Immune response to cancer and its regulation in regional lymph nodes.

Regional draining lymph nodes (LNs) play a pivotal role in initiating immune responses. However, the presence of metastases may compromise their normal immunological function. Preclinical studies indicate that despite metastases, early tumor-draining LNs are still a rich source of sensitized T cells. Recently, we found that dendritic (DC)-tumor fusion hybrids were capable of stimulating therapeutic T-cell generation in the LN. However, this response is regulated by a tumor-specific suppression mechanism(s). Reversal of these dysfunctions would help the success of immunotherapy.

STAT3- and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34pos cells into tumor-competent dendritic cells.

The clinical outcomes of dendritic cell (DC)-based immunotherapy remain disappointing, with DCs often displaying a tenuous capacity to complete maturation and DC1 polarization in the tumor host. Surprisingly, we observed that the capacity for successful DC1 polarization, including robust IL12p70 production, could be regulated by STAT-dependent events even prior to DC differentiation. Exposure of CD34(pos) cells to single-agent granulocyte-macrophage colony-stimulating factor (GMCSF) induced multilineage, STAT5-dependent differentiation, including DCs that failed to mature in the absence of further exogenous signals. In contrast, Flt3L induced nearly global differentiation of CD34(pos) cells into spontaneously maturing DCs. IL-6 synergized with Flt3L to produce explosive, STAT3-dependent proliferation of phenotypically undifferentiated cells that nevertheless functioned as committed DC1 precursors. Such precursors not only resisted many tumor-associated immunosuppressants, but also responded to tumor contact or TGFbeta with facilitated DC maturation and IL12p70 production, and displayed a superior capacity to reverse tumor-induced T-cell tolerance. GMCSF preempted Flt3L or Flt3L plus IL-6 licensing by blocking STAT3 activation and promoting STAT5-dependent differentiation. Paradoxically, following overt DC differentiation, STAT5 enhanced whereas STAT3 inhibited DC1 polarization. Therefore, nonoverlapping, sequential activation of STAT3 and STAT5, achievable by sequenced exposure to Flt3L plus IL-6, then GMCSF, selects for multilog expansion, programming, and DC1 polarization of tumor-competent DCs from CD34(pos) cells.

Tumor-induced CD11b+Gr-1+ myeloid cells suppress T cell sensitization in tumor-draining lymph nodes.

Suppression of tumor-specific T cell sensitization is a predominant mechanism of tumor escape. To identify tumor-induced suppressor cells, we transferred spleen cells from mice bearing progressive MCA205 sarcoma into sublethally irradiated mice. These mice were then inoculated subdermally with tumor cells to stimulate T cell response in the tumor-draining lymph-node (TDLN). Tumor progression induced splenomegaly with a dramatic increase (22.1%) in CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) compared with 2.6% of that in normal mice. Analyses of therapeutic effects by the adoptive immunotherapy revealed that the transfer of spleen cells from tumor-bearing mice severely inhibited the generation of tumor-immune T cells in the TDLN. We further identified MDSC to be the dominant suppressor cells. However, cells of identical phenotype from normal spleens lacked the suppressive effects. The suppression was independent of CD4(+)CD25(+) regulatory T cells. Intracellular IFN-gamma staining revealed that the transfer of MDSC resulted in a decrease in numbers of tumor-specific CD4(+) and CD8(+) T cells. Transfer of MDSC from MCA207 tumor-bearing mice also suppressed the MCA205 immune response indicating a lack of immunologic specificity. Further analyses demonstrated that MDSC inhibited T cell activation that was triggered either by anti-CD3 mAb or by tumor cells. However, MDSC did not suppress the function of immune T cells in vivo at the effector phase. Our data provide the first evidence that the systemic transfer of MDSC inhibited and interfered with the sensitization of tumor-specific T cell responses in the TDLN.

Immunogenicity of dendritic-tumor fusion hybrids and their utility in cancer immunotherapy.

