Potent activity of soluble B7-IgG fusion proteins in therapy of established tumors and as vaccine adjuvant.

Fusion proteins consisting of the extracellular region of murine B7.1 or B7.2 and the Fc portion of murine IgG2a (B7-IgG) were evaluated for their ability to promote antitumor responses. Therapeutic administration of soluble B7-IgG in mice with established tumors induced complete regression of the tumor and increased the survival of mice. In three models, MethA, P815, and MB49, mice with 7-day-old established tumors were cured with two to three treatment cycles of B7-IgG, given twice a week. Even in mice with an established B16/F10 tumor (a poorly immunogenic melanoma), therapeutic treatment with B7-IgG alone slowed tumor growth and increased survival significantly. Still stronger antitumor activity was achieved when B7-IgG was used as a vaccine adjuvant mixed with irradiated tumor cells. In 80% of mice with 7-day-old B16 tumors, tumors regressed completely, and mice survived for at least 80 days. In all tumor models, B7.1-IgG and B7.2-IgG had similar antitumor activity. B7-IgG-mediated tumor rejection was dependent on T cells, specifically CD8 cells, as demonstrated by the failure of B7-IgG to induce tumor regression in severe combined immunodeficient or CD8-depleted mice. In addition, mice that were cured of an established tumor were protected against a rechallenge with the same tumor for at least 4 months, suggesting the generation of memory responses. Surprisingly, the antitumor activity of B7-IgG was independent of IFN-gamma, as demonstrated by tumor rejection in IFN-gamma knockout mice. Our findings demonstrate the potent capacity of B7-IgG to generate or enhance antitumor immune responses and suggest the clinical value of B7-IgG.     

Two mechanisms for tumor evasion of preexisting cytotoxic T-cell responses: lessons from recurrent tumors.

Tumors evade host immunity at both the induction and effector phases Most studies have focused on tumor evasion at the induction phase, and, due in part to poor antitumor CTL responses to most tumors, the mechanism for evasion of CTL effector function is less clear. Here we have taken advantage of the strong CTL responses to a costimulator B7-1-transfected tumor to study the mechanism for tumor evasion of preexisting host immunity. We have investigated six independent recurrent tumors isolated from mice that were challenged with and had rejected B7-1-transfected J558 (J558-B7) tumors. Because the mice had developed strong antitumor CTL responses, these recurrent tumors must have evaded preexisting antitumor CTLs. Indeed, whereas the parental J558-B7 cell line is efficiently lysed by the ex vivo tumor-infiltrating lymphocytes, all of the recurrent tumors are resistant to such lysis. Interestingly, the recurrent tumors can be divided into two groups. The group 1 tumors have vastly reduced levels of cell surface MHC class I with a concurrent reduction in the expression of multiple genes devoted to MHC class I antigen presentation. In contrast, the group 2 tumors have lost the expression of costimulatory molecule B7-1 while retaining cell surface MHC class I and expression of all antigen presentation genes studied. These results demonstrate that tumors can evade preexisting CTLs either by avoiding presentation of the tumor antigen or, surprisingly, by down-regulation of costimulatory molecules. The paradoxical requirements of both antigen and costimulatory molecules at the effector phase raised an interesting question on the nature of antitumor immunity.     

Immunogene therapy against mouse leukemia using B7 molecules

B7 costimulatory molecules play an important role in T-cell activation. It is well known that tumor cells that express B7 molecules can elicit antitumor immunity, but little is known regarding which B7 molecule, B7-1 (CD80) or B7-2 (CD86), can do so more efficiently. To address this issue, we have introduced B7-1 or B7-2 into 8709 cells, a radiation-induced mouse myelocytic leukemic cell line, and have compared their potentials regarding the induction of antitumor immunity. Either B7-1- or B7-2-transduced monoclonal sublines, 8709/B7-1 or 8709/B7-2, respectively, diminished tumorigenicity in syngeneic C3H mice. Some reports have indicated that B7-1 is superior to B7-2 in the induction of antitumor immunity. Contrary to these results, the 8709/B7-2 lines are superior to the 8709/B7-1 lines in their capacity to induce antitumor immunity. In vivo depletion of lymphocyte subsets demonstrated that both CD4+ and CD8+ T cells were indispensable for B7-1- or B7-2-dependent antitumor immunity, whereas natural killer cells were not. These results suggest that in some circumstances, B7-2 molecule is more effective than B7-1 molecule in eliciting antitumor immunity.     

