The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells.

Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8+ T cells. We found that 2'3'-cGAMP and 3'3'-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8+ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2'3'-cGAMP-adjuvanted vaccination elicited protective antitumor or antiviral CD8+ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

No need for constant help: human IgG2 antibodies have an autonomous agonistic activity for immunotherapy of cancer.

Agonistic antibodies specific for members of the tumor necrosis factor receptor protein family hold great promise for immunotherapy of cancer. In this issue of Cancer Cell, White and colleagues provide evidence that the human IgG2 subclass may represent a superior backbone for the use of these antibodies in human therapy.

Conformation of the human immunoglobulin G2 hinge imparts superagonistic properties to immunostimulatory anticancer antibodies.

Monoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment.

Does the mode of surgical resection affect the prognosis/recurrence in patients with thymoma?

BACKGROUND: Among the various controversies in the treatment strategies for patients with thymoma, the optimal mode of resection needs to be defined. To explore whether or not the mode of resection affects the prognosis/recurrence in patients with thymoma, we evaluated the treatment outcome of patients with resected thymoma. METHODS: One hundred seventy-three nonmyasthenic patients with stage I or II resected thymoma were studied. Patients were divided into two groups: a thymomectomy (resection of thymoma without total thymectomy) group (n = 100) and a thymothymomectomy (resection of thymoma with total thymectomy) group (n = 73). The differences in the clinicopathological characteristics and prognosis between the two groups were examined. RESULTS: Myasthenia gravis developed postoperatively in three patients (3%) in the thymomectomy group and in 6 (8%) in the thymothymomectomy group. The 5- and 10-year overall survival rates in the thymomectomy group were 96.7% and 92.2%, and those in the thymothymomectomy group were 94.0% and 86.2%, respectively (P = 0.755). Two patients (2%) in the thymomectomy group and 4 (5%) in the thymothymomectomy group experienced recurrence. CONCLUSIONS: There was no difference in prognosis/recurrence between the two groups. Thymothymomectomy might not always be necessary for nonmyasthenic patients with stage I or II thymoma.

Vaccines delivered by integration-deficient lentiviral vectors targeting dendritic cells induces strong antigen-specific immunity.

We report a study of an integration-deficient lentiviral vector (IDLV) enveloped with a Sindbis virus glycoprotein mutant (SVGmu) capable of selectively binding to dendritic cells (DCs) for its potential as a vaccine carrier. The in vitro assays showed that the D64V point mutation in the catalytic domain of HIV-1 integrase efficiently inhibited the integration of the transgene upon vector transduction, while the targeting specificity of the vector to preferentially transduce and mediate durable expression in DCs was maintained. Substantial immune responses in C57BL/6 mice and complete protection against a challenge with the C57BL/6 thymoma EG.7 tumor expressing a delivered ovalbumin (OVA) antigen in mice have been achieved through the direct injection of the DC-directed IDLV encoding OVA. Thus, this DC-directed IDLV system represents a promising and efficient vector platform with remarkably improved safety for the future development of DC-based immunotherapy.

Peripheral 4-1BB signaling negatively regulates NK cell development through IFN-gamma.

Stimulation of 4-1BB (CD137) was shown to produce strong anticancer effects in vivo. In contrast, 4-1BB-deficient (4-1BB(-/-)) B6 mice are remarkably resistant to tumor growth. We set out to determine the mechanisms involved in these seemingly contradictory observations. We found that the therapeutic effects of 4-1BB triggering were mainly dependent on CD8(+) T cells and partially on NK cells, whereas CD8(+) T and NK cells were equally needed to suppress tumor growth in 4-1BB(-/-) mice. Cellular analysis showed that the frequency and number of NK cells in the spleen and bone marrow were decreased by 4-1BB triggering but were increased in the absence of 4-1BB signaling in tumor-challenged mice. The 4-1BB-mediated downregulation of NK cell development was primarily dependent on IFN-gamma, which was produced by peripheral CD8(+) T and NK cells. The suppression of NK cell development by 4-1BB-mediated IFN-gamma production occurred in the bone marrow. As 4-1BB signaling increased in the periphery, more CD8(+) T cells but fewer NK cells contributed to the antitumor immunity. As 4-1BB signaling decreased, more NK cells participated in the antitumor immunity. We conclude that 4-1BB signaling results in a shift of the dominant type of immune cell in antitumor immunity from the innate NK cell to the adaptive CD8(+) T cell and that the level of IFN-gamma is critical for this 4-1BB-mediated shift.