Cancer immunotherapy using fusion hybrid cells generated from dendritic cells (DCs) and tumor cells may be more effective than other DC-based vaccines. DC-tumor fusion potentially confers not only the DCs' antigen-presenting functionality but also a continuing source of endogenous tumor antigens for major-histocompatibility-complex-restricted T-cell sensitization. In animal models, many investigators demonstrated that vaccination with fusion hybrids was protective against tumor challenge and therapeutic, resulting in the regression of established tumors. In clinical trials for patients with a variety of metastatic diseases, fusion hybrid vaccines were well tolerated, but the overall objective response rate was only 10.9%. Careful scrutiny of a large number of publications revealed that, in most cases, no definitive evidence of heterokaryonic fusion cell formation was found. Further corroboration of this conclusion comes from reports that fusion hybrids generated from autologous (syngeneic) and allogeneic DCs displayed equivalent immunological function and therapeutic effects in vitro and in vivo. This puzzling finding suggests that effective fusion immunotherapy depends on tumor antigen scavenging and presentation by antigen-presenting cells (APCs) of host origin and is in violation of the basic tenet of the principle of DC function. We believe that conclusions drawn from reported clinical trials have not properly evaluated the efficacy of the DC-tumor hybrid vaccine, and therefore, they neither confirm nor disclaim the potential benefits that may be derived from this form of immunotherapy.

Host lymphodepletion augments T cell adoptive immunotherapy through enhanced intratumoral proliferation of effector cells.

T-cell adoptive immunotherapy for stringent murine tumor models, such as intracranial, s.c., or advanced pulmonary metastases, routinely uses lymphodepletive conditioning regimens before T-cell transfer, like recent clinical protocols. In this study, we examined whether host lymphodepletion is an obligatory component of curative T-cell therapy; we also examined the mechanism by which it augments therapy. Mice bearing intracranial, s.c., or 10-day pulmonary metastases of MCA 205 received total body irradiation conditioning or were nonirradiated before i.v. transfer of tumor-reactive T cells. Total body irradiation was not required for immunologically specific curative therapy and induction of memory provided that a 3- to 12-fold higher T-cell dose was administered. The mechanism involved enhanced intratumoral proliferation of T-effector cells in total body irradiation-conditioned recipients. In this tumor model, intratumoral T(reg) cells were not detected; consequently, intratumoral T-effector cells produced identical amounts of IFN-gamma upon ex vivo antigen stimulation irrespective of total body irradiation conditioning. Thus, host lymphodepletion augments T-cell immunotherapy through enhanced antigen-driven proliferation of T-effector cells, but curative therapy can be achieved in nonconditioned hosts by escalation of T-cell dose. These data provide a rationale for dose escalation of T-effector cells in situations where single or repeated lymphodepletion regimens are contraindicated.

Everolimus (RAD) inhibits in vivo growth of murine squamous cell carcinoma (SCC VII).

OBJECTIVE: Everolimus (RAD) is an mTOR inhibitor closely related to rapamycin. A potent immunosuppressive agent, it has also shown evidence of antineoplastic properties. SCC VII is a spontaneously arising murine squamous cell carcinoma line. This study examines the effect of everolimus on SCC VII proliferation. The data may provide support for the use of everolimus in transplant recipients with a history of malignancy. METHODS: A dose efficacy study was conducted that used a murine model of intradermal tumor growth and pulmonary metastases. The development of intradermal tumors and pulmonary metastases were studied. Of 80 total mice, 40 received intradermal injection of 1 x 10 SCC VII cells and 40 received intravenous injection of 1 x 10 cells to establish pulmonary metastases. Within each group, animals were subdivided into four subgroups that received 1) 1 mg/kg everolimus twice a day, 2) 0.5 mg/kg everolimus twice a day, 3) 7.5 mg/kg cyclosporine per day, and 4) no treatment. Intradermal tumors were measured three times per week. Animals receiving an intravenous tumor injection were killed after 17 days and pulmonary metastases were quantified. Medication trough levels were measured in all treated animals. RESULTS: Everolimus showed statistically significant tumor inhibition at 1.0 mg/kg twice a day and 0.5 mg/kg twice a day when compared with animals treated with cyclosporine and with untreated animals (P < .0001). Tumor inhibition was evident in both models studied (intradermal tumors and pulmonary metastasis generation). CONCLUSIONS: Everolimus provides potent tumor inhibition in animals inoculated with SCC VII cells. Inhibition of both local and distant spread of disease is evident. Although most immunosuppressives are known to potentiate neoplastic disease, this study supports the use of everolimus immunosuppression in the face of prior malignancy. This data has significant implication for laryngeal transplantation after laryngectomy.

