Search

ImportanceHealth systems play a central role in the delivery of health care, but relatively little is known about these organizations and their performance.ObjectiveTo (1) identify and describe health systems in the United States; (2) assess differences between physicians and hospitals in and outside of health systems; and (3) compare quality and cost of care delivered by physicians and hospitals in and outside of health systems.Evidence ReviewHealth systems were defined as groups of commonly owned or managed entities that included at least 1 general acute care hospital, 10 primary care physicians, and 50 total physicians located within a single hospital referral region. They were identified using Centers for Medicare & Medicaid Services administrative data, Internal Revenue Service filings, Medicare and commercial claims, and other data. Health systems were categorized as academic, public, large for-profit, large nonprofit, or other private systems. Quality of preventive care, chronic disease management, patient experience, low-value care, mortality, hospital readmissions, and spending were assessed for Medicare beneficiaries attributed to system and nonsystem physicians. Prices for physician and hospital services and total spending were assessed in 2018 commercial claims data. Outcomes were adjusted for patient characteristics and geographic area.FindingsA total of 580 health systems were identified and varied greatly in size. Systems accounted for 40% of physicians and 84% of general acute care hospital beds and delivered primary care to 41% of traditional Medicare beneficiaries. Academic and large nonprofit systems accounted for a majority of system physicians (80%) and system hospital beds (64%). System hospitals were larger than nonsystem hospitals (67% vs 23% with >100 beds), as were system physician practices (74% vs 12% with >100 physicians). Performance on measures of preventive care, clinical quality, and patient experience was modestly higher for health system physicians and hospitals than for nonsystem physicians and hospitals. Prices paid to health system physicians and hospitals were significantly higher than prices paid to nonsystem physicians and hospitals (12%-26% higher for physician services, 31% for hospital services). Adjusting for practice size attenuated health systems differences on quality measures, but price differences for small and medium practices remained large.Conclusions and RelevanceIn 2018, health system physicians and hospitals delivered a large portion of medical services. Performance on clinical quality and patient experience measures was marginally better in systems but spending and prices were substantially higher. This was especially true for small practices. Small quality differentials combined with large price differentials suggests that health systems have not, on average, realized their potential for better care at equal or lower cost.

Background: While checkpoint inhibitors (CPIs) such as anti-CTLA-4 and anti-PD-1/L1 have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal response observed. We aimed to use a proprietary machine learning/artificial intelligence platform to identify novel stromal targets to relieve this immunosuppressive barrier and increase CPI responsiveness in difficult to treat indications. Methods: Based on bioinformatic analysis using our single cell RNA atlas, we assessed cancer-associated fibroblasts (CAFs)/fibroblastic cells in cancer tissue for identification of novel targets, including proteoglycans. Antibodies were generated by immunization of humanized mice, and lead antibodies were tested for activity in inhibiting cell adhesion and were further characterized for staining of both CAFs as well as tumor cells. ADCs were developed and tested in vitro for selective tumor cell killing. Results: Bioinformatic analysis identified a unique subset of cancer-associated fibroblasts, termed ecmCAFs, which demonstrated selective expression of Collagen Triple Helix Repeat Containing 1 (CTHRC1). This highly-selective expression pattern suggests it may be ideal as a target for alternative modalities, including ADC targeting or specific T cell activation. In addition, we identified that in certain tumor types, such a triple negative breast cancer and pancreatic ductal adenocarcinoma (PDAC), CTHRC1 is also highly expressed by cancer cells within the tumor and shows a more favorable expression profile for ADC targeting when compared to other stromal proteins such as FAP and LRRC15. We have confirmed surface expression and binding of CTHRC1 by our lead antibodies and have observed robust internalization on both human and mouse cancer cell lines. In vitro killing of tumor cells by ADCs and in vivo PD and efficacy will be presented on both ADCs and naked antibodies. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both ecmCAFs and tumor cells that appears to be an ideal target for both direct inhibition of stromal barrier function as well as targeting of cytotoxic payloads as an ADC. CTHRC1 expression is more selective than the classical markers FAP and LRRC15, both of which have been previously developed as ADCs. Citation Format: Elizabeth Koch, Max London, Amy Berkley, Allison Nixon, Sean Phippen, Kerry White, Amanda Hanson, Samuel Cooper, Christopher Harvey, Michael Briskin. AI/ML-driven discovery of a novel proteoglycan for precision targeting of ADCs for disruption of stromal barriers and direct anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 388.