Adoptively transferred tumor-specific T cells stimulated ex vivo using herpes simplex virus amplicons encoding 4-1BBL persist in the host and show antitumor activity in vivo

4-1BB is a T-cell costimulatory receptor which binds its ligand 4-1BBL, resulting in prolonged T cell survival. We studied the antitumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV) amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed HSV amplicons encoding either the 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7.1, or control HSV amplicons and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1/GFP) ex vivo. Naive or ex vivo stimulated OT-1/GFP cells were adoptively transferred into LLC/OVA tumor-bearing mice. Higher percentages of OT-1/GFP cells were seen in the peripheral blood, spleen, and tumor bed of the HSV.4-1BBL-stimulated OT-1/GFP group compared with all other experimental groups. OT-1 cells identified within the tumor bed and draining lymph nodes of the HSV.4-1BBL-stimulated OT-1 group showed enhanced bromodeoxyuridine (BrdUrd) incorporation, suggesting ongoing expansion in vivo. Mice receiving HSV.4-1BBL-stimulated OT-1/GFP had significantly decreased tumor volumes compared with untreated mice (P<0.001) or to mice receiving naive OT-1/GFP (P<0.001). Transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Mice that received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against LLC/OVA and higher percentages of Ly-6C+ OT-1/GFP in the spleen and tumor bed compared with controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and the generation of tumor-specific CD44+Ly-6C+CD62L- effector memory T cells.     

Protein transfer of glycosyl-phosphatidylinositol-B7-1 into tumor cell membranes: a novel approach to tumor immunotherapy.

Modification of tumor cells with one or more costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy. Expression of B7-1 (CD80) in tumors by gene transfer creates an immunogenic tumor cell that induces antitumor immunity and protects mice from further challenge with wild-type tumor cells. In this report, we demonstrate that protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored costimulatory molecules into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. Incubation of isolated tumor membranes with purified GPI-anchored B7-1 results in stable incorporation of B7-1 on tumor cell membranes within a few hours. Immunization of C57BL/6 mice with EG7 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition, immunization with these EG7 membranes protects mice from parental tumor challenge. The protein transfer approach used here does not require foreign vectors or live tumor cells and is completed within a matter of hours. Irradiated cells or membrane preparations from fresh or frozen tumor tissue can be used. Therefore, protein transfer of glycolipid-anchored molecules provides an efficient and novel approach to modify tumor membranes for human immunotherapy. This approach is not limited to costimulatory molecules because any cell surface protein can be converted to a GPI-anchored form by recombinant techniques.     

Tumor regression induced by intratumor administration of adenovirus vector expressing CD40 ligand and naive dendritic cells

We have previously shown that in vivo genetic modification of tumors with an adenovirus (Ad) vector engineered to express CD40 ligand (AdmCD40L) induces tumor-specific CTLs and suppresses tumor growth in vivo. In the present study, we investigate the hypothesis that this treatment can be made more efficient with 10(2)-fold less Ad vector by also administering bone marrow-generated dendritic cells (DCs) to the tumor. Using AdmCD40L and the number of DCs that alone had no effect on tumor growth, the data show that the growth of CT26 (colon adenocarcinoma; H-2d) and B16 (melanoma; H-2b) murine s.c. tumors is significantly suppressed by direct administration of DCs into s.c. established tumors that had been pretreated with AdmCD40L 2 days previously. The antitumor effect produced by the combination therapy AdmCD40L + DCs correlated with in vivo priming of tumor-specific CTLs. The antitumor cell-mediated immunity was transferable to naive mice by spleen cells from AdmCD40L + DC-treated animals. The interactions between CD40L and CD40 within treated tumors were critical because tumor suppression was abrogated by coadministration to the tumors of neutralizing monoclonal antibody against CD40L along with the DCs. Finally, in vivo depletion and knockout mice experiments demonstrated that tumor regression produced by this combination therapy depends on CD8+ T cells, but not on CD4+ T cells. These findings should be useful in designing strategies for use of DCs and AdmCD40L in cancer immunotherapy.     

