Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia

Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4(+) T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4(+)CD25(+)FoxP3(+) regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3(+) Tregs from naive CD4(+)CD25(-)FoxP3(-) T cells by an interferon-γ-dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.     

An NKT-mediated autologous vaccine generates CD4 T-cell dependent potent antilymphoma immunity

Relapses occurring in most patients with lymphoma after antibody or chemotherapy highlight a need for effective vaccination approaches. Autologous tumors are ideal sources of patient-specific tumor antigens for vaccines; however, their poor immunogenicity has been a major obstacle in practice. Natural killer T (NKT) cells have recently emerged as crucial regulators of autoimmunity and tumor immunosurveillance. Here, we show that an autologous lymphoma vaccine that activates NKT cells generated tumor-specific protective immunity in experimental mice. Single vaccination with alpha-galactosylceramide (alphaGC)-loaded A20 lymphoma cells elicited effective antitumor immunity against tumor challenge. This vaccination strategy also induced significant tumor regression in A20-bearing mice. Importantly, the survivors from primary tumor inoculation were all resistant to tumor rechallenge, indicative of established adaptive memory immunity. Depletion as well as adoptive transfer studies revealed an exclusive role of conventional CD4(+) but not CD8(+) T cells in mediating antitumor immunity. In addition, we found normal hematopoietic compartments in the vaccinated mice. Therefore, NKT ligand-loaded lymphoma elicits long-lasting and effective antitumor immunity, which can be further developed as patient- and tumor-specific immunotherapy against human lymphomas.     

PD-1-mediated attrition of polyfunctional memory CD8+ T cells in chronic toxoplasma infection

We reported earlier that during chronic toxoplasmosis CD8(+) T cells become functionally exhausted with concomitant PD-1 upregulation, leading to eventual host mortality. However, how immune exhaustion specifically mediates attrition of CD8 polyfunctionality, a hallmark of potent T-cell response, during persistent infections has not been addressed. In this study, we demonstrate that PD-1 is preferentially expressed on polyfunctional memory CD8(+) T cells, which renders them susceptible to apoptosis. In vitro blockade of the PD-1-PD-L1 pathway dramatically reduces apoptosis of polyfunctional and interferon γ(+)/granzyme B(-) memory but not effector CD8(+) T cells. In summary, the present report underscores the critical role of the PD-1-PD-L1 pathway in mediating attrition of this important CD8(+) T-cell subset and addresses the mechanistic basis of how αPD-L1 therapy reinvigorates polyfunctional CD8 response during chronic infections. The conclusions of this study can have profound immunotherapeutic implications in combating recrudescent toxoplasmosis as well other chronic infections.     

Adoptive transfer of allogeneic tumor-specific T cells mediates effective regression of large tumors across major histocompatibility barriers

Graft-versus-tumor effects can be achieved after allogeneic bone marrow transplantation in patients with malignancies of the kidney or hematopoietic system but are often accompanied by severe graft-versus-host-disease (GVHD). We sought to maximize graft-versus-tumor while minimizing GVHD using tumor-specific allogeneic effector T cells rather than open-repertoire T cells. We transferred allogeneic CD8(+) pmel-1 or CD4(+) TRP-1 T cells specific for the melanoma-associated antigens, glycoprotein 100 (gp100) and tyrosinase-related protein-1 (TRP-1), respectively, into B16-melanoma-bearing mice. Mice receiving a preparative regimen of nonmyeloablating (5 Gy) total body irradiation experienced the rapid rejection of tumor-specific allogeneic lymphocytes with no impact on tumor growth. However, when mice were given more intense total body irradiation conditioning regimens combined with autologous bone marrow transplantation, adoptively transferred allogeneic tumor-specific T lymphocytes persisted at detectable levels for several weeks and mediated significant regression of large, vascularized tumors. We found that the risk of GVHD was low when tumor-specific T cells were transferred and significant toxicity was observed only when substantial numbers of open repertoire allogeneic naive T cells were mixed with the tumor-specific lymphocytes. Taken together, these data indicate that the use of tumor-specific allogeneic CD8(+) T cells or CD4(+) can result in significant antitumor effects in the absence of measurable GVHD.     

