bluuebunny/crossref_metadata_2025 huggingface dataset:
B cell transcription factors define a novel tumor suppressor gene network In ALL
Authors: Heltemes Harris Lynn M , Hubbard Gregory , LaRue Rebecca , Munro Sarah A. , Yang Rendong , Henzler Christine , Sarver Aaron , Farrar Michael A
Published: January 2023

The transcription factors EBF1 and PAX5 are frequently mutated in B cell acute lymphoblastic leukemia (B-ALL). We demonstrated that Pax5+/− x Ebf1+/− compound heterozygous mice develop leukemia with high penetrance. Similar results were seen in Pax5+/− x Ikzf1+/− and Ebf1+/− x Ikzf1+/− mice for B-ALL, or in Tcf1+/− x Ikzf1+/− mice for T cell leukemia. To identify genetic defects that cooperate with Pax5 and Ebf1 compound heterozygosity to initiate leukemia, we carried out a Sleeping Beauty transposon screen. This screen identified a number of cooperating partners including gain-of-function mutations in Stat5 (~65%) and Jak1(~68%), as well as loss-of-function mutations in Cblb (61%)and Myb (32%). These findings highlight the key role of JAK/STAT5 signaling in cooperating with Pax5 and Ebf1 compound haploinsufficiency to drive B cell transformation. Moreover, these studies pointed to unexpected roles for loss of function mutations in CBL-b and MYB in B cell transformation. Subsequent RNA-Seq studies on WT, Pax5+/−, Ebf1+/−, Pax5+/− x Ebf1+/− pre-leukemic, Pax5+/− x Ebf1+/− leukemic cells and Pax5+/− x Ebf1+/− Sleeping Beauty leukemic cells demonstrated upregulation of a PDK1>PLK1>MYC pathway; treatment of Pax5+/− x Ebf1+/− leukemias with PDK1 specific inhibitors blocked their proliferation in vitro. Finally, we identified conserved transcriptional variation in a subset of genes between human leukemias and our mouse B-ALL models. Thus, compound haploinsufficiency for B cell transcription factors likely plays a critical role in transformation of human B cells and suggest that newly developed PDK1 inhibitors may be effective for treating patients characterized by such defects.
Multivariate Gene Expression Analysis Identifies Novel Signatures and Therapeutic Targets for CLL Relapse
Authors: Griffen Tiara L , Dammer Eric D , Dill Courtney D , Carey Kaylin M , Young Corey D , Nunez Sha’Kayla K , Lillard James W
Published: January 2023

Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of immature B cells that is characterized by episodes of relapse. CLL relapse is supported by pro-survival factors, such as chemokines, secreted CLL cells of the CLL microenvironment. The CXCL13-CXCR5 axis has been previously shown to contribute to the progression of several malignancies and possibly CLL relapse. CXCR5 is highly expressed by CLL cells and CXCL13 serum levels were found to be significantly higher in CLL patients versus healthy donors. There is a gap in the understanding of the molecular mechanisms following CXCL13-CXCR5 interactions during CLL. The presence of CXCR5, CXCL13, and associated genes were investigated by using a novel weighted gene co-expression network analysis (WGCNA) method as well as examining overrepresentation of upstream regulators and biological functions found in the transcriptomes (24,658 genes) of CLL patients (n = 203). The WGCNA-designated yellow (p = 0.003), brown (p = 0.05), purple (p < 0.001), black (p = 0.006), and salmon (p = 0.001) modules were significantly correlated with relapse. The fourth-largest, yellow co-expression module contained CXCR5 and 1,315 other genes. The biological processes associated with this module included lipid metabolism, protein catabolism, positive regulation of cell cycle arrest, and cell cycle checkpoint signal transduction. Systems pharmacology analysis revealed drugs targeting ATPases, proteasomes, and microtubules, could disrupt CLL relapse signatures, found in the yellow module. Taken together, the relapse associated gene networks have provided new insights on the complex biology of CLL and identified new targets for the treatment CLL relpase.
The role of the splenic microenvironment in Chronic Lymphocytic Leukemia
Authors: Bondada Subbarao , Collard James P. , McKenna Mary Kathryn , Noothi Sunil K. , Alhakeem Sara S. , Rivas Jacqueline R. , Broeking Shelbi
Published: January 2023