B7-2 expression above a threshold elicits anti-tumor immunity as effective as interleukin-12 and prolongs survival in murine B-cell lymphoma

The costimulatory molecule, B7-2, is expressed by various lymphomas, but this level of expression is not sufficient to generate effective anti-tumor immunity in vivo. To determine whether up-regulated expression of the costimulatory molecule, B7-2, leads to more effective anti-tumor immunity in vivo, the A20 murine model of B-cell lymphoma was used. A20 tumor cells express major histocompatibility complex (MHC) I and II molecules and moderate constitutive levels of B7-2. While B7-1 and B7-2 have been introduced into tumor cells lacking these molecules, studies have not been conducted to determine whether tumors that constitutively express B7-1 or B7-2 can be made more immunogenic by increasing the expression of these molecules. In this report, A20/B7-2 transfectants expressing greater levels of B7-2 were rejected in syngeneic mice, and systemic immunity against the A20 parental cells was generated. Treatment with the A20/B7-2 variant cells significantly improved the survival of tumor-bearing mice. Coinjection with IL-12 secreting variants did not further augment the anti-tumor immunity observed for B7-2 therapy alone. Both CD8(+) T cells and natural killer (NK) cells mediated the anti-tumor immune response observed in A20/B7-2 immunized mice. In mice that developed tumors after immunization with the A20/B7-2 variant cells, resected tumor cells were shown to express lower levels of B7-2 than the transfected variants. These results suggest that the level of costimulation is important for the generation of anti-tumor immunity and for host survival. In addition, tumors appear to be able to evade the immune response by downregulating the expression of B7-2 below a threshold level.     

Cryptotanshinone Induces Inhibition of Breast Tumor Growth by Cytotoxic CD4+ T Cells through the JAK2/STAT4/ Perforin Pathway

Cryptotanshinone (CPT), is a quinoid diterpene isolated from the root of the Asian medicinal plant, Salviamiotiorrhiza bunge. Numerous researchers have found that it could work as a potent antitumor agent to inhibittumor growth in vitro, buith there has been much less emphasis on its in vivo role against breast tumors. Usinga mouse tumor model of MCF7 cells, we showed that CPT strongly inhibited MCF7 cell growth in vivo withpolarization of immune reactions toward Th1-type responses, stimulation of naive CD4+ T cell proliferation,and also increased IFN-γ and perforin production of CD4+ T cells in response to tumor-activated splenocytes.Furthermore, data revealed that the cytotoxic activity of CD4+ T cells induced by CPT was markedly abrogatedby concanamycin A(CMA), a perforin inhibitor, but not IFN-γ Ab. On the other hand, after depletion of CD4+T cells or blocked perforin with CMA in a tumor-bearing model, CPT could not effectively suppress tumorgrowth, but this phenomenon could be reversed by injecting naive CD4+ T cells. Thus, our results suggestedthat CPT mainly inhibited breast tumor growth through inducing cytotoxic CD4+ T cells to secrete perforin.We further found that CPT enhanced perforin production of CD4+ T cells by up-regulating JAK2 and STAT4phosphorylation. These findings suggest a novel potential therapeutic role for CPT in tumor therapy, anddemonstrate that CPT performs its antitumor functions through cytotoxic CD4+ T cells.     