B7-H4 (VTCN1, B7x, B7S1) is a transmembrane protein belonging to the B7 family of costimulatory proteins and has been shown to inhibit T cell proliferation, cytokine secretion, and cytotoxic lymphocyte (CTL) induction. B7-H4 expressed on tumor cells or macrophages has been associated with poor prognosis and impaired T cell function in renal cell and ovarian cancers. Here we show B7-H4 is abundantly expressed in human breast cancer with triple negative breast cancer (TNBC) having the highest overall B7-H4 mRNA expression. We developed a specific and sensitive immunohistochemistry (IHC) assay for evaluation of B7-H4 protein and quantified B7-H4 expression in 156 breast tumor samples. Approximately 70% of the breast tumor samples had detectable B7-H4 expression whereas none of the normal or benign breast tissues stained positive for B7-H4. Multiplex IHC and flow cytometry studies showed that the majority of B7-H4 expression was restricted to the tumor epithelial cells, the CD45+ immune cells were negative for B7-H4 expression. Interestingly none of the TNBC samples that were positive for B7-H4 showed detectable expression of PD-L1 suggesting that B7-H4 and PD-L1 checkpoint proteins may act in a mutually exclusive manner. To evaluate the role of B7-H4 on tumor immune evasion, we overexpressed murine or human B7-H4 on the mouse colon-26 (CT26) tumor cell line and injected these cells intravenously into Balb/c mice. By day 14 we observed significantly more tumors as well as larger percent tumor area in the lungs of mice given CT26 cells transduced with human or mouse B7-H4 as compared to vector control transduced cells. These data suggest B7-H4 expression in tumors can accelerate tumor growth in immune competent mice and that targeting B7-H4 may provide therapeutic benefit. Given the mutually exclusive expression patterns of B7-H4 and PD-L1 a B7-H4 targeting agent may provide particular benefit in those patients where current anti-PD-1/PD-L1 therapies are not effective. Citation Format: Shaffer DR, Nagashima K, Cortez-Retamozo V, Feldman I, Smith J, Zafari M, Larson R, Mabry R, Novorantseva T, Briskin M, Sathyanaryananan S. Mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1 in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-07.

Adoptive cell therapy with chimeric antigen receptor (CAR) modified T cells has demonstrated remarkable clinical efficacy in the treatment of certain B cell malignancies, and more recently in multiple myeloma. However, CAR-T therapy has been less successful in treating solid tumors due to multiple obstacles, including the lack of robust CAR-T cell expansion, the immunosuppressive tumor microenvironment, and tumor escape due to the loss of targeted antigen. Engineering CAR-T cells to produce immunomodulatory factors such as Interleukin 12 (IL12) and Cluster of Differentiation 40 Ligand (CD40L) has been shown to enhance functional activity by driving T cell expansion, conferring resistance to immunosuppression, improving antigen presentation, and inducing antigen spread. However, the clinical utility of both IL12 and activators of the CD40 signaling pathway have been limited by systemic toxicity associated with their potent pharmacological activities. Providing precise tuning of the timing and level of expression of these immunomodulatory factors in CAR-T cells could significantly enhance safety and therapeutic efficacy, in particular against solid tumor malignancies. We describe here the implementation of ligand-controlled regulation of IL12 and CD40L in vitro and in vivo in engineered primary human T cells via the use of destabilizing domain (DD) technology. DDs are small protein domains that are misfolded and inherently unstable in the cell, but which can be reversibly stabilized by the binding of approved pharmacologic agents. This conditional stability of DDs can be readily conferred to any protein of interest by fusing it to the DD, thus providing fine-tuned, exogenous regulation of protein expression and function. We have identified mutations in several human protein substrates, including phosphodiesterase 5 (PDE5), dihydrofolate reductase (DHFR), and estrogen receptor (ER), which convey DD activity and can be regulated by FDA-approved drugs. We show that transduction of human T cells with either DD-IL12 or DD-CD40L fusion constructs yields low expression levels in the basal state and a rapid, dose-dependent induction of IL12 or CD40L protein in the presence of the corresponding stabilizing ligand. Moreover, kinetically precise, on-demand production of either factor from CAR-T cells can be achieved in mice by oral dosing of stabilizing drugs. A CD19 CAR-T Nalm6 mouse model has been established that measures potent CAR-T expansion and enhanced anti-tumor efficacy with armored immunomodulatory constructs. Studies are underway in this model, as well as in solid tumor models, to test for enhanced CAR-T activity via drug-induced activation of IL12 and CD40L towards the development of next generation cell therapies with more favorable efficacy and safety profiles. Citation Format: Michelle Ols, Michael Schebesta, Emily Brideau, Kutlu Elpek, Michelle Fleury, Jennifer Gori, Scott Heller, Dan Jun Li, Benjamin Primack, Christopher Reardon, Dhruv Sethi, Alex Storer, Dexue Sun, Karen Tran, Elizabeth Weisman, Michael Briskin, Celeste Richardson, Vipin Suri, Steven Shamah. CAR-Ts armored with small molecule-regulated IL12 or CD40L cassettes for enhanced activity against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-013.