Long-lasting antitumor protection by anti-CD20 antibody through cellular immune response.

The anti-CD20 monoclonal antibody (mAb) rituximab has been used successfully for lymphoma therapy for more than 10 years. Although several direct mechanisms by which anti-CD20 mAbs act have been characterized in vitro, their specific role in clinical efficacy is still debated. Little is known about the possible antitumor immune response that they may induce in patients, despite clinical data suggesting a "vaccinal" effect. We show here that an initial treatment with anti-CD20 induces protection against human CD20-expressing tumor cells and allows immunocompetent mice to survive tumor challenge. This long-lasting protection requires the presence of the Fc portion of the anti-CD20 mAb and is achieved through the induction of a cellular immune response. Only CD4(+) cells were needed at the beginning of the treatment, but both CD4(+) and CD8(+) cells were required after tumor challenge to achieve protection. Finally, we show that interleukin-2 treatment, given after tumor challenge, improves the overall survival rate, compared with that obtained by anti-CD20 treatment alone. These findings demonstrate that anti-CD20 mAbs exert therapeutic effects through the induction of an adaptive cellular immune response, aside from any direct mechanisms involving effectors from innate immunity.

Notch2 signaling is required for potent antitumor immunity in vivo.

CD8(+) T cells play a central role in cancer immunosurveillance, and the efficient induction of CTLs against tumor Ags is required for successful immunotherapy for cancer patients. Notch signaling directly regulates the transcription of effector molecules in CTLs. However, it remains unclear whether Notch signaling in CD8(+) T cells is required for antitumor CTL responses and whether modulation of Notch signaling can augment antitumor CTL responses. In this study, we demonstrate that signaling by Notch2 but not Notch1 in CD8(+) T cells is required for antitumor CTL responses. Notch2(flox/flox) mice crossed with E8I-cre transgenic (N2F/F-E8I) mice, in which the Notch2 gene is absent only in CD8(+) T cells, die earlier than control mice after inoculation with OVA-expressing EG7 thymoma cells. In contrast, Notch1(flox/flox) mice crossed with E8I-cre transgenic mice inoculated with EG7 cells die comparable to control mice, indicating that Notch2 is crucial for exerting antitumor CTL responses. Injection of anti-Notch2 agonistic Ab or delta-like 1-overexpressing dendritic cells augmented the antitumor response in C57BL/6 mice inoculated with EG7 cells. These findings indicate that Notch2 signaling in CD8(+) T cells is required for generating potent antitumor CTLs, thus providing a crucial target for augmenting tumor immune responses.

Induction of protective cytotoxic T-cell responses by a B-cell-based cellular vaccine requires stable expression of antigen.

B-cell-based cellular vaccines represent a promising approach to active immunotherapy of cancer complementing the use of dendritic cells, especially in pediatric patients and patients with low bone marrow reserves. B cells can be easily prepared in large numbers and readily home to secondary lymphoid organs, the primary site of induction of cytotoxic T lymphocyte (CTL) responses. However, most B-cell-based vaccines tested so far failed to induce functional and protective CTLs in in vivo models. Here, we show that B-cells activated through the toll-like receptor-9 (TLR-9) and CD40 up-regulate surface expression of major histocompatibility complex and costimulatory molecules, produce IL-12, and exhibit potent antigen-presenting properties in vitro. Importantly, although administration of peptide-coated or transiently transfected B cells fails to induce immune responses, therapeutic immunization with low numbers of genetically modified B cells stably expressing antigen results in an induction of functional CTLs and protection against the growth of tumor in an animal model. After activation, B cells partially loose their ability to home to organized lymphoid tissue because of the shedding of CD62L; however, this property can be restored by expression of protease-resistant mutant of CD62L. In summary, the data presented in this report suggest that genetically modified activated B cells represent a promising candidate for a cancer vaccine eliciting functional systemic CTLs.

