Systemic Cancer Therapy Does Not Significantly Impact Early Vaccine-Elicited SARS-CoV-2 Immunity in Patients with Solid Tumors.

mRNA vaccines have been shown to be safe and effective in individuals with cancer. It is unclear, however, if systemic anti-cancer therapy impacts the coordinated cellular and humoral immune responses elicited by SARS-CoV-2 mRNA vaccines. To fill this knowledge gap, we assessed SARS-CoV-2 mRNA vaccine-elicited immunity in a cohort of patients with advanced solid tumors either under observation or receiving systemic anti-cancer therapy. This analysis revealed that SARS-CoV-2 mRNA vaccine-elicited cellular and humoral immunity was not significantly different in individuals with cancer receiving systemic anti-cancer therapy relative to individuals under observation. Furthermore, even though some patients exhibited suboptimal antibody titers after vaccination, SARS-CoV-2 specific cellular immune responses were still detected. These data suggest that antibody titers offer an incomplete picture of vaccine-elicited SARS-CoV-2 immunity in cancer patients undergoing active systemic anti-cancer therapy, and that vaccine-elicited cellular immunity exists even in the absence of significant quantities of SARS-CoV-2 specific antibodies.

Interleukin-2-inducible T-cell kinase (Itk) signaling regulates potent noncanonical regulatory T cells.

Regulatory T cells (Tregs) play an important role in controlling autoimmunity and limiting tissue damage and inflammation. IL2-inducible T cell kinase (Itk) is part of the Tec family of tyrosine kinases and is a critical component of T cell receptor mediated signaling. Here, we showed that either genetic ablation of Itk signaling or inhibition of Itk signaling pathways resulted in increased frequency of "noncanonical" CD4+ CD25- FOXP3+ Tregs (ncTregs), as well as of "canonical" CD4+ CD25+ FOXP3+ Tregs (canTregs). Using in vivo models, we showed that ncTregs can avert the formation of acute graft-versus-host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft-versus-leukaemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in Itk-/- ncTregs. Furthermore, disrupting the SLP76: ITK interaction using a specific peptide inhibitor led to enhanced Treg development in both mouse and primary human cells. This peptide inhibitor also significantly reduced inflammatory cytokine production in primary GVHD patient samples and mouse T cells without causing cell death or apoptosis. We provide evidence that specifically targeting Itk signaling could be a therapeutic strategy to treat autoimmune disorders.

Human Wnt/ß-Catenin Regulates Alloimmune Signaling during Allogeneic Transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most widely applied forms of adoptive immunotherapy for the treatment of hematological malignancies. Detrimental graft-versus-host disease (GVHD), but also beneficial graft-versus-leukemia (GVL) effects occurring after allo-HSCT are largely mediated by alloantigen-reactive donor T cells in the graft. Separating GVHD from GVL effects is a formidable challenge, and a greater understanding of donor T cell biology is required to accomplish the uncoupling of GVHD from GVL. Here, we evaluated the role of ß-catenin in this process. Using a unique mouse model of transgenic overexpression of human ß-catenin (Cat-Tg) in an allo-HSCT model, we show here that T cells from Cat-Tg mice did not cause GVHD, and surprisingly, Cat-Tg T cells maintained the GVL effect. Donor T cells from Cat-Tg mice exhibited significantly lower inflammatory cytokine production and reduced donor T cell proliferation, while upregulating cytotoxic mediators that resulted in enhanced cytotoxicity. RNA sequencing revealed changes in the expression of 1169 genes for CD4, and 1006 genes for CD8+ T cells involved in essential aspects of immune response and GVHD pathophysiology. Altogether, our data suggest that ß-catenin is a druggable target for developing therapeutic strategies to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also mediated by these cells. Here, we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in GVL vs. GVHD effects after allo-HSCT. CD8+ and CD4+ donor T cells from mice expressing a Y145F mutation in SLP-76 did not cause GVHD but preserved GVL effects against B-ALL cells. SLP76Y145FKI CD8+ and CD4+ donor T cells also showed less inflammatory cytokine production and migration to GVHD target organs. We developed a novel peptide to specifically inhibit SLP76:ITK interactions, resulting in decreased phosphorylation of PLCγ1 and ERK, decreased cytokine production in human T cells, and separation of GVHD from GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to separate GVHD from GVL effects after allo-HSCT treatment.

Longitudinal Analysis of Peripheral and Colonic CD161+ CD4+ T Cell Dysfunction in Acute HIV-1 Infection and Effects of Early Treatment Initiation.