Tumors utilize many different escape mechanisms to evade anti-tumor immune responses. Xios Therapeutics has screened X-Chem’s proprietary 200-billion molecule DNA-encoded library against several immuno-oncology (IO) targets addressing T-cell centric, myeloid immunity and onco-metabolite pathways. Adenosine, for example, is a potent immunosuppressive metabolite, and the ecto-5-nucleotidase (a.k.a. CD73), which catalyzes the conversion of AMP to adenosine, is the rate limiting enzyme for the production of extracellular adenosine in the tumor microenvironment. Hence, CD73 and/or the downstream adenosine receptors are considered attractive targets for IO drug discovery. Likewise, the characteristics of tumor-associated macrophages (TAMs) have fueled interest in therapeutically targeting the colony-stimulating factor 1 axis (CSF1R). Here, we exemplify and enumerate the diversity, selectivity and physiochemical properties of selected hit-to-lead compounds identified from our DNA-encoded library screens of CD73, the adenosine A2A receptor and CSF1R. In the context of the immune-suppressive purinergic pathway, we have integrated structural biology, medicinal chemistry and clinical pathology evaluation of target expression across tumor types and developed both A2A selective and dual A2A/A2B selective receptor antagonists. From a DNA-encoded library screen, we have identified novel sub-micromolar ligands, which binds to CD73 in an ‘open conformation’ revealed by the co-crystal structure of X6034 (EC50 = 310 nM) in complex with CD73. Interestingly, an inorganic phosphate molecule (Pi) that is structurally shown to be co-present in the active site, illustrates the novelty of these inhibitors, which are chemically distinct from currently reported ADP/AMP substrate analogs. Finally, using tumor tissue microarrays and in situ cell hybridization (RNA-Scope; Advanced Cell Diagnostics), we have explored the co-expression pattern of pathway targets across a subset of immune cells. Informed by the tissue expression pattern of A2AR (primarily CD3+ T-Cells) and A2BR (primarily CD33+ myeloid cells), we compared the ability of equipotent A2A and dual A2A/A2B adenosine receptor antagonists to reverse the effect of NECA, a nonhydrolyzable analog of adenosine, on the maturation and activation of dendritic cells (DC). Starting with the differentiation of human immature DC, we demonstrate the added benefit of dual A2A/A2B inhibitors vs A2A selective inhibitors on relieving immunosuppression of myeloid cells. To conclude, the combination of all these data, together with the productivity of the DNA-encoded library screens for identifying novel, drug like chemical matter on challenging targets like CD73, provide a unique opportunity for potentially harnessing the full power of immunotherapy for cancer. Citation Format: Andrew J. McRiner, Jannik N. Andersen, Lynette A. Fouser, Junyi Zhang, Kaan Certel, John Cuozzo, Betty Chan, Ragunath Chandran, Matt Clark, Diana Gikunju, Christopher D. Hupp, Anthony D. Keefe, Julie Liu, Yanbin Liu, Michael Monteiro, Allison Olszewski, Moritz Von Rechenberg, Daniel Resnicow, Heather A. Thomson, Dawn M. Troast, Zooey Wang, Neil Westlund, Ying Zhang, Fei Zhou, Xiaotian Zhu, Michael Briskin, Diala Ezzeddine. Novel, potent, and selective small-molecule inhibitors modulating immuno-oncology targets CD73, A2A/A2B adenosine receptors and CSF1R discovered via DNA-encoded library screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4453.