Chronic Lymphocytic Leukemia (CLL), the most prevalent adult leukemia, is characterized by the clonal expansion of CD5+ CD19+ B cells within lymphoid organs and accumulation of quiescent cells in the blood. Eμ-Tcl1 transgenic mice expressing the Tcl1 oncogene in a B cell-specific manner develop CLL and adoptive transfer of CLL cells from these mice induces CLL growth in C57BL/6 mice. These models typically cause splenomegaly as the CLL burden increases, and CLL cells localize mainly in the spleen within the first 10 days post-injection. We hypothesized that the splenic microenvironment is vital for CLL expansion. Accordingly, we found that splenectomy significantly delayed and decreased CD5+ CD19+ CLL cell burden. The role of the splenic microenvironment was further explored by isolating splenic stromal cells from Eμ-Tcl1 mice and establishing stable cell lines (EMST cells). In cell culture, CLL cells divide poorly and remain mostly quiescent. The EMST cells induced proliferation in a majority of mouse and human CLL cells without the addition of any cytokines or growth factors commonly used to accomplish in vitro proliferation of CLL cells. They do not express CD40L, an important stimulatory molecule for B cell proliferation. EMST cells lack conventional immune cell markers, but they express the cytokines and chemokines BAFF, CCL2, CXCL12, and Wnt4. CCL2 levels dramatically increased upon co-culture with CLL. EMST cells also enhance IL-10 secretion by the CLL cells and thus may enhance an immunosuppressive environment to prevent anti-CLL immunity from developing. Our studies support the hypothesis that the spleen is vital for the establishment of CLL and EMST cells produce specific factors that may play a role in CLL growth.
Elucidating nurse-like cell development as a new therapeutic strategy for chronic lymphocytic leukemia
Authors: Reyes Giovanna Merchand , Santhanam Ramasamy , Weigel Christoph , Woyach Jennifer , Tridandapani Susheela , Oakes Christopher , Butchar Jonathan P
Published: January 2023

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, with the NIH estimating 20,720 new cases and 3,930 deaths in 2019. Despite advances in treatment, some patients still relapse (eg. up to 10 to 18% of ibrutinib-treated patients). CLL cells depend on interactions with supportive cells for survival, and nurse-like cells (NLCs) are the major such cell type. However, little is known about how NLCs develop. Thus, our objective is to elucidate NLC development for the purpose of identifying novel therapeutic targets that may aid in the treatment of CLL.

We examined DNA methylation patterns of NLCs and found unusually strong MAPK and AP-1 transcription factor signatures. qPCR showed upregulation of MAF, MAFF and JUN in NLCs, supporting this epigenetics analysis. To test the role of the high MAPK signature we treated NLC cultures with inhibitors of Mek, p38 or Jnk. Results showed that Mek inhibition dramatically reduced NLC development. Of note, we did not detect phosphorylated Erk in CLL cells within treated or control cultures, suggesting that the Mek inhibitor acted directly against the CD14+-derived cells. We then tested Mek inhibition in vivo with the Eμ-TCL1 mouse model of CLL and found that it led to significantly longer survival time and lower white-blood-cell counts.

In conclusion, we found that NLC development is dependent on Mek signaling and that Mek inhibition extends survival in vivo. This suggests that targeting the Mek/Erk pathway may be an effective treatment strategy for CLL.
Regulation of macrophages in tumor microenvironment by microRNA in T cell lymphoma-bearing mice
Authors: Abdulla Osama Azeldeen , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

The tumor microenvironment is a complex niche created by cancer cells to survive and evade the immune system. Macrophages (Mϕs) are considered to be one of the most abundant cells in the cancer microenvironment. Studies have shown that cancer cells have predilection for the M2 cell phenotype compared to M1 in the microenvironment, because M2 Mϕs promote immunosuppression and subsequently enhance tumor cell proliferation and angiogenesis. The aim of this study was to compare the gene expression profile of tumor-associated Mϕs (TAMs) and splenic Mϕs from tumor-bearing hosts (TBHs) and determine the epigenetic regulation of altered genes. For this purpose, C57BL/6 mice were injected with 1×106 EL4 cancer cells subcutaneous to induce tumor growth and 10 days later, TAMs and splenic Mϕs were isolated. Cell phenotyping by flow cytometry showed that there was an increase in M2 cells in TAMs when compared to splenic Mϕs in TBHs. TAMs also exhibited increase in IL-10, IL-6, VEGFA, and ARG-1 whereas there was a decrease in the expression of MYD88, PTEN, TGFβ3 and P53 when compared to splenic Mϕs in TBHs. Analysis of the microRNA profile of the Mϕs showed differential regulation and using ingenuity pathway analysis (IPA), we found various miRs (miR-21a, miR-30a, miR-30c, miR-125b, miR-155) were upregulated. These miRs targeted MYD88, PTEN and P53, which leads to the overexpression of IL10, known to skew Mϕ polarization to an M2 phenotype. In summary, these studies demonstrate that several miRs regulate IL10 to polarize TAMs from an M1 to M2 phenotype in the tumor microenvironment. (Supported by NIH P01AT003961, P20GM103641, R01AI129788, R01 ES030144 and R01AI123947)
Altering the dendritic cell signature in the lung: a new adjuvant to immune checkpoint inhibitor therapies?
Authors: Brassard Julyanne , Bernatchez Emilie , Gill Meredith Elizabeth , Joubert Philippe , Blanchet Marie-Renée
Published: January 2023

