ALBERT

Description: Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct “reprogramming” of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1–presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Description: Metabolic bone disease affects hundreds of millions of people worldwide, and as a result, in vitro models of bone tissue have become essential tools to help analyze bone pathogenesis, develop drug screening, and test potential therapeutic strategies. Drugs that either promote or impair bone formation are in high demand for the treatment of metabolic bone diseases. These drugs work by targeting numerous signaling pathways responsible for regulating osteogenesis such as Hedgehog, Wnt/β-catenin, and PI3K-AKT. In this study, differentiated bone marrow-derived mesenchymal stem cell (BM-MSC) scaffold-free 3D bioprinted constructs and 2D monolayer cultures were utilized to screen four drugs predicted to either promote (Icariin and Purmorphamine) or impair osteogenesis (PD98059 and U0126). Osteogenic differentiation capacity was analyzed over a four week culture period by evaluating mineralization, alkaline phosphatase (ALP) activity, and osteogenesis related gene expression. Responses to drug treatment were observed in both 3D differentiated constructs and 2D monolayer cultures. After four weeks in culture, 3D differentiated constructs and 2D monolayer cultures treated with Icariin or Purmorphamine showed increased mineralization, ALP activity, and the gene expression of bone formation markers (BGLAP, SSP1, and COL1A1), signaling molecules (MAPK1, WNT1, and AKT1), and transcription factors (RUNX2 and GLI1) that regulate osteogenic differentiation relative to untreated. 3D differentiated constructs and 2D monolayer cultures treated with PD98059 or U0126 showed decreased mineralization, ALP activity, and the expression of the aforementioned genes BGLAP, SPP1, COL1A1, MAPK1, AKT1, RUNX2, and GLI1 relative to untreated. Differences in ALP activity and osteogenesis related gene expression relative to untreated cells cultured in a 2D monolayer were greater in 3D constructs compared to 2D monolayer cultures. These findings suggest that our bioprinted bone model system offers a more sensitive, biologically relevant drug screening platform than traditional 2D monolayer in vitro testing platforms.

Description: PD-1 is a T cell inhibitor for which blocking agents have achieved success as anti-cancer therapeutics. The current view is that cancer limits host immune responses by upregulating PD-L1 in the tumor microenvironment (TME) thereby causing PD-1 ligation and inactivation of CD8+ Teff cells. However, PD-L1 expression in the TME does not always correlate with therapeutic response. Thus, the mechanism(s) by which PD-1 blockade reverses compromised anti-tumor immunity are poorly understood. The rapid increase in hematopoietic cell output that occurs in response to immunologic stress is known as emergency myelopoiesis. Low-level stimulation by cancer-generated factors induces modest but continuous expansion of myeloid progenitors (MP) (common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP)) albeit with hindered differentiation, leading to output of tumor-promoting myeloid-derived suppressor cells (MDSCs). We determined that myeloid cells expanding during cancer-driven emergency myelopoiesis in tumor-bearing mice express PD-1 and PD-L1. Using PD-1 KO mice we found that PD-1 deletion prevented the accumulation of GMP and stimulated the output of Ly6Chi effector monocytes, macrophages and dendritic cells (DC). To determine whether these outcomes were mediated by a myeloid-intrinsic impact of PD-1 ablation or by the effects of PD-1neg T cells on myeloid cells, we generated mice with conditional targeting of the Pdcd1 gene (PD-1f/f) and selectively eliminated PD-1 in myeloid cells (PD-1f/fLysMcre) or T cells (PD-1f/fCD4cre). Myeloid-specific, but not T cell-specific PD-1 ablation, prevented the accumulation of GMP while promoting the output of effector-like myeloid cells expressing CD80, CD86, CD16/32 (FcRII/III) and CD88 (C5aR). Myeloid cells with PD-1 ablation had elevated expression of IRF8 that drives monocyte and DC differentiation and decreased expression of the MDSC hallmark markers IL-4R, CD206, ARG1 and CD38. Nutrient utilization has a decisive role on the fate of hematopoietic progenitors (HP) and MP. Stemness and pluripotency are regulated by maintenance of glycolysis whereas switch to mitochondrial metabolism is associated with differentiation. To examine whether PD-1 ablation affected these metabolic proceces, bone marrow (BM) from PD-1f/f and PD-1f/fLysMcre mice was cultured with G-CSF/GM-CSF/IL-6, key drivers of emergency myelopoiesis. MP differentiation was documented by decrease of Linneg and increase of Linpos cells, which was more prominent in PD-1f/fLysMcre BM cultures. This coincided with increase of CD45+CD11b+ and dominance of Ly6C+ monocytic cells consistent with a cell-intrinsic mechanism of monocytic lineage commitment. PD-1f/fLysMcre MP had elevated mTORC1, Erk1/2 and Stat1 activation, and enhanced glucose uptake and mitochondrial biogenesis. Bioenergetics studies showed robust development of a mitochondrial-dominant profile, consistent with metabolism-driven enhanced differentiation of MP. Mass spectrometry revealed enhanced intermediates of glycolysis, PPP and TCA cycle, but the most prominent difference was the increased cholesterol. Because mTORC1 signaling, which was enhanced in PD-1f/fLysMcre MP, activates de novo lipid and cholesterol synthesis via SREBP1, we examined the mevalonate pathway of cholesterol synthesis. mRNA for genes mediating cholesterol synthesis and uptake was increased whereas mRNA for genes mediating cholesterol metabolism was decreased. Cholesterol induces a proinflammatory program in myeloid cells, drives differentiation of monocytes, macrophages and DC and promotes antigen-presenting function. We examined how such changes in myeloid cells might affect the function of T cells, which are key anti-tumor mediators. Compared to tumor-bearing PD-1f/f mice, PD-1f/fLysMcre tumor-bearers had no quantitative T cell differences but had an increase in IFNγ- IL-17-, and IL-10-expressing CD8+ Teff-mem and IL-2-expressing Tcentral-mem cells, consistent with superior functionality. These changes correlated with enhanced anti-tumor protection despite preserved PD-1 expression in T cells. Our findings reveal a previously unidentified role of PD-1 in metabolism-driven myeloid cell lineage fate commitment and differentiation and suggest that switch to effector myeloid cells might be a key mechanism by which PD-1 blockade mediates systemic anti-tumor immunity. Disclosures No relevant conflicts of interest to declare.