Dendritic cells loaded with apoptotic antibody-coated tumor cells provide protective immunity against B-cell lymphoma in vivo

The in vitro priming of tumor-specific T cells by dendritic cells (DCs) phagocytosing killed tumor cells can be augmented in the presence of antitumor monoclonal antibody (mAb). We investigated whether DCs phagocytosing killed lymphoma cells coated with tumor-specific antibody could elicit antitumor immunity in vivo. Irradiated murine 38C13 lymphoma cells were cocultured with bone marrow-derived DCs in the presence or absence of tumor-specific mAb. Mice vaccinated with DCs cocultured with mAb-coated tumor cells were protected from tumor challenge (60% long-term survival), whereas DCs loaded with tumor cells alone were much less effective. The opsonized whole tumor cell-DC vaccine elicited significantly better tumor protection than a traditional lymphoma idiotype (Id) protein vaccine, and in combination with chemotherapy could eradicate preexisting tumor. Moreover, the DC vaccine protected animals from both wild-type and Id-negative variant tumor cells, indicating that Id is not a major target of the induced tumor immunity. Protection was critically dependent upon CD8(+) T cells, with lesser contribution by CD4(+) T cells. Importantly, opsonized whole tumor cell-DC vaccination did not result in tissue-specific autoimmunity. Since opsonized whole tumor cell-DC and Id vaccines appear to target distinct tumor antigens, optimal antilymphoma immunity might be achieved by combining these approaches.     

Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines

The fate of tumor-specific CD4(+) T cells is central to the outcome of the host immune response to cancer. We show that tumor antigen recognition by a subset of CD4(+) T cells led to their differentiation into cells capable of suppressing naive and Th1 effector cells. Such tumor-induced regulatory T cells (TMTregs) arose both from precommitted "natural" regulatory T cells and CD4(+)CD25(-)GITR(low) precursors. Once induced, TMTregs were capable of maintaining suppressor activity long after transfer into antigen-free recipients. Suppression was mediated by GITR(high) cells residing within both CD25(+) and CD25(-) subsets. Vaccination of the tumor-bearing host concomitantly expanded TMTregs and effector cells, but suppression was dominant, blunting the expansion of naive tumor-specific T cells and blocking the execution of effector function in vitro and in vivo. These studies illustrate the possibility that therapeutic vaccination could actually worsen host tolerance to tumor antigens and support treatment paradigms that seek to not only increase the frequency of tumor-specific T cells, but to do so in conjunction with strategies that inactivate or remove regulatory T-cell populations.     

Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer

The ligands for programmed cell death 1 (PD-1), an immunoinhibitory receptor belonging to CD28/cytotoxic T lymphocyte antigen 4 family, are PD-1 ligand 1 and 2 (PD-Ls). Recent reports suggest that the aberrant expression of PD-Ls on tumor cells impairs antitumor immunity, resulting in the immune evasion of the tumor cells. Although an inverse correlation between the expression level of PD-Ls and patients' prognosis has been reported for several malignant tumors, the follow-up period was limited because of the lack of the antibody (Ab) applicable to paraffin-embedded specimens. Here we generated a new Ab against PD-1 ligand 1 (PD-L1) and analyzed the expression level of PD-Ls in human ovarian cancer using paraffin-embedded specimens. Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression. Although patients with higher expression of PD-1 ligand 2 also had a poorer prognosis, the difference was not statistically significant. A significant inverse correlation was observed between PD-L1 expression and the intraepithelial CD8(+) T lymphocyte count, suggesting that PD-L1 on tumor cells directly suppresses antitumor CD8(+) T cells. Multivariate analysis showed the expression of PD-L1 on tumor cells and intraepithelial CD8(+) T lymphocyte count are independent prognostic factors. The PD-1/PD-L pathway can be a good target for restoring antitumor immunity in ovarian cancer.     