Antitumor immunity after vaccination with B lymphoma cells overexpressing a triad of costimulatory molecules

BACKGROUND: The costimulatory molecules B7-1, intercellular adhesion molecule-1 (ICAM-1), and leukocyte function-associated antigen-3 (LFA-3) play pivotal roles in the activation of T cells. We investigated whether in vivo vaccination with lymphoma cells infected with a recombinant, nonreplicating fowlpox (FP) virus encoding this triad of costimulatory molecules (TRICOM) could stimulate lymphoma-specific immunity. METHODS: TRICOM-infected A20 B lymphoma cells were analyzed for expression of B7-1, ICAM-1, and LFA-3. Mice (10 per group) were vaccinated with irradiated A20 cells infected with either the TRICOM vector or the wild-type FP virus (WT-FP), challenged with live A20 tumor cells, and followed for survival. Mice with established A20 tumors were also treated with irradiated TRICOM-infected A20 cells. Survival curves were compared with the log-rank statistic. The mechanism of the antitumor effect was studied by in vivo depletion of CD4(+) and CD8(+) T cells and in vitro cytotoxicity assays. All statistical tests were two-sided. RESULTS: A20 tumor cells infected with TRICOM expressed high levels of B7-1, ICAM-1, and LFA-3. Mice vaccinated with irradiated TRICOM-infected A20 cells had prolonged survival relative to mice vaccinated with WT-FP-infected cells (80% versus 20% survival at 110 days; P<.001). In mice with established tumors, tumor growth was slower in those treated with TRICOM-infected tumor cells than in those treated with WT-FP-infected cells, and this treatment provided a survival advantage (P<.001). Depletion of CD4(+) or CD8(+) T cells reduced the antitumor immunity provided by the tumor cell-TRICOM vaccine, and lymphocytes from vaccinated mice displayed in vitro cytotoxic activity toward A20 cells. CONCLUSIONS: Increasing expression of costimulatory molecules on B lymphoma cells by infection with a recombinant FP virus encoding B7-1, ICAM-1, and LFA-3 stimulates antitumor immune responses in vivo and may provide a novel strategy for treating patients with B-cell malignancies.     

ANTITUMOR EFFECTS INDUCED BY B7-1 GENE MODIFIED EL-4 LYMPHOMA COOPERATED WITH IL-2 IN VIVO AND IN VITRO

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Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies

It is becoming increasingly evident that radiotherapy may benefit from coincident or subsequent immunotherapy. In this study, we examined whether the antitumor effects of radiotherapy, in established triple-negative breast tumors could be enhanced with combinations of clinically relevant monoclonal antibodies (mAb), designed to stimulate immunity [anti-(α)-CD137, α-CD40] or relieve immunosuppression [α-programmed death (PD)-1]. While the concomitant targeting of the costimulatory molecules CD137 and CD40 enhanced the antitumor effects of radiotherapy and promoted the rejection of subcutaneous BALB/c-derived 4T1.2 tumors, this novel combination was noncurative in mice bearing established C57BL/6-derived AT-3 tumors. We identified PD-1 signaling within the AT-3 tumors as a critical limiting factor to the therapeutic efficacy of α-CD137 therapy, alone and in combination with radiotherapy. Strikingly, all mice bearing established orthotopic AT-3 mammary tumors were cured when α-CD137 and α-PD-1 mAbs were combined with single- or low-dose fractionated radiotherapy. CD8+ T cells were essential for curative responses to this combinatorial regime. Interestingly, CD137 expression on tumor-associated CD8+ T cells was largely restricted to a subset that highly expressed PD-1. These CD137+PD-1High CD8+ T cells, persisted in irradiated AT-3 tumors, expressed Tim-3, granzyme B and Ki67 and produced IFN-γ ex vivo in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Notably, radiotherapy did not deplete, but enriched tumors of functionally active, tumor-specific effector cells. Collectively, these data show that concomitant targeting of immunostimulatory and inhibitory checkpoints with immunomodulatory mAbs can enhance the curative capacity of radiotherapy in established breast malignancy.     

Inhibitory effects of B cells on antitumor immunity

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments, spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors), IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells.     