Description: Umbilical cord blood transplantation (UCBT) has extended the availability of hematopoietic stem cell transplantation to patients without compatible adult donors. Studies in zebrafish and mouse models have shown that the prostaglandin compound, 16,16 dimethyl prostaglandin E2 (PGE2), increases HSC number and homing. In a human clinical trial of double UCBT, PGE2 decreased time to engraftment, promoted early T cell chimerism, favored generation of long-lived memory CD8+ cells and reduced the incidence of CMV viremia. To obtain mechanistic insight of how PGE2 affects the differentiation program of antigen-specific T cells, we used a well-established model of T effector (TEFF) vs. T memory (TM) differentiation of TCR-transgenic OTI T cells. OTI cells were stimulated with Ova257-264 plus APC with or without PGE2, followed by incubation with either IL-2 or IL-7, which are critical for the development of CD8+ TEFF and TM, respectively. Antigen-specific stimulation with or without PGE2 followed by IL-2 resulted in differentiation to CD44+CD62L- TEFF cells. In contrast, antigen-specific stimulation followed by IL-7 resulted in generation of CD44+CD62L+ central memory T cells. Under these conditions, PGE2 treatment gave rise to a phenotype of CD44-CD62L+Bcl2+Sca1+ cells, consistent with T stem cell memory. Two key pathways regulated by AMPK and mTOR have a decisive role on TM differentiation. AMPK can promote the generation of TM cells. However, we found that PGE2 inhibited AMPK activation indicating that differentiation of TM by PGE2 is not mediated by AMPK. Because the mTORC1-specific inhibitor rapamycin can promote the generation of TM, we focused our studies on the effects of PGE2 on mTORC1. Two classes of direct downstream targets of mTORC1 have been well characterized. mTOR phosphorylates the ribosomal protein S6 kinases (S6K1/2) and the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BP1/2), both of which control specific steps in the initiation of cap-dependent translation. mTORC1 activation can stimulate glycolysis as well as lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia inducible factor 1a (HIF1a) and sterol regulatory element-binding protein (SREBP1/2). HIF1a is regulated downstream of 4E-BP whereas SREBP is regulated downstream of S6K1. Although the mTORC1 inhibitor rapamycin can promote differentiation of TM and Treg cells, recent studies revealed that mTORC1, via its effects on lipid metabolism has a mandatory role on Treg differentiation. We found that PGE2 treatment during antigen-specific stimulation of OTI cells with Ova257-264 resulted in activation of mTOR as determined by phosphorylation of mTOR, 4E-BP and S6K and phosphorylation of Akt on the mTOR-specific site. Surprisingly, when antigen-specific stimulation was followed by IL-7, PGE2 treatment inhibited expression of the 4E-BP downstream targets Myc, HIF1a, the glycolysis genes Glut1, HK2, LDH-A, and the uptake of glucose. Phosphorylation of S6K was also impaired and lipid biosynthesis was suppressed as determined by decreased expression of fatty acid synthase FASN. In contrast, a metabolic program of lipid utilization was activated, characterized by increase of CPT1a, which promotes fatty acid transport in the mitochondria for b-oxidation, and the lipid oxidase Acox1. Mitochondria biogenesis analyzed by Mitotracker staining and expression of mitochondrial genes MTOC-1, TIMM50 and COX5a were also enhanced. In bioenergetics studies control-treated antigen-specific T cells had a glycolytic phenotype with elevated extracellular acidification rate (ECAR) whereas PGE2-treated cells had elevated oxygen consumption rate (OCR) and increased OCR/ECAR ratio, indicating preferential use of oxidative phosphorylation to generate energy. Thus, mTORC1 might regulate differentiation of antigen-specific T cells to TM by promoting lipid biosynthesis upon engaging distinct downstream targets in response to extracellular cues, thereby providing the required fuel for the bioenergetic demands of TM cells. Our studies reveal an unexpected mechanism by which PGE2 regulates the functional fate of T cells by modifying mTORC1 downstream signals and altering T cell metabolic imprints. These findings have implications for harnessing immune memory in the context of tumor-specific and pathogen-specific immunity. Disclosures No relevant conflicts of interest to declare.