Active immunotherapy for advanced intracranial murine tumors by using dendritic cell-tumor cell fusion vaccines.

OBJECT: Immunotherapy for malignant brain tumors by active immunization or adoptive transfer of tumor antigen-specific T lymphocytes has the potential to make up for some of the limitations of current clinical therapy. In this study, the authors tested whether active immunotherapy is curative in mice bearing advanced, rapidly progressive intracranial tumors. METHODS: Tumor vaccines were created through electrofusion of dendritic cells (DCs) and irradiated tumor cells to form multinucleated heterokaryons that retained the potent antigen processing and costimulatory function of DCs as well as the entire complement of tumor antigens. Murine hosts bearing intracranial GL261 glioma or MCA 205 fibrosarcoma were treated with a combination of local cranial radiotherapy, intrasplenic vaccination with DC/tumor fusion cells, and anti-OX40R (CD134) monoclonal antibody (mAb) 7 days after tumor inoculation. Whereas control mice had a median survival of approximately 20 days, the treated mice underwent complete tumor regression that was immunologically specific. Seven days after vaccination treated mice demonstrated robust infiltration of CD4+ and CD8+ T cells, which was exclusively confined to the tumor without apparent neurological toxicity. Cured mice survived longer than 120 days with no evidence of tumor recurrence and resisted intracranial tumor challenge. CONCLUSIONS: These data indicate a strategy to achieve an antitumor response against tumors in the central nervous system that is highly focused from both immunological and anatomical perspectives.

Tumor-dendritic cell fusion as a basis for cancer immunotherapy.

OBJECTIVE: To establish the basis for use of allogeneic dendritic-tumor fusion cells. STUDY DESIGN: Fusion cells were created by electrofusion. We used 2 allogeneic murine tumor lines (D5 and 4T1) that were virally transduced to express the antigen (beta-galactosidase) as a surrogate tumor marker. RESULTS: Cross-immunization was achieved with irradiated allogenic tumor cells. Successful electrofusion of dendritic cells and tumor cells was confirmed by using fluorescence-activated cell sorting and cytospin. Significant responses were shown in immunized mice against tumor challenge and established 3-day pulmonary metastasis with fusion cells. CONCLUSIONS: Allogeneic tumor sharing a common tumor antigen can immunize against syngeneic tumor challenge. Fusion cells showed successful immunization against tumor challenge and showed regression of 3-day established pulmonary metastasis. SIGNIFICANCE: These preclinical studies provide evidence that an allogenic tumor-dendritic cell fusion vaccine is a valid approach for head and neck cancer immunotherapy.

Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells.

T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vbeta families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.

Considerations on clinical use of T cell immunotherapy for cancer.

The recognition by effector T lymphocytes of novel antigenic targets on tumor cells is the premise of specific, targeted immunotherapy of cancer. With the molecular characterization of peptide epitopes from melanoma antigens and, more recently, broadly expressed tumor antigens, there has been considerable enthusiasm for clinical evaluation of peptide tumor vaccines. Immunologic monitoring of vaccinated patients has demonstrated an expansion of CD8+ T cells that react with the relevant peptide and, more importantly, with native tumor. In most instances, however, vaccine-induced CD8+ T cell responses alone have not been sufficiently robust or sustained to translate into a high percentage of durable clinical responses. Vaccine strategies have also utilized dendritic cells (DCs) that have been modified to present tumor antigens. The superior antigen-processing capacity and co-stimulatory function of DCs convey a powerful stimulatory signal to both CD4+ and CD8+ T cells. Several strategies are attempting to broaden the immune response beyond single antigens by introducing the entire complement of tumor antigens into DCs. Adoptive immunotherapy is a promising strategy to recover tumor-reactive precursor T cells from patients, stimulate them to induce numerical expansion, and then re-infuse them. Ex vivo manipulation of the tumor-reactive T cells also permits cytotoxic therapy to be administered to the patient without damaging the effector cells. Recently, host lymphodepletion prior to adoptive transfer of effector T cells has resulted in an extremely high and sustained frequency of effectors that has achieved therapeutic efficacy against bulky metastatic disease in a substantial fraction of treated patients.