Lung cancer is the leading cause of cancer-related death. While the recent introduction of immune checkpoint immunotherapies significantly improves patient outcome, many do not respond to this treatment. Dendritic cells (DCs) activate CD8+ T cells to promote the anticancer immune response. Also, in mice, pulmonary CD103+ DC1s specialize in tumour antigen presentation. However, cancer induces an immunosuppressive microenvironment that alters immune function to promote tumor development. Yet, the impact of lung tumor development on DCs remains misunderstood. To study the impact of lung tumour development on the lung dendritic cell signature, Lewis lung carcinoma (LLC) and B16F10 cells (melanoma lung metastasis) were injected intravenously, and lung DC populations analysed by flow cytometry. We observed that in cancer, the proportions of CD103+ DC1 are largely reduced, while an uncharacterized lung CD103+CD11b+ DC population is induced. The latest express surface markers and transcription factors associated with the DC2 population and high levels of PD-L1 and PD-L2 regulatory molecules. This is of crucial importance to immune checkpoint inhibitor therapies, as they rely on the efficient presentation of tumour antigen by DCs to induce T cell responses. In order to promote the anti-tumor capacity of the DC signature, CD103+ DC1s were injected in combination with an immune checkpoint inhibitor (anti-PD-1) in the B16F10 model of resistance to anti-PD1 therapy. The co-injection led to improved sensitivity to immunotherapy. Thus, promoting the replenishment of an anticancer DC environment could be an interesting therapeutic avenue to increase the efficiency of existing immune checkpoint inhibitor therapies.
Depletion of tumor-associated macrophages by melittin-dKLA enhances anti PD-L1 mediated anti-tumor effects in breast cancer models
Authors: LEE HYUNJI , KIM SO YOUNG , CHOI ILSEOB
Published: January 2023

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment and known to have an M2-like pro-metastatic phenotype. M2-like TAMs directly support migration, adhesion and invasion of tumor cells. Therefore, M2-like TAMs are promising targets for inhibiting cancer. Anti-PD-1/PD-L1 immunotherapies have been used for an inhibition of PD-1/PD-L1 mediated T cell exhaustion in various cancers such as lymphoma, melanoma, and renal cancer with remarkable clinical success, however, most breast cancer patients have been reported to show resistances or short response durations. A lot of researches have been reported that targeting TAMs from tumor microenvironment synergized the effect of PD-1/PD-L1 therapies, suggesting that TAMs are major causes of PD-1/PD-L1 resistance. Previously, I have developed MEL-dKLA, a hybrid peptide that was conjugated with melittin (MEL) and pro-apoptotic peptide dKLA to eliminate M2 like TAM. In this study, I investigated whether removal of M2-like TAMs by MEL-dKLA enhanced the anti-cancer effect of PD-L1 antibodies in 4T1 breast cancer. Combination therapy with MEL-dKLA and anti PD-L1 delays tumor growth and increases survival. It inhibits the exhausted CD8+ T cell population in tumors, enabling them to perform cytotoxic functions. Simultaneous treatment with MEL-dKLA and anti PD-L1 reduced the contact area between TAM and CD8+ T cells in the tumor microenvironment. Therefore, these results suggested that MEL-dKLA and anti PD-L1 combination therapy will be promising for breast cancer treatments.
Targeting AXL-JAK1 in tumor and immune ecosystem of lung cancer
Authors: Taverna Josephine Amalia , Hung Chia-Nung , Dearmond Daniel , Chen Meizhen , Lin Chun-Lin , Osmulski Pawel , Gaczynska Maria , Wang Chiou-Miin , Lucio Nicholas Daniel , Chou Chih-Wei , Chen Chun-Liang , Bearss David , Warner Steve , Whatcott Clifford , Mouritsen Lars , Wade Mark , Weitman Steven , Mesa Ruben , Kirma Nameer , Huang Tim
Published: January 2023