Depletion of radio-resistant regulatory T cells enhances antitumor immunity during recovery from lymphopenia

Cytotoxic lymphodepletion therapies augment antitumor immune responses. The generation and therapeutic efficacy of antitumor effector T cells (T(E)s) are enhanced during recovery from lymphopenia. Although the effects of lymphodepletion on naive T cells (T(N)s) and T(E)s have been studied extensively, the influence of lymphodepletion on suppressor cells remains poorly understood. In this study, we demonstrate a significant increase of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in sublethally irradiated lymphopenic mice. These radio-resistant Tregs inhibited the induction of T(E)s in tumor-draining lymph-nodes (TDLNs) during recovery from lymphopenia. The transfer of T(N)s into lymphopenic tumor-bearing mice resulted in some antitumor effects; however, Treg depletion after whole-body irradiation and reconstitution strongly inhibited tumor progression. Further analyses revealed that tumor-specific T cells were primed from the transferred T(N)s, whereas the Tregs originated from irradiated recipient cells. As in irradiated lymphopenic mice, a high percentage of Tregs was observed in cyclophosphamide-treated lymphopenic mice. The inhibition of Tregs in cyclophosphamide-treated mice significantly reduced tumor growth. These results indicate that the Tregs that survive cytotoxic therapies suppress antitumor immunity during recovery from lymphopenia and suggest that approaches to deplete radio and chemo-resistant Tregs can enhance cancer immunotherapies.     

Role of SEREX-defined immunogenic wild-type cellular molecules in the development of tumor-specific immunity.

Recognition of altered self-antigens in tumor cells by lymphocytes forms the basis for antitumor immune responses. The effector cells in most experimental tumor systems are CD8(+) T cells that recognize MHC class I binding peptides derived from molecules with altered expression in tumor cells. Although the need for CD4(+) helper T cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumor responses remain unclear. We examined whether broadly expressed wild-type molecules in murine tumor cells eliciting humoral immunity contributed to the generation of CD8(+) T cells and protective antitumor immune responses to unrelated tumor-specific antigens [mutated ERK2 (mERK2) and c-erbB2/HER/neu (HER2)]. The immunogenic wild-type molecules, presumably dependent on recognition by CD4(+) helper T cells, were defined by serological analysis of recombinant cDNA expression libraries (SEREX) using tumor-derived lambda phage libraries screened with IgG antibodies of hosts bearing transplanted 3-methylchoranthrene-induced tumors. Coimmunization of mice with plasmids encoding SEREX-defined murine wild-type molecules and mERK2 or HER2 led to a profound increase in CD8(+) T cells specific for mERK2 or HER2 peptides. This heightened response depended on CD4(+) T cells and copresentation of SEREX-defined molecules and CD8(+) T cell epitopes. In tumor protection assays, immunization with SEREX-defined wild-type molecules and mERK2 resulted in an inhibition of pulmonary metastasis, which was not achieved by immunization with mERK2 alone.     

CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) delivers inhibitory signals to activated T cells. CTLA4 is constitutively expressed on regulatory CD4(+) T cells (Tregs), but its role in these cells remains unclear. CTLA4 blockade has been shown to induce antitumor immunity. In this study, we examined the effects of anti-CTLA4 antibody on the endogenous CD4(+) T cells in cancer patients. We show that CTLA4 blockade induces an increase not only in the number of activated effector CD4(+) T cells, but also in the number of CD4(+) FoxP3(+) Tregs. Although the effects were dose-dependent, CD4(+) FoxP3(+) regulatory T cells could be expanded at lower antibody doses. In contrast, expansion of effector T cells was seen only at the highest dose level studied. Moreover, these expanded CD4(+) FoxP3(+) regulatory T cells are induced to proliferate with treatment and possess suppressor function. Our results demonstrate that treatment with anti-CTLA4 antibody does not deplete human CD4(+) FoxP3(+) Tregs in vivo, but rather may mediate its effects through the activation of effector T cells. Our results also suggest that CTLA4 may inhibit Treg proliferation similar to its role on effector T cells. This study is registered at http://www.clinicaltrials.gov/ct2/show/NCT00064129, registry number NCT00064129.     

Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia

Tumor-associated immune suppression can lead to defective T cell-mediated antitumor immunity. Here, we identified a unique phenotype of exhausted T cells in mice with advanced acute myelogenous leukemia (AML). This phenotype is characterized by the coexpression of Tim-3 and PD-1 on CD8(+) T cells in the liver, the major first site of AML metastases. PD-1 and Tim-3 coexpression increased during AML progression. PD-1(+)Tim-3(+) CD8(+) T cells were deficient in their ability to produce IFN-γ, TNF-α, and IL-2 in response to PD-1 ligand (PDL1) and Tim-3 ligand (galectin-9) expressing AML cells. PD-1 knockout (KO), which were partially resistant to AML challenge, up-regulated Tim-3 during AML progression and such Tim-3(+)PD-1- KO CD8(+) T cells had reduced cytokine production. Galectin-9 KO mice were more resistant to AML, which was associated with reduced T-regulatory cell accumulation and a modest induction of PD-1 and Tim-3 expression on CD8(+) T cells. Whereas blocking the PD-1/PDL1 or Tim-3/galectin-9 pathway alone was insufficient to rescue mice from AML lethality, an additive effect was seen in reducing-albeit not eliminating-both tumor burden and lethality when both pathways were blocked. Therefore, combined PD-1/PDL1 and Tim-3/galectin-9 blockade may be beneficial in preventing CD8(+) T-cell exhaustion in patients with hematologic malignancies such as advanced AML.     

Interleukin-10 promotes the maintenance of antitumor CD8(+) T-cell effector function in situ

Interleukin-10 (IL-10) is a multifunctional cytokine that can exert suppressive and stimulatory effects on T cells. It was investigated whether IL-10 could serve as an immunostimulant for specific CD8(+) cytotoxic T cell (CTL) in vivo after vaccination and, if so, under what conditions. In tumor prevention models, administration of IL-10 before, or soon after, peptide-pulsed primary dendritic cell immunization resulted in immune suppression and enhanced tumor progression. Injection of IL-10, however, just after a booster vaccine significantly enhanced antitumor immunity and vaccine efficacy. Analysis of spleen cells derived from these latter animals 3 weeks after IL-10 treatment revealed that the number of CD8(+) CD44(hi) CD122(+) T cells had increased and that antigen-specific proliferation in vitro was enhanced. Although cytotoxicity assays did not support differences between the various treatment groups, 2 more sensitive assays measuring antigen-specific interferon-gamma production at the single-cell level demonstrated increases in the number of antigen-specific responder T cells in animals in the vaccine/IL-10 treatment group. Thus, IL-10 may maintain the number of antitumor CD8(+) T cells. In adoptive transfer studies, the ability of IL-10 to maintain CTL function could be enhanced by the depletion of CD4(+) T cells. This suggests that IL-10 mediates contrasting effects on both CD4(+) and CD8(+) T cells that result in either immune dampening or immune potentiation in situ, respectively. Appreciation of this dichotomy in IL-10 immunobiology may allow for the design of more effective cancer vaccines designed to activate and maintain specific CD8(+) T-cell effector function in situ.     

CD8(+) T-cell reconstitution in recipients of umbilical cord blood transplantation and characteristics associated with leukemic relapse