[Immunotherapeutic reactivity of dendritic cells loaded with a variety of antigen preparations].

The findings summarized here provide a direct comparison of the immunogenicity of various DC loading strategies included pulsing with protein, peptide, tumor cell lysate, irradiated tumor cells and electrofusion of DCs and tumor cells. For the treatment of 3-day established pulmonary metastases, electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. Consistent with their therapeutic activity, fusion hybrids stimulated the release of the largest amount of interferon-gamma from immune T cells. However, IL-10 secretion did not correlate with in vivo therapeutic reactivity. In conclusion, DC-tumor fusion hybrids were the most effective vaccine to eradicate existing tumors. These data support the use of DC-tumor electrofusion cells for the treatment of human cancer.

Extracellular ATP and Toll-like receptor 2 agonists trigger in human monocytes an activation program that favors T helper 17.

Strategically-paired Toll-like receptor (TLR) ligands induce a unique dendritic cell (DC) phenotype that polarizes Th1 responses. We therefore investigated pairing single TLR ligands with a non TLR-mediated danger signal to cooperatively induce distinct DC properties from cultured human monocytes. Adenosine triphosphate (ATP) and the TLR2 ligand lipoteichoic acid (LTA) selectively and synergistically induced expression of IL-23 and IL-1ß from cultured monocytes as determined by ELISA assays. Flow cytometric analysis revealed that a sizable sub-population of treated cells acquired DC-like properties including activated surface phenotype with trans-well assays showing enhanced migration towards CCR7 ligands. Such activated cells also preferentially deviated, in an IL-23 and IL-1-dependent manner, CD4(pos) T lymphocyte responses toward the IL-22(hi), IL-17(hi)/IFN-γ(lo) Th17 phenotype in standard in vitro allogeneic sensitization assays. Although pharmacological activation of either ionotropic or cAMP-dependent pathways acted in synergy with LTA to enhance IL-23, only inhibition of the cAMP-dependent pathway antagonized ATP-enhanced cytokine production. ATP plus atypical lipopolysaccharide from P. gingivalis (signaling through TLR2) was slightly superior to E. coli-derived LPS (TLR4 ligand) for inducing the high IL-23-secreting DC-like phenotype, but greatly inferior for inducing IL-12 p70 production when paired with IFN-γ, a distinction reflected in activated DCs' ability to deviate lymphocytes toward Th1. Collectively, our data suggest TLR2 ligands encountered by innate immune cells in an environment with physiologically-relevant levels of extracellular ATP can induce a distinct activation state favoring IL-23- and IL-1ß-dependent Th17 type response.

Effect of multiple activation stimuli on the generation of Th1-polarizing dendritic cells.

It was originally reported that only a small fraction of total matured dendritic cells (DCs) produced interleukin (IL)-12, but it has never been determined whether different combinations of activating signals now shown to maximize secreted IL-12 do so through increasing output by the same IL-12 producers, or by recruiting additional cytokine-secreting cells. We therefore tested all combinations of bacterial lipopolysaccharide (LPS) (TLR4 ligand), R848 (TLR8 ligand), interferon (IFN)-γ, and CD40L for activating human monocyte-derived dendritic cells (DC), and determined by intracellular flow cytometry that enhanced IL-12 secretion was accomplished in large part by markedly increasing the proportion of cells producing IL-12, with the triple and quadruple combinations recruiting the most DC. This optimization requirement for multiple signals was not reflected in differential Toll-like receptor (TLR) expression by the cells. Interestingly, DCs activated with single TLR ligands plus IFN-γ were capable of responding with a second burst of IL-12 upon later CD40L stimulation, whereas DCs activated with R848 plus LPS were not, despite the trend of the latter for superior polarization of naive T cells toward IFN-γ-secreting Th1. These results have implications for the biology of IL-12-secreting DCs and choice of activation regimen for prospective use in DC-based immunotherapy.