Adenoviral B7-H3 therapy induces tumor specific immune responses and reduces secondary metastasis in a murine model of colon cancer

Current cancer gene therapies aim at the induction of systemic antitumor immune responses. Tumors may deliver antigens to T-cells, but may lack the costimulatory signals necessary for mounting an effective response. The purpose of this study was to evaluate the efficacy of an adenoviral delivery of the B7-H3 costimulatory molecule in mice to induce antitumor immune responses. Colon cancers were established by orthotopic injection of syngeneic colon cancer cells into the cecum on Balb/c mice. After two weeks, these mice were treated by intratumoral injection of an adenovirus expressing mouse B7-H3 (Ad-B7-H3-GFP) or a control virus (Ad-GFP). Ad-B7-H3-GFP treatment resulted in a reduction of tumor size compared to the controls. In addition, the occurrence of secondary metastasis was significantly reduced in B7-H3 treated mice compared to control animals (lymph node 7/10 vs. 10/10; liver 2/10 vs. 8/10, p相似文献   

In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity

In vivo delivery of immunomodulatory genes is a promising strategy for solid tumor vaccination. A drawback is that it necessitates induction of a large effect from transgene expression in a small percentage of tumor cells. Although the B7 family is known to be the most potent of the costimulatory molecules, gene transduction of B7 alone has not been effective in inducing antitumor immunity in nonimmunogenic tumors by ex vivo methods, much less in vivo. We have developed a novel approach where a gene encoding soluble B7-1, a fusion protein of the extracellular domain of murine B7-1 and the Fc portion of human IgG1, is delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus, and the gene product is secreted by tumor cells rather than expressed on the cell surface. Defective herpes simplex virus vectors containing the B7-1-immunoglobulin (B7-1-Ig) fusion transgene (dvB7Ig) were generated using G207 as a helper virus and tested in the poorly immunogenic murine neuroblastoma, Neuro2a, in syngeneic A/J mice. Intraneoplastic inoculation of dvB7Ig/G207 at a low titer successfully inhibited the growth of established s.c. tumors, despite the expression of B7-1-Ig being detected in only 1% or fewer of tumor cells at the inoculation site, and prolonged the survival of mice bearing intracerebral tumors. Immunohistochemistry of dvB7Ig/G207-inoculated tumors revealed a significant increase in CD4+ and CD8+ T-cell infiltration compared with control tumors inoculated with defective vector expressing alkaline phosphatase (dvAP/G207). The antitumor effect of dvB7Ig/G207 was not manifested in athymic mice. In vivo depletion of immune cell subsets in A/J mice further revealed that CD8+ T cells, but not CD4+ T cells, were required. Animals cured of their tumors by dvB7Ig/G207 treatment were protected against rechallenge with a lethal dose of Neuro2a cells but not SaI/N cells. The results demonstrate that the use of soluble B7-1 for immune gene therapy is a potent and clinically applicable means of in situ cancer vaccination.     

Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand

Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and flt3-ligand (FL) induce the development of dendritic cells (DCs). To compare the functional properties of DCs stimulated by these cytokines in vivo, we used retroviral-mediated gene transfer to generate murine tumor cells secreting high levels of each molecule. Injection of tumor cells expressing either GM-CSF or FL resulted in the dramatic increase of CD11c+ cells in the spleen and tumor infiltrate. However, vaccination with irradiated, GM-CSF-secreting tumor cells stimulated more potent antitumor immunity than vaccination with irradiated, FL-secreting tumor cells. The superior antitumor immunity elicited by GM-CSF involved a broad T cell cytokine response, in contrast to the limited Thl response elicited by FL. DCs generated by GM-CSF were CD8alpha- and expressed higher levels of B7-1 and CD1d than DCs cells generated by FL. Injection sites of metastatic melanoma patients vaccinated with irradiated, autologous tumor cells engineered to secrete GM-CSF demonstrated similar, dense infiltrates of DCs expressing high levels of B7-1. These findings reveal critical differences in the abilities of GM-CSF and FL to enhance the function of DCs in vivo and have important implications for the crafting of tumor vaccines.     