CD161 expression on CD4+ T cells is associated with a Th17 functional phenotype, as well as with an innate capacity to respond to interleukin (IL)-12 and IL-18 without T cell receptor (TCR) stimulation. Chronic HIV-1 infection is associated with loss of the CD161+ CD4 T cell population, and non-human primate studies suggest that their depletion is associated with disease progression. However, the dynamics of the CD161+ CD4+ T cell population during acute HIV-1 infection remains unknown. In this study, we characterize peripheral blood CD161+ CD4+ T cells in detail, and examine how they are affected during the earliest stages of HIV-1 infection. Unbiased surface proteome screening and principal component analysis indicated that CD161+ CD4+ T cells are relatively phenotypically homogeneous between donors, and are intermediates between conventional CD4 T cells and innate-like T cells. In acute untreated HIV-1 infection, the circulating CD161+ CD4+ T cell population decreased in frequency, as did absolute cell counts starting from peak viral load, with elevated levels of activation and exhaustion markers expressed throughout acute HIV-1 infection. The capacity of these cells to respond to stimulation with IL-12 and IL-18 was also reduced. Early initiation of anti-retroviral treatment (ART) during acute HIV-1 infection restored the functionality of peripheral blood CD161+ CD4+ T cells, but not their frequency. In contrast, early ART initiation prevented the decline of colonic CD161+ CD4+ T cells that otherwise started during acute infection. Furthermore, loss of peripheral and colonic CD161+ CD4+ T cells in untreated infection was associated with levels of viral load. These results suggest that acute HIV-1 infection has profound effects on the CD161+ CD4+ T cell population that could not be completely prevented by the initiation of ART.

Analysis of cell-associated DENV RNA by oligo(dT) primed 5' capture scRNAseq.

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5' capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5' to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5' end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5' capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5' capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.

IL7 receptor signaling in T cells: A mathematical modeling perspective.

Interleukin-7 (IL7) plays a nonredundant role in T cell survival and homeostasis, which is illustrated in the severe T cell lymphopenia of IL7-deficient mice, or demonstrated in animals or humans that lack expression of either the IL7Rα or γ c chain, the two subunits that constitute the functional IL7 receptor. Remarkably, IL7 is not expressed by T cells themselves, but produced in limited amounts by radio-resistant stromal cells. Thus, T cells need to constantly compete for IL7 to survive. How T cells maintain homeostasis and further maximize the size of the peripheral T cell pool in face of such competition are important questions that have fascinated both immunologists and mathematicians for a long time. Exceptionally, IL7 downregulates expression of its own receptor, so that IL7-signaled T cells do not consume extracellular IL7, and thus, the remaining extracellular IL7 can be shared among unsignaled T cells. Such an altruistic behavior of the IL7Rα chain is quite unique among members of the γ c cytokine receptor family. However, the consequences of this altruistic signaling behavior at the molecular, single cell and population levels are less well understood and require further investigation. In this regard, mathematical modeling of how a limited resource can be shared, while maintaining the clonal diversity of the T cell pool, can help decipher the molecular or cellular mechanisms that regulate T cell homeostasis. Thus, the current review aims to provide a mathematical modeling perspective of IL7-dependent T cell homeostasis at the molecular, cellular and population levels, in the context of recent advances in our understanding of the IL7 biology. This article is categorized under: Models of Systems Properties and Processes > Organ, Tissue, and Physiological Models Biological Mechanisms > Cell Signaling Models of Systems Properties and Processes > Mechanistic Models Analytical and Computational Methods > Computational Methods.

Distinct IL-7 signaling in recent thymic emigrants versus mature naïve T cells controls T-cell homeostasis.

IL-7 is essential for T-cell survival but its availability is limited in vivo. Consequently, all peripheral T cells, including recent thymic emigrants (RTEs) are constantly competing for IL-7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T-cell pool. However, it remains unknown how RTEs successfully compete with resident mature T cells for IL-7. Moreover, RTEs express low levels of IL-7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL-7 than mature naïve T cells as demonstrated by markedly increased STAT5 phosphorylation upon IL-7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL-7-induced homeostatic proliferation and diminished expansion compared to naïve donor T cells. Mechanistically, we found that IL-7 signaling in RTEs preferentially upregulated expression of Bcl-2, which is anti-apoptotic but also anti-proliferative. In contrast, naïve T cells showed diminished Bcl-2 induction but greater proliferative response to IL-7. Collectively, these data indicate that IL-7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of T cells rich in diversity but not in frequency.

mTORC1 and mTORC2 selectively regulate CD8⁺ T cell differentiation.

Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4+ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8+ T cell effector and memory populations. Evaluation of mice with a T cell-specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8+ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8+ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8+ T cell effector responses, mTORC2 activity regulated CD8+ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8+ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8+ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity.