Ly49D engagement on T lymphocytes induces TCR-independent activation and CD8 effector functions that control tumor growth.

Recent data showing expression of activating NK receptors (NKR) by conventional T lymphocytes raise the question of their role in the triggering of TCR-independent responses that could be damaging for the host. Transgenic mice expressing the activating receptor Ly49D/DAP12 offer the opportunity to better understand the relevance of ITAM signaling in the biology of T cells. In vitro experiments showed that Ly49D engagement on T lymphocytes by a cognate MHC class I ligand expressed by Chinese hamster ovary (CHO) cells or by specific Ab triggered cellular activation of both CD4 and CD8 populations with modulation of activation markers and cytokine production. The forced expression of the ITAM signaling chain DAP12 is mandatory for Ly49D-transgenic T cell activation. In addition, Ly49D stimulation induced T lymphocyte proliferation, which was much stronger for CD8 T cells. Phenotypic analysis of anti-Ly49D-stimulated CD8 T cells and their ability to produce high levels of IFN-gamma and to kill target cells indicate that Ly49D ligation generates effector cytotoxic CD8 T cells. Ly49D engagement by itself also triggered cytotoxic activity of activated CD8 T cells. Adoptive transfer experiments confirmed that Ly49D-transgenic CD8 T cells are able to control growth of CHO tumor cells or RMA cells transfected with Hm1-C4, the Ly49D ligand normally expressed by CHO. In conclusion, Ly49D engagement on T cells leads to T cell activation and to a full range of TCR-independent effector functions of CD8 T cells.

Neutrophil-dependent tumor rejection and priming of tumoricidal CD8+ T cell response induced by dendritic cells overexpressing CD95L.

Overexpression of CD95 (Fas/Apo-1) ligand (CD95L) has been shown to induce T cell tolerance but also, neutrophilic inflammation and rejection of allogeneic tissue. We explored the capacity of dendritic cells (DCs) genetically engineered to overexpress CD95L to induce an antitumor response. We first found that DCs overexpressing CD95L, in addition to MHC class I-restricted OVA peptides (CD95L-OVA-DCs), induced increased antigen-specific CD8(+) T cell responses as compared with DCs overexpressing OVA peptides alone. The enhanced T cell responses were associated with improved regression of a tumor expressing OVA, allowing survival of all animals. When DCs overexpressing CD95L (CD95L-DCs) were injected with the tumor expressing OVA, in vivo tumor proliferation was strikingly inhibited. A strong cellular apoptosis and a massive neutrophilic infiltrate developed in this setting. Neutrophil depletion prevented tumor regression as well as enhanced IFN-gamma production induced by CD95L-OVA-DCs. Furthermore, the CD8(+) T cell response induced by the coadministration of tumor cells and CD95L-DCs led to rejection of a tumor implanted at a distance from the DC injection site. In summary, DCs expressing CD95L promote tumor rejection involving neutrophil-mediated innate immunity and CD8(+) T cell-dependent adaptative immune responses.

Oligomannose-coated liposomes as a therapeutic antigen-delivery and an adjuvant vehicle for induction of in vivo tumor immunity.

In the present study, the adjuvant capacity of oligomannose-coated liposomes (OMLs) was evaluated in mice, and an OML-based vaccine was shown to induce effective anti-tumor immunity. C57BL/6 mice were immunized subcutaneously with OML-encased ovalbumin (OVA) and challenged with OVA-expressing E.G7-OVA tumor cells. All mice that received OVA in OMLs completely rejected the E.G7-OVA tumor. Spleen cells from the immunized mice showed strong cytotoxic activity against E.G7-OVA cells, but not against the parental EL4 cells. The therapeutic efficacy of OML-encased OVA against established E.G7-OVA tumors was then investigated. When the tumor mass became palpable (8-10 mm in length), the mice were treated with a single injection of 1 microg of OML-encased OVA. Tumor growth was reduced significantly in mice treated with OML-encased OVA and tumors were completely eliminated in about 40% of these mice. Similar results were obtained using EL4 tumors, with the EL4 cell lysate used as an antigen. These results indicate that an OML-based vaccine with an encased tumor antigen might be useful clinically to raise an effective immune response against a tumor.

Efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway.

It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8(+) T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.

High-affinity interactions between peptides and heat shock protein 70 augment CD8+ T lymphocyte immune responses.

Exogenously delivered antigenic peptides complexed to heat shock proteins (HSPs) are able to enter the endogenous Ag-processing pathway and prime CD8+ CTL. It was determined previously that a hybrid peptide containing a MHC class I-binding epitope and HSP70-binding sequence Javelin (J0) in complex with HSP70 could induce cytotoxic T cell responses in vivo that were more robust than those induced by the minimal epitope complexed with HSP70. The present study introduces a novel, higher-affinity HSP70-binding sequence (J1) that significantly enhances binding of various antigenic peptides to HSP70. A competition binding assay revealed a dissociation constant that was 15-fold lower for the H2-K(b) OVA epitope SIINFEKL-J1 compared with SIINFEKL-J0, indicating a substantially higher affinity for HSP70. Further, modifying the orientation of the hybrid epitope and introducing a cleavable linker sequence between the Javelin and the epitope results in even greater immunogenicity, presumably by greater efficiency of epitope processing. The enhanced immunogenicity associated with Javelin J1 and the cleavable linker is consistently observed with multiple mouse and human epitopes. Thus, by creating a series of epitopes with uniform, high-affinity binding to HSP70, successful multiple epitope immunizations are possible, with equal delivery of each antigenic epitope to the immune system via HSP70. These modified epitopes have the potential for creating successful multivalent vaccines for immunotherapy of both infectious disease and cancer.

Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination, CpG-oligodeoxynucleotide adjuvant, and CTLA-4 blockade.

Although peptide immunization often leads to the induction of strong T-cell responses, it is seldom effective against established tumors. One possibility is that these T-cell responses are not strong enough or do not last sufficiently long to have an effect in tumor eradication. Here, we examined the role of synthetic oligodeoxynucleotide (ODN) adjuvants containing unmethylated cytosine-guanine motifs (CpG-ODN) and CTLA-4 blockade in enhancing the antitumor effectiveness of peptide vaccines intended to elicit CTL responses. The results show that combination immunotherapy consisting of vaccination with a synthetic peptide corresponding to an immunodominant CTL epitope derived from tyrosinase-related protein-2 administered with CpG-ODN adjuvant and followed by systemic injection of anti-CTLA-4 antibodies increased the survival of mice against the poorly immunogenic B16 melanoma. Interestingly, whereas this combination therapy was effective when administered to tumor-bearing mice (therapeutic protocol), it had no significant effect when applied in the prophylactic mode (i.e., before the tumor challenge). Moreover, the antitumor effect of the combination immunotherapy required the participation of CD4+ and CD8+ T lymphocytes and was accompanied by the induction of antitumor CD4+ T-cell responses. The overall results suggest that peptide vaccination of tumor-bearing mice, applied in combination with a strong adjuvant and CTLA-4 blockade, is capable of eliciting durable antitumor T cell responses that provide survival benefit. These findings bear clinical significance for the design of peptide-based therapeutic vaccines for human cancer patients.

CD8+ T-cell mediated tumor protection by Pseudomonas exotoxin fused to ovalbumin in C57BL/6 mice.