The expression of CD70 and CD80 by gene-modified tumor cells induces an antitumor response depending on the MHC status

The expression of costimulatory molecules such as CD70 or CD80 by gene-modified tumor cells has been shown to enhance the antitumor immune response based mainly on T lymphocytes. However, most human tumors show defects of major histocompatibility complex (MHC) expression, preventing them from being recognized by MHC-restricted T cells. To investigate if coexpression of CD70 and CD80 costimulatory molecules induces comparable antitumor responses in low and high MHC-expressing tumor cells, we used two low immunogenic murine tumor models, the B16.F10 melanoma and the TS/A mammary adenocarcinoma cell lines expressing, respectively, low and high levels of MHC class I molecules. Transfection of both CD70 and CD80 genes resulted in an increased capacity of gene-modified tumor cells to costimulate in vitro the proliferation and cytokine production of optimally activated lymphoid cells. Coexpression of CD70 and CD80 by the two tumor cell lines, TS/A and B16.F10, resulted in both cases in partial regression of subcutaneous tumors. Immunochemical analysis and studies in nude mice showed that, even in the B16.F10 model, T cells had a significant role in the antitumor response induced by combining CD70 and CD80. However, rejection of the CD70/CD80-transfected tumor cells appeared more effective in the MHC class I high TS/A model, leading to a protection against parental tumor cells. B16.F10 and TS/A transfectants were then tested with fibroblasts genetically modified to secrete interleukin-12 (IL-12) as a therapeutic vaccine in mice bearing parental tumors. In the two models tested, the injections of irradiated IL-12 and CD70/CD80 gene-modified cells generated an antitumor response to established tumors leading to the slowing down of the tumor growth rate. Although the mechanisms remain to be defined, these findings suggest that the combination of several immuno-modulatory molecules could provide additional strategies for cancer immuno-gene therapy, even for MHC expression-deficient tumors.     

Coexpression of CD40L and CD70 by semiallogenic tumor cells induces anti-tumor immunity

The immune system is potentially qualified to detect and eliminate tumor cells, but various mechanisms developed by tumor cells allow tumor escape. Strategies selected to promote antitumor responses have included genetic modifications of tumor cells to induce expression of costimulatory molecules. Moreover, alloantigens can also act as strong enhancers of the immune response. In this work, we have associated the expression of two costimulatory members of the TNF superfamily, CD40L and CD70 along with an allogenic MHC Class I (H-2K(d)) molecule expression on melanoma cells (B16F10, H-2(b)) to favor the antitumor immune response. B16F10 tumor growth slows significantly when CD40L and CD70 are coexpressed by tumor cells and the association with the allogenic molecule (H-2K(d)) enhances this effect. Growth kinetics of mock and CD40L-CD70-H-2K(d)-expressing B16F10 tumors in immunocompetent versus nu/nu and beige mice suggested that CD8(+) T lymphocytes and NK cells were involved in this antitumor immunity. A delay in mock tumor growth was observed when CD40L-CD70-H-2K(d)-expressing B16F10 cells and mock tumor cells were injected simultaneously and contralaterally. It was also shown that in vivo immunization of immunocompetent mice with CD40L-CD70-H-2K(d) B16F10 tumor cells improved the generation of cytotoxic lymphocytes against the wild-type melanoma cells expressing the syngenic MHC Class I molecule H-2K(b) (B16K1). These observations lay a path for new immunotherapeutic trials using semiallogenic fibroblasts expressing costimulatory molecules and tumor-associated antigens.     

Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo

We have investigated the role of the host's CD1d-dependent innate antitumor immune response in a murine T-cell lymphoma model in vivo. We found that C57BL/6 wildtype (WT) mice inoculated with RMA/S cells transfected with murine CD1d1 died at the same rate as mice inoculated with vector-transfected cells. In contrast, natural killer T (NKT) cell-deficient CD1d or Jalpha18 knockout mice inoculated with CD1d-transfected RMA/S cells survived significantly longer than mice inoculated with vector-transfected RMA/S cells, implicating the involvement of invariant NKT (iNKT) cells in inhibiting antitumor activity in vivo. In contrast to the mutant mice, which produced more of the proinflammatory cytokines IFN-gamma and GM-CSF, WT mice produced significantly elevated amounts of IL-13. Antitumor activity in the knockout mice was not due to the development of CD1d-specific cytotoxic T lymphocytes or circulating antibodies. However, iNKT cell numbers were elevated in tumor-bearing mice. Thus, iNKT cells may be playing a negative role in the host's antitumor immune response against T-cell lymphomas in a CD1d-dependent manner.     

