Vaccine-draining Lymph Nodes Research Articles

Evaluation of a Preclinical Model of Bone Metastasis for the Study of Adoptive Immunotherapy

The development of new therapeutic strategies for the treatment of bone metastases strongly depends on the availability of valid animal models. In this paper, we evaluate a preclinical model of bone metastases using a technique of tumor cell injection into the left heart ventricle of mice to study the efficacy of adoptive immunotherapy. Using flow cytometric analysis and histopathological and radiological examination, we investigated whether this experimental model of bony metastases using two murine cell lines of melanoma and breast cancer would be suitable for the study of adoptive immunotherapy for these diseases. We further report that anti-CD3-activated and IL-2-expanded tumor vaccine draining lymph node cells cause regression of tumor metastases, including bone metastases, following adoptive transfer to mice bearing 3-day metastases from the D5 melanoma cell line. These promising early results lead us to conclude the following: (1) this model of experimental bone metastases is suitable for the study of immunotherapy, and (2) further studies are warranted to extend these promising early findings of the therapeutic effects of adoptive immunotherapy in this animal model.

Tumour-induced polarization of tumour vaccine-draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumour and correlates with therapeutic efficacy in adoptive transfer studies.

Previously we have shown that vaccination with the poorly immunogenic B16BL6-D5 melanoma (D5) elicits a dominant type 2 (T2) cytokine response that fails to protect the host from a subsequent tumour challenge. Here we investigated whether the inherent immunogenicity of a tumour can be correlated with its ability to bias the anti-tumour cytokine response towards either a type 1 (T1) or a T2 profile. The immune response to six tumours of different inherent immunogenicity was assayed. By isolating l-selectinlow T cells from tumour vaccine draining lymph nodes (TVDLN), it was possible to detect tumour-specific cytokine responses from both immunogenic, poorly immunogenic and non-immunogenic tumours. Immunogenic tumours (MCA-304, MCA-309, MPR-4) induced a predominant tumour-specific T1 cytokine response. In contrast, weakly (MCA-310, MPR-3) and poorly/non-immunogenic tumours (MPR-5, D5) sensitized T cells with a predominant tumour-specific T2 cytokine response. A significant correlation (P < 0.025) between immunogenicity and the ratio of tumour-specific interferon-gamma : interleukin-4 (IL-4) secretion by TVDLN T cells was identified. We then documented that non-therapeutic T cells primed by the poorly immunogenic D5, recognized "tumour-rejection" antigens and that reprogramming their cytokine response, by in vitro culture with IL-12 and anti-IL-4, to a T1 profile uncovered therapeutic efficacy. In contrast, TVDLN T cells primed by a therapeutic vaccine lose therapeutic efficacy when cultured with IL-4. These results provide insights into the development of a protective anti-tumour immune response and strengthen the hypothesis that a T1 cytokine response is critical for T-cell-mediated tumour regression.

CD28, TNF Receptor, and IL-12 Are Critical for CD4-Independent Cross-Priming of Therapeutic Antitumor CD8+ T Cells

Previously, we have shown that priming of therapeutic CD8(+) T cells in tumor vaccine-draining lymph nodes of mice vaccinated with GM-CSF secreting B16BL6 melanoma cells occurs independent of CD4 T cell help. In this study, we examined the contribution of the major costimulatory molecules, CD40 ligand (CD40L), CD80, and CD86, in the priming of CD8(+) T cells. Priming of therapeutic CD8(+) T cells by a GM-CSF-transduced tumor vaccine did not require CD40 and CD40L interactions, as therapeutic T cells could be generated from mice injected with anti-CD40L Ab and from CD40L knockout mice. However, costimulation via either CD80 or CD86 was required, as therapeutic T cells could be generated from mice injected with either anti-CD80 or anti-CD86 Ab alone, but administration of both Abs completely inhibited the priming of therapeutic T cells. Blocking experiments also identified that priming of therapeutic T cells in MHC class II-deficient mice required TNFR and IL-12 signaling, but signaling through CD40, lymphotoxin-betaR, or receptor activator of NF-kappaB was not essential. Thus, cross-priming of therapeutic CD8(+) T cells by a tumor vaccine transduced with GM-CSF requires TNFR, IL-12, and CD28 signaling.