BACKGROUND: Pseudomonas exotoxin (PE) is a 66 kDa bacterial toxin that is able to bind to mammalian cells, undergo receptor mediated endocytosis, and translocate its C-terminal catalytic domain into the cytosol. We investigated whether PE could be used in vivo to deliver CD8+ T-cell epitopes to the MHC-class I antigen presentation pathway to trigger a specific cytotoxic T-lymphocyte (CTL) response. METHODS: Amino acid 553 of PE was deleted to eliminate toxin catalytic activity, and amino acids 204-386 of ovalbumin were fused near the nontoxic PE C-terminus to produce PE(D)-OVA200. Mice were vaccinated with 100 microg of PE(D)-OVA200 3 times at 21 day intervals. Splenocytes were harvested 1 week later, and stimulated in vitro with ovalbumin expressing EG7 murine thymoma cells. In vivo tumor protection experiments were performed by vaccinating groups of mice as before, followed by a lethal dose of ovalbumin expressing tumor cells (MO5) injected subcutaneously. RESULTS: Splenocytes from PE(D)-OVA200 vaccinated mice lysed (51)Cr labeled EG7 cells but not the untransfected EL4 parent cell line, whereas splenocytes from mice immunized with PBS, PE(D), or ovalbumin were unable to lyse EG7 cells. Cytotoxicity in vitro was mediated by CD8+ T-cells. PE(D)-OVA200 vaccinated mice survived (88%) a lethal subcutaneous challenge of ovalbumin expressing MO5 cells. Depletion of CD8+ cells from PE(D)-OVA200 vaccinated mice abolished this protection, indicating that this cell population is required for tumor rejection in vivo. CONCLUSIONS: Our results indicate that PE(D) may be used as a vehicle to stimulate a protective CTL response to heterologous antigen in vivo.

Targeting apoptotic tumor cells to Fc gamma R provides efficient and versatile vaccination against tumors by dendritic cells.

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcgammaRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcgammaRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.

In vivo augmentation of tumor-specific CTL responses by class I/peptide antigen complexes on microspheres (large multivalent immunogen).

Tumor membrane Ag immobilized on cell size microspheres (large multivalent immunogen (LMI)) was previously shown to augment tumor-specific CTL activity and reduce tumor growth, and a clinical trial examining this approach is in progress. In the current study, LMI treatment has been examined using adoptive transfer of TCR-transgenic CD8 T cells to visualize Ag-specific cells during the response. OT-I T cells specific for H-2K(b)/OVA(257-264) were transferred into mice that were then challenged with LMI made by immobilizing H-2K(b)/OVA(257-264) on microspheres (K(b)/OVA(257-264)-LMI) alone, or along with i.p. challenge with OVA-expressing E.G7 tumor. K(b)/OVA(257-264)-LMI caused significant reduction of tumor growth when administered to E.G7-bearing mice. When administered alone, the K(b)/OVA(257-264)-LMI caused only weak clonal expansion of OT-I cells in the spleen and lymph nodes, although most of the OT-I cells up-regulated expression of CD44 and VLA-4. In contrast, K(b)/OVA(257-264)-LMI administration to E.G7-bearing mice stimulated no detectable expansion of OT-I cells in the spleen and lymph nodes but caused a rapid increase in the number of OT-I cells in the peritoneal cavity, the site of the growing tumor. These results demonstrate the potential for using class I/tumor peptide complexes for immunotherapy. In addition, they suggest a model for the mechanism of CTL augmentation in which recognition of the LMI Ag results in altered trafficking of the tumor-specific CD8 T cells so that they reach the site of a growing tumor more rapidly and in greater numbers, where they may further expand and acquire effector function.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein.

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1Kb) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

T cell immunity to lymphoma following treatment with anti-CD40 monoclonal antibody.

In this study we demonstrate that treatment with anti-CD40 mAb eradicates a range of mouse lymphomas (BCL(1), A31, A20, and EL4), but only when used against i.v. tumor doses in excess of 10(7) cells. Only partial protection was seen against smaller tumor loads. We saw no evidence that anti-CD40 mAb changed the phenotype of the lymphomas or inhibited their growth in the initial period following treatment, but it did result in a rapid expansion of cytotoxic CD8(+) cells that was able to clear the neoplastic disease and provide long-term protection against tumor rechallenge. The CTL responses were blocked by mAb against a range of coreceptors and cytokines, including CD8, B7-1, B7-2, LFA-1, and IFN-gamma, but not CD4 or CTLA-4, indicating the presence of a conventional cellular Th1 response. Furthermore, we found evidence of cross-recognition between lymphomas (BCL(1) and A20) as measured by cytotoxicity and IFN-gamma responses in vitro and using tumor rechallenge experiments, suggesting common target Ags. Finally, although anti-CD40 was shown to stimulate NK cell killing, we could find no role for these cells in controlling tumor growth. These data underline the ability of anti-CD40 mAb to potentiate CTL responses and the potency of cellular immunity in eradicating large quantities of syngeneic tumor.