CD8+ T cells in cutaneous T-cell lymphoma: expression of cytotoxic proteins, Fas Ligand, and killing inhibitory receptors and their relationship with clinical behavior.

PURPOSE: We investigated the number, phenotype, and prognostic significance of CD8+ T cells in patients with mycosis fungoides (MF) and CD30- primary cutaneous large T-cell lymphoma (PCLTCL). PATIENTS AND METHODS: Immunohistochemical stainings for CD8, granzyme B (GrB), T cell-restricted intracellular antigen (TIA-1), Fas ligand (FasL), and killer-cell inhibitory receptors (KIRs; CD95, CD158a, and CD158b) were performed on 83 first-diagnostic biopsy samples obtained from patients with plaque-stage MF (n = 42), tumor-stage MF (n = 20), and CD30- PCLTCL (n = 21). RESULTS: Serial sections and double-staining experiments showed that the large majority of CD8+ T cells in MF and CD30- PCLTCL expressed TIA-1 and FasL, whereas only a minority expressed GrB, which suggested that these CD8+ T cells were partly activated cytotoxic T lymphocytes (CTLs). These CD8+ CTLs never or rarely expressed KIRs. This phenotype was a constant feature of CD8+ CTLs and did not alter with disease progression. In contrast, the median percentage of CD8+ CTLs in plaque-stage MF (22%), tumor-stage MF (7%), and CD30- PCLTCL (3%) differed significantly (P < .0001) and was associated with a significant decrease in 5-year survival. Also within the group of tumor-stage MF, a significant relation between CD8+ CTLs and survival was found. Multivariate analysis in the total group of MF demonstrated that both skin stage and percentage of CD8+ CTLs were independent parameters of survival. CONCLUSION: Our results demonstrated that partly activated CD8+ CTLs were present in CTCL and that high proportions of these cells correlated with a better prognosis. This suggested that these CD8+ CTLs could play an important role in the antitumor response in these conditions.     

In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (4-1BB)

Human T cells engineered to express a chimeric antigen receptor (CAR) specific for folate receptor-α (FRα) have shown robust antitumor activity against epithelial cancers in vitro but not in the clinic because of their inability to persist and home to tumor in vivo. In this study, CARs were constructed containing a FRα-specific scFv (MOv19) coupled to the T-cell receptor CD3ζ chain signaling module alone (MOv19-ζ) or in combination with the CD137 (4-1BB) costimulatory motif in tandem (MOv19-BBζ). Primary human T cells transduced to express conventional MOv19-ζ or costimulated MOv19-BBζ CARs secreted various proinflammatory cytokines, and exerted cytotoxic function when cocultured with FRα(+) tumor cells in vitro. However, only transfer of human T cells expressing the costimulated MOv19-BBζ CAR mediated tumor regression in immunodeficient mice bearing large, established FRα(+) human cancer. MOv19-BBζ CAR T-cell infusion mediated tumor regression in models of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly, tumor response was associated with the selective survival and tumor localization of human T cells in vivo and was only observed in mice receiving costimulated MOv19-BBζ CAR T cells. T-cell persistence and antitumor activity were primarily antigen-driven; however, antigen-independent CD137 signaling by CAR improved T-cell persistence but not antitumor activity in vivo. Our results show that anti-FRα CAR outfitted with CD137 costimulatory signaling in tandem overcome issues of T-cell persistence and tumor localization that limit the conventional FRα T-cell targeting strategy to provide potent antitumor activity in vivo.