Recipients of umbilical cord blood (UCB) transplantation (UCBT) face a high risk of morbidity and mortality related to opportunistic infections (OI) and leukemic relapse. To understand the molecular basis of these UCBT-related complications, the characteristics of UCB-derived antigen-specific CD8(+) T cells were examined in a group of pediatric UCBT recipients. Compared with the UCB graft inoculum and the late post-UCBT period (12-36 months), declining clonal diversity of UCB-derived CD8(+) T cells specific for the Melan-A(26-35) A27L peptide and high frequencies of PD-1-expressing CD8(+) T cells were observed in the first 3 months after UCBT, a period during which OIs are most frequent. The CD8(+) T-cell compartment predominantly comprised CD45RA(+) CCR7(-) terminally differentiated effector-memory T cells until 6 months after UCBT, at which time the polyfunctionality of antigen-specific CD8(+) T cells was reestablished. Finally, the frequency of PD-1(+) CD8(+) T cells was significantly higher in subjects who subsequently experienced leukemic relapse. This study informs the biologic properties of UCB-derived CD8(+) T cells and provides a rationale for the characteristics of UCBT in terms of immune reconstitution and OI. These results also suggest that the elevated frequency of PD-1(+) CD8(+) T cells could be associated with leukemic relapse in pediatric UCBT recipients.     

CD4 cells can be more efficient at tumor rejection than CD8 cells

Researchers designing antitumor treatments have long focused on eliciting tumor-specific CD8 cytotoxic T lymphocytes (CTL) because of their potent killing activity and their ability to reject transplanted organs. The resulting treatments, however, have generally been surprisingly poor at inducing complete tumor rejection, both in experimental models and in the clinic. Although a few scattered studies suggested that CD4 T "helper" cells might also serve as antitumor effectors, they have generally been studied mostly for their ability to enhance the activity of CTL. In this mouse study, we compared monoclonal populations of tumor-specific CD4 and CD8 T cells as effectors against several different tumors, and found that CD4 T cells eliminated tumors that were resistant to CD8-mediated rejection, even in cases where the tumors expressed major histocompatibility complex (MHC) class I molecules but not MHC class II. MHC class II expression on host tissues was critical, suggesting that the CD4 T cells act indirectly. Indeed, the CD4 T cells partnered with NK cells to obtain the maximal antitumor effect. These findings suggest that CD4 T cells can be powerful antitumor effector cells that can, in some cases, outperform CD8 T cells, which are the current "gold standard" effector cell in tumor immunotherapy.     

CTLA-4 dysregulation of self/tumor-reactive CD8+ T-cell function is CD4+ T-cell dependent

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) maintains peripheral tolerance by suppressing T-cell activation and proliferation but its precise role in vivo remains unclear. We sought to elucidate the impact of CTLA-4 expression on self/tumor-reactive CD8(+) T cells by using the glycoprotein (gp) 100-specific T-cell receptor (TCR) transgenic mouse, pmel-1. pmel-1 CLTA-4(-/-) mice developed profound, accelerated autoimmune vitiligo. This enhanced autoimmunity was associated with a small but highly activated CD8(+) T-cell population and large numbers of CD4(+) T cells not expressing the transgenic TCR. Adoptive transfer of pmel-1 CLTA-4(-/-) CD8(+) T cells did not mediate superior antitumor immunity in the settings of either large established tumors or tumor challenge, suggesting that the mere absence of CTLA-4-mediated inhibition on CD8(+) T cells did not directly promote enhancement of their effector functions. Removal of CD4(+) T cells by crossing the pmel-1 CLTA-4(-/-) mouse onto a Rag-1(-/-) background resulted in the complete abrogation of CD8(+) T-cell activation and autoimmune manifestations. The effects of CD4(+) CLTA-4(-/-) T cells were dependent on the absence of CTLA-4 on CD8(+) T cells. These results indicated that CD8(+) CLTA-4(-/-) T-cell-mediated autoimmunity and tumor immunity required CD4(+) T cells in which the function was dysregulated by the absence of CTLA-4-mediated negative costimulation.     

