Hong-Ming Hu

Divergent Roles for CD4+ T Cells in the Priming and Effector/Memory Phases of Adoptive Immunotherapy

The requirement for CD4+ Th cells in the cross-priming of antitumor CTL is well accepted in tumor immunology. Here we report that the requirement for T cell help can be replaced by local production of GM-CSF at the vaccine site. Experiments using mice in which CD4+ T cells were eliminated, either by Ab depletion or by gene knockout of the MHC class II β-chain (MHC II KO), revealed that priming of therapeutic CD8+ effector T cells following vaccination with a GM-CSF-transduced B16BL6-D5 tumor cell line occurred independently of CD4+ T cell help. The adoptive transfer of CD8+ effector T cells, but not CD4+ effector T cells, led to complete regression of pulmonary metastases. Regression of pulmonary metastases did not require either host T cells or NK cells. Transfer of CD8+ effector T cells alone could cure wild-type animals of systemic tumor; the majority of tumor-bearing mice survived long term after treatment (>100 days). In contrast, adoptive transfer of CD8+ T cells to tumor-bearing MHC II KO mice improved survival, but eventually all MHC II KO mice succumbed to metastatic disease. WT mice cured by adoptive transfer of CD8+ T cells were resistant to tumor challenge. Resistance was mediated by CD8+ T cells in mice at 50 days, while both CD4+ and CD8+ T cells were important for protection in mice challenged 150 days following adoptive transfer. Thus, in this tumor model CD4+ Th cells are not required for the priming phase of CD8+ effector T cells; however, they are critical for both the complete elimination of tumor and the maintenance of a long term protective antitumor memory response in vivo.

TNF Plays an Essential Role in Tumor Regression after Adoptive Transfer of Perforin/IFN-γ Double Knockout Effector T Cells

We have recently shown that effector T cells (TE) lacking either perforin or IFN-γ are highly effective mediators of tumor regression. To rule out compensation by either mechanism, TE deficient in both perforin and IFN-γ (perforin knockout (PKO)/IFN-γ knockout (GKO)) were generated. The adoptive transfer of PKO/GKO TE mediated complete tumor regression and cured wild-type animals with established pulmonary metastases of the B16BL6-D5 (D5) melanoma cell line. PKO/GKO TE also mediated tumor regression in D5 tumor-bearing PKO, GKO, or PKO/GKO recipients, although in PKO/GKO recipients efficacy was reduced. PKO/GKO TE exhibited tumor-specific TNF-α production and cytotoxicity in a 24-h assay, which was blocked by the soluble TNFRII-human IgG fusion protein (TNFRII:Fc). Blocking TNF in vivo by administering soluble TNFR II fusion protein (TNFRII:Fc) significantly reduced the therapeutic efficacy of PKO/GKO, but not wild-type TE. This study identifies perforin, IFN-γ, and TNF as a critical triad of effector molecules that characterize therapeutic antitumor T cells. These insights could be used to monitor and potentially tune the immune response to cancer vaccines.

Immunotherapy of Melanoma: A Dichotomy in the Requirement for IFN-γ in Vaccine-Induced Antitumor Immunity Versus Adoptive Immunotherapy

The mechanism by which tumors are rejected following the adoptive transfer of tumor-specific T cells is not well characterized. Recent work has challenged the requirement for cytotoxicity mediated by either the perforin/granzyme or Fas/Fas ligand pathway in T cell-mediated tumor regression. Many reports, including ours, suggest that tumor-specific production of IFN-γ is critical for T cell-mediated tumor regression. However, in most of these studies the evidence to support the role for IFN-γ is only indirect. We have directly examined the requirement for IFN-γ using IFN-γ knockout (GKO) mice. The results show an interesting dichotomy in the requirement for IFN-γ: Antitumor immunity induced by active-specific immunotherapy (vaccination) required IFN-γ, whereas adoptive immunotherapy did not. In GKO mice vaccination with the GM-CSF gene-modified B16BL6-D5 tumor (D5-G6) failed to induce protective immunity against parental D5 tumor. However, adoptive transfer of effector T cells from GKO mice cured 100% of GKO mice with established pulmonary metastases and induced long term antitumor immunity and depigmentation of skin. Furthermore, in vivo neutralization of IFN-γ by mAb treatment or adoptive transfer into IFN-γ receptor knockout mice failed to block the therapeutic efficacy of effector T cells generated from wild-type or perforin knockout mice. Analysis of regressing metastases revealed similar infiltrates of macrophages and granulocytes in both wild-type and GKO mice. These results indicate that in this adoptive immunotherapy model, neither a direct effect on the tumor nor an indirect effect of IFN-γ through activation of myeloid or lymphoid cells is critical for therapeutic efficacy.

Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines

BackgroundThe mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5) melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-γ or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site.MethodsThese studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN), were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-γ or TNF-α was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO) was determined using GRIES reagent.ResultsWe observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1α and MIP-1β following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-γ or TNF-α, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC), MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin-deficient (PKO) effector T cells induced macrophages to secrete nitric oxide (NO), providing an additional effector mechanism for T cell-mediated tumor regression.ConclusionThese data suggest two possible sources for chemokine secretion that stimulates macrophage recruitment to the site of tumor metastases. Both appear to be initiated by T cell recognition of specific antigen, but one is dependent on the tumor cells to produce the chemokines that recruit macrophages.

Tumor-specific T cells signal tumor destruction via the lymphotoxin β receptor

BackgroundPreviously, we reported that adoptively transferred perforin k/o (PKO), and IFN-γ k/o (GKO), or perforin/IFN-γ double k/o (PKO/GKO) effector T cells mediated regression of B16BL6-D5 (D5) pulmonary metastases and showed that TNF receptor signaling played a critical role in mediating tumor regression. In this report we investigated the role of lymphotoxin-α (LT-α) as a potential effector molecules of tumor-specific effector T cells.MethodsEffector T cells were generated from tumor vaccine-draining lymph node (TVDLN) of wt, GKO, LT-α deficient (LKO), or PKO/GKO mice and tested for their ability to mediate regression of D5 pulmonary metastases in the presence or absence of LT-βR-Fc fusion protein or anti-IFN-γ antibody. Chemokine production by D5 tumor cells was determined by ELISA, RT-PCR and Chemotaxis assays.ResultsStimulated effector T cells from wt, GKO, or PKO/GKO mice expressed ligands for LT-β receptor (LT-βR). D5 tumor cells were found to constitutively express the LT-βR. Administration of LT-βR-Fc fusion protein completely abrogated the therapeutic efficacy of GKO or PKO/GKO but not wt effector T cells (p < 0.05). Consistent with this observation, therapeutic efficacy of effector T cells deficient in LT-α, was greatly reduced when IFN-γ production was neutralized. While recombinant LT-α1β2 did not induce apoptosis of D5 tumor cells in vitro, it induced secretion of chemokines by D5 that promoted migration of macrophages.ConclusionThe contribution of LT-α expression by effector T cells to anti-tumor activity in vivo was not discernable when wt effector T cells were studied. However, the contribution of LT-β R signaling was identified for GKO or PKO/GKO effector T cells. Since LT-α does not directly induce killing of D5 tumor cells in vitro, but does stimulate D5 tumor cells to secrete chemokines, these data suggest a model where LT-α expression by tumor-specific effector T cells interacts via cross-linking of the LT-βR on tumor cells to induce secretion of chemokines that are chemotactic for macrophages. While the contribution of macrophages to tumor elimination in our system requires additional study, this model provides a possible explanation for the infiltration of inate effector cells that is seen coincident with tumor regression.

Utilizing quantitative immunohistochemistry for relationship analysis of tumor microenvironment of head and neck cancer patients

Meeting abstractsnnAnalysis of tumor-infiltrating immune cells using quantitative immunohistochemistry (IHC) has proved to be a powerful prognostic biomarker in colon cancer [[1][1], [2][2]]. Similar observations have been made in patients with oral, head and neck squamous cell carcinoma (OHNSCC),

Entwicklung einer anti-Tumor Immuntherapie gegen Magenkarzinom im Tiermodell

Vakzinierung mit DCs welche mit DRibbles aus Proteasominhibitor-behandelten Tumorzellen beladen werden, fuhrt zur Induktion tumorspezifischer therapeutisch wirksamer T-Zellen, welche die Regression subkutan etablierter Tumore bewirkt.