Immunotherapy using allogeneic squamous cell tumor-dendritic cell fusion hybrids.

BACKGROUND: Tumor-associated antigens (TAAs) are known to be immunotherapy targets; thus tumor-sharing TAA may be used as a fusion hybrid partner to confer protection against subsequent tumor challenge. METHODS: The squamous cell carcinomas (SCCs), SCCVII and B4B8, were used in C3H/HEN mice: SCCVII (H-2(k)) is syngeneic, B4B8 (H-2(d)) is allogeneic. Experiments using tumor alone included hyperimmunization schedule, subdermal and intranodal routes. Mice were challenged 2 weeks later. Fusion hybrids were created from both SCC tumor cell lines and syngeneic dendritic cells (DCs). These were delivered intranodally for immunization, and mice were challenged with tumor 2 weeks later. RESULTS: Only syngeneic tumor given subdermally was able to protect after tumor challenge 2 weeks later. Hyperimmunization schedule did not alter these findings. However, fusion hybrid immunization from both allogeneic and syngeneic SCCs conferred protection after tumor challenge. CONCLUSIONS: Allogeneic tumor-DC fusion hybrids targeting TAA can protect against subsequent tumor challenge.

Toll-like receptor agonists as third signals for dendritic cell-tumor fusion vaccines.

BACKGROUND: The aim of the present study was to evaluate the therapeutic efficacy of dendritic cell (DC)-tumor fusion hybrids with Toll-like receptor (TLR) agonists. METHODS: DC-tumor fusion hybrids were generated by electrofusion and injected into the inguinal lymph nodes of C57BL/6 mice with 3-day established pulmonary metastases. Paired TLR agonists polyinosine:polycytadilic acid [poly(I:C)] and cytosine-phosphate-guanine (CpG) were then injected intraperitoneally. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate interleukin (IL)-12 production from the DC-tumor fusion hybrids in vitro. RESULTS: Fusion + TLR agonists (60 metastases) had significantly fewer metastases than did the untreated control (262 metastases, p = .0001) and fusion alone (150 metastases, p = .02). ELISA showed that the DC-tumor fusion hybrids yielded 90 pg of IL-12 after TLR stimulation compared with 1610 pg from dendritic cells alone. CONCLUSIONS: CpG and poly(I:C) administered as a third signal with fusion hybrids as described significantly reduce melanoma metastasis compared with fusion hybrids alone. Fusion hybrids do not appear to be a significant source for IL-12 secretion.

IL-12 is required for anti-OX40-mediated CD4 T cell survival.

Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rbeta2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rbeta2 on OX40-stimulated CD4 T cells was tightly regulated and peaked approximately 4-6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rbeta2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.

Effective treatment of spontaneous metastases derived from a poorly immunogenic murine mammary carcinoma by combined dendritic-tumor hybrid vaccination and adoptive transfer of sensitized T cells.

Curative immunotherapy against spontaneous metastases of poorly immunogenic tumors has been difficult to demonstrate, but it is highly relevant to clinical disease conditions. The 4T1 mammary carcinoma shares many characteristics of human mammary cancer. Here, mice with 4T1 spontaneous metastases were treated effectively with a combination of dendritic (DC)-tumor hybrid vaccination and adoptive transfer of tumor-draining lymph node-derived immune T cells. This strategy significantly prolonged survival and cured some mice. In this model, the combined immunotherapy induced a dramatic increase of T cells in the lung where metastases were located and in the spleen where tumor was not present. The mechanism of increasing numbers of T cells is likely attributed to the ability of DC-tumor hybrids to stimulate vigorous proliferation of adoptively transferred T cells rather than to promote their infiltration into tumor-harboring and lymphoid organs. Taken together, the combined approach may be useful for clinical development of cancer immunotherapy.