Description: Programmed death-1 (PD-1) is a checkpoint receptor expressed on activated T-cells. PD-1 has a key role in maintenance of peripheral tolerance but also restrains anti-viral and anti-tumor immunity. Although PD-1 blockade leads to durable clinical responses in a significant fraction of patients, the majority of patients have only transient responses, emphasizing the need for better understanding of the mechanism of PD-1-mediated T cell inhibition. PD-1 consists of a single N-terminal IgV-like domain, a 20 amino acid stalk separating the IgV domain from the plasma membrane, a transmembrane domain, and a cytoplasmic tail containing two tyrosine-based structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). SHP-2 tyrosine phosphatase interacts with the ITSM and has a critical role in PD-1-mediated inhibition but the precise mechanism is poorly understood. We sought to determine how PD-1: SHP-2 interaction leads to inhibition of T-cell responses. SHP-2 contains two SH2 domains, a phosphatase (PTP) domain and a C-terminus tail (C-tail), forming a structure of N-SH2-C-SH2-PTP-C-tail. We generated five GST-fusion proteins in which GST was fused with either SHP-2 full length, N-SH2, C-SH2 C-SH2-PTP (lacking the N-terminus SH2 domain), or PTP. Pull-down assays using lysates from human T cells revealed that PD-1 interacted with GST-SHP-2 fusion protein only after TCR/CD3-mediated activation with simultaneous PD-1 ligation, and the interaction of PD-1 with SHP-2 was mediated via the SH2 domains of SHP-2. The SH2 domains of SHP-2 have a crucial and distinct role in regulating SHP-2 PTPase activity. In the absence of a tyrosine-phosphorylated ligand, N-SH2 binds the PTP domain leading to an auto-inhibitory closed conformation that blocks the PTP site. Phosphorylation of Y542 in the SHP-2 C-tail leads to an intramolecular interaction of Y542 with the N-SH2 domain that relieves N-SH2 binding to the PTP domain and thereby reverses basal inhibition of the PTPase. Phosphorylation of Y580 in the SHP-2 C-tail relieves the auto-inhibitory closed conformation by interaction with the C-SH2 domain. Subsequent high affinity intermolecular interaction of the N-SH2 with a phosphorylated protein partner completely disrupts its PTP recognition surface, reversing the auto-inhibitory conformation and activating the PTPase activity, whereas the C-SH2 domain contributes to binding energy and specificity. We found that in activated T cells, PD-1-associated SHP-2 was phosphorylated in the tyrosines of the C-tail. To determine whether PD-1 selectively interacts with a specific SH2 domain of SHP-2, we mutagenized the functional sites of N-SH2 and C-SH2 domains at arginines 32 and 138, respectively, to alanine (R32A and R138A) and transfected COS cells with cDNA of SHP-2 wild type or each SH2 mutant together with PD-1 and TCR proximal kinase Fyn, which is required for PD-1 phosphorylation. Immunoprecipitation and immunoblot showed that mutagenesis of either SH2 domain abrogated interaction of SHP-2 with PD-1, indicating that both SH2 domains of SHP-2 are involved in the interaction with PD-1. Surprisingly, each SH2 domain of SHP-2 interacted with tyrosine phosphorylated ITSM of PD-1, as determined by immunoblot with a phopho-PD-1 antibody specific for the phosphorylated tyrosine residue Y245 and by disruption of both N-SH2:PD-1 and C-SH2:PD-1 interaction by mutation of PD-1 ITSM tyrosine residue Y245. These results indicate that SHP-2 brings together two tyrosine phosphorylated PD-1 molecules by interaction with N-SH2 and C-SH2 domains. To determine the functional implications of PD-1 homodimerization, we cultured human T cells in the presence of a soluble dimeric PD-L1 or a monomeric PD-L1. Although dimeric PD-L1 inhibited T cell proliferation and IFN-g production, monomeric PD-L1 had the opposite effect. Our results reveal a previously unidentified mechanism of PD-1: SHP-2 interaction and have implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or to enhance T cell activation by disrupting PD-1 homodimerization. Our findings open new avenues for the development of selective PD-1-binding compounds in order to augment T cell responses for the induction of antitumor immunity or to suppress aberrant T cell activation in autoimmunity and graft versus host disease. Disclosures No relevant conflicts of interest to declare.

Description: PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248+ (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1+CD8+ T cells were identified in human peripheral blood and had impaired effector function. pPD-1+ T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1+ T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.