Die Chemokine RANTES, MIP-1α und MIP-1ß spielen eine entscheidende Rolle bei T-Zell vermittelter Tumorregression

Introduction: Understanding the mechanisms involved in T-cell-mediated tumor regression is critical to develop and improve immunotherapy trials. Recently we could show that mice could be cured of established pulmonary metastases of the murine melanoma B16BL6 (D5) by adoptively transferred tumor-specific effector T-cells (TE) from wild-type (wt), perform k/o (PKO), FasL mutant (gld) and IFN-γ k/o (GKO) mice. These observations question the importance of direct cytotoxicity or type-1 cytokines (IFN-γ) in T-cell-mediated tumor regression. Therefore, the aim of our present study was to identify factors, involved in tumor regression after adoptive transfer of tumor-specific TE. Methods: For the induction of tumor-specific TE wt and GKO mice were vaccinated s.c. with 106 D5-G6. D5-G6 is a stable GM-CSF-transduced clone of the murine melanoma D5. T-cells generated from the tumor vaccine draining lymph nodes (TVDLN) were stimulated in vitro with anti-CD3 and expanded in low doses of IL-2. TE were adoptively transferred into wt and GKO mice with established pulmonary D5 metastases. 24- and 48 hours after adoptive transfer the lungs of the treated animals were examined immunohistochemically. The tumor-specific chemokine expression of T-cells and of D5 tumor cells were determined by RT-PCR and ELIS A. Furthermore, the secretion of chemotactic factors by TE was determined by investigating the migration of macrophages (DJ2pm) in transwell plates after stimulation of TE with D5 or control tumor. Results: The lungs of treated wt and GKO animals show an infiltration of macrophages most pronounced around the metastases 24 hours after adoptive transfer of wt and GKO T-cells. TE from wt and GKO mice secrete RANTES tumor specifically (p < 0,05). TE express tumorspecifically the chemokines Rantes, MlP-lα and MIP-lβ. After stimulation of TE with D5 the migration of macrophages was significantly induced. Summary: Adoptive transfer of TE induces the infiltration of macrophages into the lungs of animals with established pulmonary metastases. TE secrete RANTES, MlP-lα and MIP-lβ tumor-specifically, which are chemoattractive for macrophages. Thus, we believe that chemokines play an important and underestimated role in T-cell mediated tumor regression and may function as adapters inbetween the innate and adaptive anti-tumor immune response.

Die Chemokine RANTES, KC und MCP-1 spielen eine mögliche Rolle bei T-Zell vermittelter Tumorregression

Introduction nUnderstanding the mechanisms involved in T-cell mediated tumor regression is critical to develop and improve immunotherapy trials. Recently we reported that adoptively transferred tumor-specific effector T-cells (TE) from wild-type (wt), perforin k/o (PKO), FasL mutant (gld) and IFN-γ k/o (GKO) mice eliminate established pulmonary metastases of the murine melanoma D5. These observations indicate that T-cell mediated tumor regression is possibly not only induced by direct T-cell dependent cytotoxicity or T1-cytokines (IFN-γ). Therefore, the aim of our present study was to identify factors involved in tumor regression after adoptive transfer of tumor-specific TE.

Die T-Zell Polarisierung (TH1/TH2) Tumorvakzin drainierender Lymphozyten korreliert mit der Immunogenität des Tumors

Introduction nPreviously we have shown that vaccination with the poorly immunogenic B16BL6 (D5) melanoma elicits a non protective type 2 immune response in the tumor vaccine draining lymph nodes (TVDLN) of C57BL/6 mice. Here we studied whether the immunogenicity of a tumor correlates with the type of immune response it induces in TVDLN.