Cellular size as a means of tracking mTOR activity and cell fate of CD4+ T cells upon antigen recognition.

mTOR is a central integrator of metabolic and immunological stimuli, dictating immune cell activation, proliferation and differentiation. In this study, we demonstrate that within a clonal population of activated T cells, there exist both mTORhi and mTORlo cells exhibiting highly divergent metabolic and immunologic functions. By taking advantage of the role of mTOR activation in controlling cellular size, we demonstrate that upon antigen recognition, mTORhi CD4+ T cells are destined to become highly glycolytic effector cells. Conversely, mTORlo T cells preferentially develop into long-lived cells that express high levels of Bcl-2, CD25, and CD62L. Furthermore, mTORlo T cells have a greater propensity to differentiate into suppressive Foxp3+ T regulatory cells, and this paradigm was also observed in human CD4+ T cells. Overall, these studies provide the opportunity to track the development of effector and memory T cells from naïve precursors, as well as facilitate the interrogation of immunologic and metabolic programs that inform these fates.

The transcription factor ThPOK suppresses Runx3 and imposes CD4(+) lineage fate by inducing the SOCS suppressors of cytokine signaling.

Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4(+) lineage fate and Runx3 promoting the CD8(+) lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8(+) lineage fate, nor is it understood why ThPOK itself imposes the CD4(+) lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4(+) lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4(+) lineage fate in the thymus.

The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex.

SGK1 is an AGC kinase that regulates the expression of membrane sodium channels in renal tubular cells in a manner dependent on the metabolic checkpoint kinase complex mTORC2. We hypothesized that SGK1 might represent an additional mTORC2-dependent regulator of the differentiation and function of T cells. Here we found that after activation by mTORC2, SGK1 promoted T helper type 2 (TH2) differentiation by negatively regulating degradation of the transcription factor JunB mediated by the E3 ligase Nedd4-2. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling expression of the long isoform of the transcription factor TCF-1. Consistent with those findings, mice with selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated substantial IFN-γ in response to viral infection and more readily rejected tumors.

An Fc domain protein-small molecule conjugate as an enhanced immunomodulator.

Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A(2A)R) has been identified as a promising target for immunotherapy, small molecule A(2A)R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A(2A)R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A(2A)R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.

Natural and inducible TH17 cells are regulated differently by Akt and mTOR pathways.

Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.

Ischemia-induced neuroinflammation is associated with disrupted development of oligodendrocyte progenitors in a model of periventricular leukomalacia.

Microglial activation in crossing white matter tracts is a hallmark of noncystic periventricular leukomalacia (PVL), the leading pathology underlying cerebral palsy in prematurely born infants. Recent studies indicate that neuroinflammation within an early time window can produce long-lasting defects in oligodendroglial maturation, myelination deficit, as well as disruption of transcription factors important in oligodendroglial maturation. We recently reported an ischemic mouse model of PVL, induced by unilateral neonatal carotid artery ligation, leading to selective long-lasting bilateral myelination deficits, ipsilateral thinning of the corpus callosum, ventriculomegaly, as well as evidence of axonopathy. Here, we report that permanent unilateral carotid ligation on postnatal day 5 in CD-1 mice induces an inflammatory response, as defined by microglial activation and recruitment, as well as significant changes in cytokine expression (increased IL-1ß, IL-6, TGF-ß1, and TNF-α) following ischemia. Transient reduction in counts of oligodendrocyte progenitor cells (OPCs) at 24 and 48 h after ischemia, a shift in OPC cell size and morphology towards the more immature form, as well as likely migration of OPCs were found. These OPC changes were topographically associated with areas showing microglial activation, and OPC counts negatively correlated with increased microglial staining. The presented data show a striking neuroinflammatory response in an ischemia-induced model of PVL, associated with oligodendroglial injury. Future studies modulating the neuroinflammatory response in this model may contribute to a better understanding of the interaction between microglia and OPCs in PVL and open opportunities for future therapies.

Macrophage A2A adenosinergic receptor modulates oxygen-induced augmentation of murine lung injury.

Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality. Exacerbating factors increasing the risk of ARDS remain unknown. Supplemental oxygen is often necessary in both mild and severe lung disease. The potential effects of supplemental oxygen may include augmentation of lung inflammation by inhibiting anti-inflammatory pathways in alveolar macrophages. We sought to determine oxygen-derived effects on the anti-inflammatory A2A adenosinergic (ADORA2A) receptor in macrophages, and the role of the ADORA2A receptor in lung injury. Wild-type (WT) and ADORA2A(-/-) mice received intratracheal lipopolysaccharide (IT LPS), followed 12 hours later by continuous exposure to 21% oxygen (control mice) or 60% oxygen for 1 to 3 days. We measured the phenotypic endpoints of lung injury and the alveolar macrophage inflammatory state. We tested an ADORA2A-specific agonist, CGS-21680 hydrochloride, in LPS plus oxygen-exposed WT and ADORA2A(-/-) mice. We determined the specific effects of myeloid ADORA2A, using chimera experiments. Compared with WT mice, ADORA2A(-/-) mice exposed to IT LPS and 60% oxygen demonstrated significantly more histologic lung injury, alveolar neutrophils, and protein. Macrophages from ADORA2A(-/-) mice exposed to LPS plus oxygen expressed higher concentrations of proinflammatory cytokines and cosignaling molecules. CGS-21680 prevented the oxygen-induced augmentation of lung injury after LPS only in WT mice. Chimera experiments demonstrated that the transfer of WT but not ADORA2A(-/-) bone marrow cells into irradiated ADORA2A(-/-) mice reduced lung injury after LPS plus oxygen, demonstrating myeloid ADORA2A protection. ADORA2A is protective against lung injury after LPS and oxygen. Oxygen after LPS increases macrophage activation to augment lung injury by inhibiting the ADORA2A pathway.