Sentinel node mapping identifies vaccine-draining lymph nodes with tumor-specific immunological activity.

Adoptive immunotherapy (AIT) with 4T07-IL2 vaccine-draining lymph node (DLN) cells induced regression of established 4T07 mammary carcinomas, but contralateral non-DLN were inactive. These experiments were performed to determine whether mapping with isosulfan blue (IB), as described for identification of sentinel nodes, would identify vaccine-DLN with antitumor activity. Ten days after vaccination with 4T07-IL-2, .1 ml of 1% IB was injected into the vaccination site (footpads or flanks). After 3 minutes, mice were euthanized, and the blue-stained nodes were collected. With flank vaccination, IB identified both an inguinal and an axillary node. We also collected DLNs blindly in mice not receiving IB dye. DLN cell suspensions were then activated with bryostatin 1, ionomycin, and IL-2, expanded in culture, and adoptively transferred to mice bearing established 4T07 flank tumors. Complete tumor regression occurred in nearly all mice treated with popliteal or inguinal DLNs collected with or without IB. IB-stained axillary DLNs cured 100% of tumor-bearing mice, whereas none of the mice treated with blindly collected axillary DLNs were cured. We have shown that IB identifies immunologically active DLNs, does not interfere with expansion of lymphocytes in vitro, and, more importantly, has no detrimental effect on the ability of lymphocytes to induce tumor regression in vivo. For axillary DLNs, use of IB mapping identified immunologically active lymph nodes that could not otherwise be found.

T cell polarization in tumor vaccine draining lymph nodes (TVDLN) correlates with the tumor immunogenicity

T cell polarization in tumor vaccine draining lymph nodes (TVDLN) correlates with the tumor immunogenicity

Tumor Regression After Adoptive Transfer of Effector T Cells Is Independent of Perforin or Fas Ligand (APO-1L/CD95L)

The adoptive transfer of tumor-specific effector T cells can result in complete regression and cure mice with systemic melanoma, but the mechanisms responsible for regression are not well characterized. Perforin- and Fas ligand (APO-1/CD95 ligand)-mediated cytotoxicity have been proposed as mechanisms for T cell-mediated tumor destruction. To determine the role of perforin and Fas ligand (FasL) in T cell-mediated tumor regression in a murine melanoma model, B16BL6-D5 (D5), we generated D5-specific effector T cells from tumor vaccine-draining lymph nodes of wild type (wt), perforin knock out (PKO), or FasL mutant (gld) mice and treated established D5 metastases in mice with the same genotype. Effector T cells from wt, PKO and gld mice induced complete regression of pulmonary metastases and significantly prolonged survival of the treated animals regardless of their genotype. Complete tumor regression induced by PKO effector T cells was also observed in a sarcoma model (MCA-310). Furthermore, adoptive transfer of PKO and wt effector T cells provided long-term immunity to D5. Therapeutic T cells from wt, PKO, or gld mice exhibit a tumor-specific type 1 cytokine profile; they secrete IFN-gamma, but not IL-4. In these models, T cell-mediated tumor regression and long-term antitumor immunity are perforin and FasL independent.

