy against RCC.

Humanized tumor mice--a new model to study and manipulate the immune response in advanced cancer therapy.

The immunological impact on antibody-based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD-scid IL2Rγ(null) mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor-cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T-cell maturation and tumor cell-specific T-cell activation. In addition, Natural-Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody-dependent cellular cytotoxicity-based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system-related target cancer therapy and resistance.