TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A2B Adenosine Receptor Signaling.

Glioblastoma (GB) is one of the deadliest brain cancers to afflict humans, and it has a very poor survival rate even with treatment. The extracellular adenosine-generating enzyme CD73 is involved in many cellular functions that can be usurped by tumors, including cell adhesion, proliferation, invasion, and angiogenesis. We set out to determine the role of CD73 in GB pathogenesis. To do this, we established a unique GB mouse model (CD73-FLK) in which we spatially expressed CD73 on endothelial cells in CD73-/- mice. This allowed us to elucidate the mechanism of host CD73 versus GB-expressed CD73 by comparing GB pathogenesis in WT, CD73-/-, and CD73-FLK mice. GB in CD73-/- mice had decreased tumor size, decreased tumor vessel density, and reduced tumor invasiveness compared with GB in WT mice. Interestingly, GBs in CD73-FLK mice were much more invasive and caused complete distortion of the brain morphology. We showed a 20-fold upregulation of A2B AR on GB compared with sham, and its activation induced matrix metalloproteinase-2, which enhanced GB pathogenesis. Inhibition of A2B AR signaling decreased multidrug resistance transporter protein expression, including permeability glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). Further, we showed that blockade of A2B AR signaling potently increased GB cell death induced by the chemotherapeutic drug temozolomide. Together, these findings suggest that CD73 and A2B AR play a multifaceted role in GB pathogenesis and progression and that targeting the CD73-A2B AR axis can benefit GB patients and inform new approaches for therapy to treat GB patients.SIGNIFICANCE STATEMENT Glioblastoma (GB) is the most devastating primary brain tumor. GB patients' median survival is 16 months even with treatment. It is critical that we develop prophylaxes to advance GB treatment and improve patient survival. CD73-generated adenosine has been implicated in cancer pathogenesis, but its role in GB was not ascertained. Here, we demonstrated that host CD73 plays a prominent role in multiple areas of glioblastoma pathogenesis, including promoting GB growth, its angiogenesis, and its invasiveness. We found a 20-fold increase in A2B adenosine receptor (AR) expression on GB compared with sham, and its inhibition increased GB chemosensitivity to temozolomide. These findings strongly indicate that blockade or inhibition of CD73 and the A2B AR are prime targets for future GB therapy.

Restoration of adenosine deaminase-deficient human thymocyte development in vitro by inhibition of deoxynucleoside kinases.

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

Inhibition of deoxynucleoside kinases in human thymocytes prevents dATP accumulation and induction of apoptosis.

Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture.

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.