Gene-Modified Tumor Vaccine with Therapeutic Potential Shifts Tumor-Specific T Cell Response from a Type 2 to a Type 1 Cytokine Profile

Vaccination with a poorly immunogenic/nonimmunogenic tumor fails to protect the host from a subsequent challenge with the same tumor. The mechanisms underlying the failure of these tumors to sensitize therapeutic T cells are not clearly understood, but the inability of host T cells to recognize tumor has been implicated. In this study, vaccination with the poorly immunogenic B16BL6-D5 (D5 H-2b) tumor fails to generate therapeutic T cells from the tumor vaccine-draining lymph nodes (TVDLN) in our adoptive immunotherapy model. However, if vaccination is performed with an allogeneic MHC class I gene (H-2 Kd)-modified tumor, the T cells obtained from the TVDLN are therapeutic after activation with anti-CD3 and IL-2. Lymph nodes (LN) draining both D5 and D5-Kd tumor vaccines contained increased numbers of cells with reduced expression of L-selectin (L-selectin(low/-)) compared with naive LN. This implies that vaccination led to sensitization of T cells even in LN draining the unmodified D5 tumor. L-selectin(low/-) cells from D5-Kd, but not D5, TVDLN were therapeutic in our animal model. No antitumor activity was seen in the high level L-selectin T cells. L-selectin(low/-) T cells exhibited tumor-specific cytokine release that was type 2 (IL-4, IL-10) following vaccination with native D5 and type 1 (IFN-gamma) following vaccination with gene-modified D5-Kd. Our data suggest that the failure of unmodified D5 to generate therapeutic T cells is not due to an inability to recognize tumor Ags, but, rather, to the induction of an immune response that is ineffective in mediating tumor regression, i.e., immune deviation.

Results of a Phase I Clinical Trial of Adoptive Immunotherapy for High Grade Gliomas Using Ex- Vivo Activated, Vaccine-Draining Lymph Node (LN) T Cells

Results of a Phase I Clinical Trial of Adoptive Immunotherapy for High Grade Gliomas Using Ex- Vivo Activated, Vaccine-Draining Lymph Node (LN) T Cells

Results of a Phase I Clinical Trial of Adoptive Immunotherapy for High Grade Gliomas Using Ex-Vivo Activated, Vaccine-Draining Lymph Node (LN) T Cells

Results of a Phase I Clinical Trial of Adoptive Immunotherapy for High Grade Gliomas Using Ex-Vivo Activated, Vaccine-Draining Lymph Node (LN) T Cells

Proliferation of vaccine draining lymph node lymphocytes (VDLNL) or IVS T Cells (IVS-T) in Culture with Bryostatin and Ionomycin

Proliferation of vaccine draining lymph node lymphocytes (VDLNL) or IVS T Cells (IVS-T) in Culture with Bryostatin and Ionomycin

Sensitizing T-lymphocytes for adoptive immunotherapy by vaccination with wild-type or cytokine gene-transduced melanoma.

For the relatively nonimmunogenic B16-F10 murine melanoma, it has been found that genetically engineered expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin (IL)-2, IL-4, or interferon-gamma (IFN-gamma) resulted in a vaccine that could induce resistance to rechallenge. Because T cells from lymph nodes draining the sites of some progressive tumors can mediate tumor regression after in vitro activation, it seemed possible that even apparently nonimmunogenic melanoma cells might induce similar preeffector cells in the vaccine-draining lymph nodes (DLNs). C57BL/6 mice were vaccinated with B16-F10 cells that were either unmodified or genetically modified to produce IL-2, IL-4, GM-CSF, or IFN-gamma. DLNs were harvested 10 days after vaccination for adoptive immunotherapy (AIT). The DLN cells were activated with bryostatin 1 and ionomycin (B/I), expanded for 10 days in culture, and transferred to mice with 3-day pulmonary metastases. Pulmonary nodules were counted 14 days after AIT. Adoptive transfer of expanded DLN lymphocytes sensitized by inoculation of WT B16-F10, or IL-4, GM-CSF, or IFN-gamma expressing cells significantly reduced pulmonary metastases. Despite the spontaneous regression of IL-2-transduced B16-F10 tumors, DLN from mice inoculated with IL-2 producing B16 cells had little or no antitumor activity. B16-F10 vaccination strategies that apparently do not induce systemic immunity can effectively sensitize DLN preeffector cells.