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, ‘fit-for-purpose’ PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable. Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

Cytokines are messenger molecules that act as regulators of innate and adaptive immunity by propagating cell-cell immune signaling. Several cytokines have been approved for the treatment of metastatic renal cell cancer, advanced melanoma, and hairy cell leukemia (HCL) and can be particularly effective when combined with adoptive cell therapy. However, systemic delivery or constitutive expression of cytokines even at moderate levels can potentially lead to significant toxicity. These hurdles to enabling cytokine-enhanced adoptive cell therapy motivated us to implement destabilizing domain (DD) technology for regulating cytokines in chimeric antigen receptor (CAR) reprogrammed T-cells. The ability of CAR-T-cells to traffic to tumor sites enables localized co-delivery of cytokines for enhanced CAR-T-cell antitumor activity while improving safety. Obsidian Therapeutics’ DD technology enables titratable and reversible regulation of a protein of interest with FDA-approved small-molecule drugs in a time- and dose-dependent manner. Protein-fused DDs are misfolded in the absence of a stabilizing small-molecule ligand and are rapidly degraded by the proteasome. However, the addition of ligand restores the folding, stability, and function of the DD-protein fusion. We have generated IL-12 and membrane-bound IL15-IL15Ra (mbIL15) fused to DDs such as FK506 binding protein (FKBP), Escherichia coli dihydrofolate reductase (ecDHFR), as well as human protein substrates (huDDs) with clinically approved ligands. DD-IL12 and DD-mbIL15 fusions displayed ligand-dependent regulation of cytokine secretion or cell surface expression, respectively, in cell lines and primary human T-cells. We then tested DD regulation of IL12 or mbIL15 in vivo by injecting T-cells engineered with cytokine-fused DDs into NSG mice, followed by oral administration of vehicle or corresponding ligand. Vehicle-treated mice displayed low level expression of the respective cytokines, whereas ligand treatment robustly induced the expression of target cytokine within 4-6 hours after treatment. Cytokine expression returned to baseline levels 24 hours following ligand administration. These data demonstrate the feasibility of exogenous control over transgene-derived protein expression in primary human T-cells for the development of next-generation CAR-T-cell products with enhanced efficacy and more favorable safety profiles. Citation Format: Karen Tran, Kutlu Elpek, Tucker Ezell, Scott Heller, Mara Inniss, Abhishek Kulkarni, Dan Jun Li, Grace Olinger, Michelle Ols, Christopher Reardon, Dexue Sun, Tariq Kassum, Michael Briskin, Celeste Richardson, Vipin Suri, Steven Shamah, Michael Gilman. Destabilizing domain technology facilitates exogenous regulation of IL15 and IL12 for adaptive T-cell therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A220.

Chlamydia trachomatis causes sexually transmitted infection and reproductive dysfunction worldwide. Identifying a population of endocervical T-cells to target in vaccine development is likely to enhance efficacy of a vaccine and reduce reproductive tract dysfunction.Endocervical samples were obtained from young women and flow cytometric analysis was used to identify lymphocytes that appeared in the genital tract in response to sexually transmitted bacterial infections caused by C. trachomatis.Increased numbers of α4β7+CLA+ memory T-cells, a unique T-cell phenotype, were found in the endocervix of human female subjects infected with C. trachomatis.A unique population of memory T lymphocytes expressing both α4β7 and CLA gain access to reproductive tract tissues during a sexually transmitted infection with C. trachomatis and should be considered in development of vaccines against sexually transmitted infections.