Skewed association of polyfunctional antigen-specific CD8 T cell populations with HLA-B genotype

We studied CD8 T cell responses against HIV-1, cytomegalovirus, Epstein-Barr virus, and influenza in 128 subjects and demonstrate that polyfunctional CD8 T cell responses, also including IL-2 production and Ag-specific proliferation, are predominantly driven by virus epitopes restricted by HLA-B alleles. Interestingly, these protective CD8 T cells are equipped with low-avidity T cell receptors (TCRs) for the cognate virus epitope. Conversely, HLA-A-restricted epitopes are mostly associated with "only effector" IFN-gamma-secreting, with cytotoxicity, and with the lack of IL-2 production and Ag-specific proliferation. These CD8 T cells are equipped with high-avidity TCR and express higher levels of the T cell exhaustion marker PD-1. Thus, the functional profile of the CD8 T cell response is strongly influenced by the extent to which there is stimulation of polyfunctional (predominantly restricted by HLA-B) versus only effector (restricted by HLA-A) T cell responses. These results provide the rationale for the observed protective role of HLA-B in HIV-1-infection and new insights into the relationship between TCR avidity, PD-1 expression, and the functional profile of CD8 T cells.     

Rebuilding immunity in cancer patients

Rebuilding and maintaining immunity are paramount to the success of cancer immunotherapy and hematopoietic stem cell transplantation. If immune surveillance indeed can protect from cancer, the very manifestation of malignancy means that the disease has prevailed over immunity. Yet, often, tumor-specific T cells can be found in cancer patients irrespective of vaccination. Interestingly, patients suffering from malignancy often harbor unexpectedly high levels of immature CD14(+)HLA-DR(-) monocytes, although the abundance of CD4(+) cells, CD8(+) cells and CD4(+)CD25(high) cells may be normal. It is plausible that in cancer such cells suppress T cell function, analogous to CD14(+)HLA-DR(-) cells in sepsis and major trauma, in addition to their likely failure to re-present tumor-associated antigens once dendritic cells have initiated the T cell response. Recent evidence indicates that tumor-borne adenosine, lactate and hypoxia in the tumor environment may modulate tumor-specific immunity to a significant extent, but their effects on myeloid cell function are unclear. Thus, understanding and controlling these factors may appreciably impact the success of rebuilding and maintaining immunity in cancer patients.     

Identification of phosphatidylinositol 4-kinase type II beta as HLA class II-restricted target in graft versus leukemia reactivity

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific CD4(+) T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II beta gene. A polyclonal CD4(+) T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4(+) T cells recognized and lysed the CD34(+) CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-gamma and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4(+) T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8(+) T cell immunity as helper cells.     

Delineation of antigen-specific and antigen-nonspecific CD8(+) memory T-cell responses after cytokine-based cancer immunotherapy

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8(+) T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8(+) CD25(-) cells within the tumor site. These findings demonstrate that memory CD8(+) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.     

Vaccination regimens incorporating CpG-containing oligodeoxynucleotides and IL-2 generate antigen-specific antitumor immunity from T-cell populations undergoing homeostatic peripheral expansion after BMT

Development of CD8(+) T-cell responses targeting tumor-associated antigens after autologous stem cell transplantations (ASCTs) might eradicate residual tumor cells and decrease relapse rates. Because thymic function dramatically decreases with aging, T-cell reconstitution in the first year after ASCT in middle-aged patients occurs primarily by homeostatic peripheral expansion (HPE) of mature T cells. To study antigen-specific T-cell responses during HPE, we performed syngeneic bone marrow transplantations (BMTs) on thymectomized mice and then vaccinated the mice with peptides plus CpG-containing oligodeoxynucleotides (CpGs) in incomplete Freund adjuvant and treated the mice with systemic interleukin-2 (IL-2). When CD8(+) T-cell responses were measured ex vivo, up to 9.1% of CD8(+) T cells were specific for tumor-associated epitopes. These large T-cell responses were generated by synergism between CpG and IL-2. When we injected mice subcutaneously with tumor cells 14 days after BMT and then treated them with peptide + CpG-containing vaccines plus systemic IL-2, survival was increased and tumor growth was inhibited in an epitope-specific manner. Depletion of CD8(+) T cells eliminated epitope-specific antitumor immunity. This is the first report to demonstrate that CD8(+) T-cell responses capable of executing antitumor immunity can be elicited by CpG-containing vaccines during HPE.