Regulation of CD4⁺ and CD8⁺ effector responses by Sprouty-1.

TCR-induced NF-AT activation leads to the expression of both activating and inhibitory proteins. Previously, we had identified Egr-2 and Egr-3 as NF-AT-induced transcription factors which promote the inhibition of T cell activation. In this report we identify Sprouty1 as a downstream target of Egr-3. CD4⁺ T cells lacking Spry1 demonstrate enhanced proliferation and cytokine production. Likewise, Spry1(Flox/Flox) Lck Cre CD8⁺ T cells display increased cytolytic activity. Mechanistically, Spry1 acts at the level of PLC-γ promoting the inhibition of both Ca⁺⁺ induced NF-AT activation and MAP-kinase induced AP-1 activation while sparing NF-κB signaling. In vivo, mice in which Spry1 is selectively deleted in T cells demonstrate enhanced responses to a tumor vaccine and subsequently reject tumors more robustly than Wt mice. These findings suggest that targeting Spry1 might prove to be a novel means of enhancing tumor immunotherapy.

CD73 is critical for the resolution of murine colonic inflammation.

CD73 is a glycosyl-phosphatidylinositol-(GPI-) linked membrane protein that catalyzes the extracellular dephosphorylation of adenosine monophosphate (AMP) to adenosine. Adenosine is a negative regulator of inflammation and prevents excessive cellular damage. We investigated the role of extracellular adenosine in the intestinal mucosa during the development of Dextran-Sulfate-Sodium-(DSS-)salt-induced colitis in mice that lack CD73 (CD73(-/-)) and are unable to synthesize extracellular adenosine. We have found that, compared to wild-type (WT) mice, CD73(-/-) mice are highly susceptible to DSS-induced colitis. CD73(-/-) mice exhibit pronounced weight loss, slower weight recovery, an increase in gut permeability, a decrease in expression of tight junctional adhesion molecules, as well as unresolved inflammation following the removal of DSS. Moreover, colonic epithelia in CD73(-/-) mice exhibited increased TLR9 expression, high levels of IL-1ß and TNF-α, and constitutive activation of NF-κB. We conclude that CD73 expression in the colon is critical for regulating the magnitude and the resolution of colonic immune responses.

Resolution of experimental lung injury by monocyte-derived inducible nitric oxide synthase.

Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS-/- mice were exposed to intratracheal LPS, early lung injury was attenuated; however, recovery was markedly impaired compared with WT mice. iNOS-/- mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS+/+) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS-/- mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule CD86 in iNOS-/- mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS-/- mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair.

Enhancement of tumor immunotherapy by deletion of the A2A adenosine receptor.

The A(2A) adenosine receptor plays a critical and non-redundant role in suppressing inflammation at sites of hypoxia and tissue damage. The tumor microenvironment has high levels of adenosine as a result of hypoxia and ectopic expression of enzymes responsible for the generation of extracellular adenosine. Thus, we sought to determine the ability of A(2A) receptor null mice to immunologically reject tumors. We observed that mice lacking the A(2A) adenosine receptor showed significantly delayed growth of lymphoma cells when compared to WT mice. Furthermore, when immunized with a low dose of tumor or with an irradiated GM-CSF-secreting tumor vaccine, A(2A) receptor null mice showed significantly enhanced protection from a subsequent high-dose challenge from both immunogenic and poorly immunogenic tumor lines. This increase in protection was accompanied by an increase in the number of tumor-antigen-specific CD8 T cells at the vaccine-site draining lymph node. Finally, we found that A(2A) receptor null mice displayed more robust anti-tumor responses than WT mice when they were treated with a soluble B7-DC/Fc fusion protein designed to antagonize B7-H1-mediated co-inhibition. This combinatorial immunotherapy strategy could also be recapitulated with pharmacological A(2A) receptor blockade paired with B7-DC/Fc administration. In light of these data, we believe that blockade of the A(2A) adenosine receptor is an attractive target for tumor immunotherapy that synergizes with other immunomodulatory approaches currently in clinical trials.