The goal of this collaboration was to enable early quantitative thought experiments and risk assessments by developing and interrogating a quantitative systems pharmacology (QSP) model of the co-modulation inhibitory receptors PD-1 and TIM-3 in immuno-oncology. The QSP model was to: (1) provide predictions of best-in-class profiles for a PD-1 and TIM-3 fixed dose combination (FDC) and dual antagonist platforms, and (2) provide biological insights. The QSP model was based on first principles as a system of elementary mass-action, mechanistic PKPD, ordinary differential equations. The model parameters and reactions were based on biophysics, and are interpretable. The model reactions include protein synthesis and elimination, ligand-receptor and drug-target formation and turnover, and drug administration and first order clearance. There were four versions of the model: PD-1 monospecific, TIM-3 monospecific, PD-1 x TIM-3 bispecific and fixed dose combination (FDC) targeting PD-1 and TIM-3. The monospecific models were then benchmarked against published data such that model parameter values were set to known values and unknown parameters were estimated. Once benchmarked, the FDC and bispecific models were analyzed by systematically investigating how tuning the model parameters (e.g., affinity, avidity, dose, half-life, target expression, etc.) impacted target inhibition, and to simulate patient variability. The model predictions were in good agreement with published clinical data from Nivolumab and Pembrolizumab, providing a hypothesis for the apparent inconsistencies in similar clinical doses for therapeutics with affinities differing by several orders of magnitude, and data from RMT3-23 in the TIM-3 driven mouse model. QSP model analysis predicted: (1) there would be diminishing returns on very tight binding biologics due to Target Mediated Drug Disposition (TMDD) that offsets potency, and (2) there is no advantage between FDC, 2-2 bispecific, and 2-1 bispecific formats, which are predicted to be roughly equivalent. Citation Format: Joshua Apgar, Jamie Wong, Ryan Phennicie, Robert Mabry, Tatiana Novobrantseva, Michael Briskin, John M. Burke. Quantitative systems pharmacology modeling and analysis provides biological insights into anti-PD-1 dosing and predicts optimal PD-1 x TIM-3 therapeutic properties for bispecifics and fixed dose combinations in immuno-oncology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5001.

Chlamydia trachomatis causes sexually transmitted infection and reproductive dysfunction worldwide. Identifying a population of endocervical T-cells to target in vaccine development is likely to enhance efficacy of a vaccine and reduce reproductive tract dysfunction.Endocervical samples were obtained from young women and flow cytometric analysis was used to identify lymphocytes that appeared in the genital tract in response to sexually transmitted bacterial infections caused by C. trachomatis.Increased numbers of α4β7+CLA+ memory T-cells, a unique T-cell phenotype, were found in the endocervix of human female subjects infected with C. trachomatis.A unique population of memory T lymphocytes expressing both α4β7 and CLA gain access to reproductive tract tissues during a sexually transmitted infection with C. trachomatis and should be considered in development of vaccines against sexually transmitted infections.

ICOS (inducible co-stimulator molecule) is a T cell encoded member of the extended B7/CD28 superfamily that is up-regulated upon early T cell activation. Previous studies have shown that higher ICOS expression on circulating T cells, post treatment with ipilimumab, is associated with better clinical outcome. In complementary pre-clinical studies, efficacy observed with a whole cell vaccine consisting of ICOS-L expressing B16 melanoma (IVAX) suggests that agonism of this pathway could provide therapeutic benefit in the cancer setting. In accordance with these observations, we generated a panel of anti-ICOS monoclonal antibodies with in vitro agonist properties. A lead series of mAbs were shown to be efficacious as monotherapies in syngeneic tumors models with enhanced tumor inhibition observed in combination with anti-PD1. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8/regulatory T (Tregs) cells and that this change is related to preferential reduction in ICOS high Tregs in the tumor microenvironment. These data provided the rationale for development of a high affinity humanized agonist monoclonal antibody to be tested in both mono therapy and in combination with other T cell checkpoints. Our lead anti-ICOS antibody is currently in IND-enabling studies and will be tested for activity in solid tumor indications. Citation Format: Kutlu Elpek, Christopher Harvey, Ellen Duong, Tyler Simpson, Jenny Shu, Lindsey Shallberg, Matt Wallace, Sriram Sathy, Robert Mabry, Jennifer Michaelson, Michael Briskin. Efficacy of anti-ICOS agonist monoclonal antibodies in preclinical tumor models provides a rationale for clinical development as cancer immunotherapeutics. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A059.

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.

Meeting abstracts ICOS (inducible co-stimulator molecule) is a T cell encoded member of the extended B7/CD28 superfamily that is up-regulated upon early T cell activation. Previous studies have shown that higher ICOS expression on circulating T cells, post treatment with ipilimumab, is associated

Meeting abstracts B7-H4 is a member of the B7 family of co-regulatory receptors. It is believed to negatively regulate T cell function and has been associated with poor prognosis in renal cell and ovarian cancers. We performed an unbiased analysis of TCGA gene expression data and identified triple

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.

B29 is a B-cell-specific member of the immunoglobulin gene superfamily that is expressed throughout B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy-chain gene segment rearrangements. We have analyzed the region upstream of the B29 gene to identify DNA sequences involved in transcriptional regulation of this gene. The B29 gene lacks a TATA box and transcription is initiated at multiple sites. The B29 gene sequence 5' of these transcription start sites contains six promoter and enhancer motifs known to control immunoglobulin gene transcription. The most notable is a perfect octamer (5'-ATTTGCAT-3'), which binds the Oct-2 B-cell-specific transcription factor and thereby can account for the tissue-specific expression of this gene.

mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.

The murine pre-B leukemic cell line 70Z/3 contains both an unrearranged immunoglobulin kappa-light-chain gene and a functionally rearranged but silent kappa-light-chain gene. Mitogenic stimulation of growing 70Z/3 cells with bacterial lipopolysaccharide (LPS) activates kappa-light-gene transcription and results in a 10- to 20-fold increase in cytoplasmic kappa-light-chain mRNA. The induction of kappa gene expression by LPS was probed by using an inhibitor of protein synthesis. Concomitant treatment of 70Z/3 cells with LPS and cycloheximide failed to block kappa-light-chain mRNA accumulation, indicating that new protein synthesis is not required for the activation of kappa-gene expression. Treatment of 70Z/3 cells with cycloheximide alone resulted in kappa-mRNA induction equivalent to those produced by LPS alone. The kappa mRNA synthesized in the presence of cycloheximide was intact and able to direct the synthesis of kappa light chains. Nuclear transcription assays revealed that cycloheximide, like LPS, activated kappa-gene transcription. These findings indicate that the trans-acting factors necessary for kappa-light-chain-gene transcription are present in pre-B cells, but their activity is blocked by short-lived inhibitory proteins.

B lymphocyte development proceeds through an ordered course of immunoglobulin gene rearrangements and expression [reviewed in 3, 24, 42)]. Pre-B cells contain rearranged immunoglobulin μ genes and express intracellular μ heavy chains. Early B lymphocytes express rearranged light chain genes and display monomeric surface IgM. Mature B lymphocytes co-express a new class of immunoglobulin, IgD, along with the IgM on their membranes. Further DNA rearrangements are not involved in the activation of IgD expression. Rather, the μ and δ heavy chains of IgM and IgD are encoded by different heavy chain mRNA species derived from the large μ + δ complex transcription unit (Figure 1). Finally, mature plasma cells actively secrete pentameric IgM formed in covalent association with J-chain.

The induction of immunoglobulin kappa light chain expression in 70Z/3 pre-B cells treated with bacterial lipopolysaccharide (LPS) requires the activation of the B cell-specific factor NF-kappa B, which binds to the kappa enhancer motif, GGGACTTTCC. This sequence alone can function as a tissue-specific enhancer for LPS-induced gene expression. A potent inhibitor of B lymphopoiesis [transforming growth factor-beta (TGF-beta)] was used to explore the mechanisms in the activation of kappa transcription by LPS and by interferon-gamma (IFN-gamma). TGF-beta inhibited LPS-induced kappa transcription but not the activation and in vitro binding of NF-kappa B. This indicates that NF-kappa B activation, while necessary, is not sufficient for LPS-induced kappa transcription. TGF-beta had no effect on IFN-gamma-induced kappa transcription, and NF-kappa B was not activated by IFN-gamma. These results reveal that LPS and IFN-gamma activate transcription through different mechanisms.