Intercellular crosstalk between lung adenocarcinoma cells (LACs) and tumor-associated macrophages (TAMs) has been implicated in promoting cancer metastasis; however, how oncogenic signaling networks corroborate in this tumorigenic process remains unexplored. Our findings suggest that coordinated activation of AXL and JAK1-pSTAT3 signaling engenders a vicious cycle of dependence between LACs and TAMs for tumor progression. AXL-overexpressing LACs polarize TAMs to an M2-like phenotype in a JAK1-STAT3-dependent manner, facilitating the development of a proinflammatory tumor microenvironment. Conversely, M2-like TAMs release Gas6 ligand to enhance AXL-mediated cancer stemness and epithelial-to-mesenchymal transition (EMT) in LACs. Cytometry by time-of-flight (CyTOF) is a single-cell detection technology that allows for measurement of multiple protein markers in diverse cell subpopulations. We designed a 34-antibody CyTOF panel to identify immune cells, stromal cells, epithelial cells, oncogenic signaling, stemness and EMT components for LAC tumor profiling. CyTOF profiling of ~642,241 single cells detected 55 subpopulations among LAC cell lines (n=4 with co-culture conditions) and lung tumors (n=12), which were further differentiated based on their cell types (LACs, TAMs, stromal cells, granulocytes, natural killer, B and T lymphocytes). High AXL expression was noticed in tumor cell subpopulation while high pSTAT3 expression was detected in the TAM subpopulation. Moreover, pSTAT3 expression levels were increased in U937 differentiated macrophage after co-culture with A549 cells. This novel platform may facilitate the optimal selection of lung cancer patients for dual targeting of AXL and JAK1-pSTAT3.
LncRNA AK036396/FcnB regulating polymorphonuclear myeloid-derived suppressor cells in tumor bearing mice
Authors: Wang Shengjun , Tian Xinyu , Wang Tingting , Yin Kai , Zhu Dongwei , Ma Jie , Xu Huaxi
Published: January 2023

Myeloid-derived suppressor cells (MDSCs), which are constituted of immature myeloid cells, represent a population of heterogeneous cells derived from bone marrow. Activated and expanded MDSCs suppress the antitumor immune response by directly inhibiting T cell by high levels of Arg1, reactive oxygen species, and inducible nitric oxide synthase. Long non-coding RNAs (lncRNAs) are transcripts with lengths >200 nts and without the capacity to encode proteins. With the development of RNA sequencing, lncRNAs that were previously considered “transcriptional noise” have been confirmed to be involved in regulating cellular biology. However, the role of lncRNAs in the development and function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) remains unclear. Here, we identified that the lncRNA AK036396 was highly expressed in PMN-MDSCs, and lncRNA AK036396 knockdown promoted the maturation and decreased the suppressive function of PMN-MDSCs. Ficolin B (Fcnb), whose expression could be used to reflect PMN-MDSC development, was the predicted target gene of lncRNA AK036396 based on microarray results. LncRNA AK036396 knockdown attenuated Fcnb protein stability in a manner dependent on the ubiquitin-proteasome system. Moreover, Fcnb inhibition downregulated the suppressive function of PMN-MDSCs. In addition, the expression of human M-ficolin, which is an orthologue of mouse Fcnb, was increased and positively correlated with arginase1 (Arg1) level in lung cancer patients. Based on these findings, lncRNA AK036396 can inhibit maturation and accelerate immunosuppression of PMN-MDSCs by enhancing Fcnb protein stability, which will be helpful for developing antitumor therapies targeting PMN-MDSCs.
IRF4 overexpression in therapeutic anti-tumor CD8 T cells inhibits tumor growth by driving functional state transition of therapeutic and endogenous CD8 T cells in tumors
Authors: yan hui , Zhang Hedong , Zou Dawei , Fu Jinfei , Zhang Xiaolong , Xiao Xiang , Guo Yang , Wang Guohua , Yu Anze , Li Xian C. , Chen Wenhao
Published: January 2023

Objective

We discovered that IRF4 inhibits CD4 T cell dysfunction after transplantation (Immunity 47:1114). Here we study the role of IRF4 in regulating anti-tumor CD8 T cell response.

Methods

In model 1, different tumor cell lines were injected s.c. into mice with T cell-specific IRF4 deletion or control mice. In model 2, B16F10 melanoma-reactive Pmel-1 CD8 T cells were transduced with IRF4-GFP or GFP-control, and injected i.v. into congenic mice at 3 days post-s.c. injection of B16F10 cells. On day 14, T cell states in tumors were defined by single cell RNA-seq and flow cytometric analyses.

Results

IRF4 deletion in T cells significantly accelerated the progression of syngeneic TRAMP-C1 prostate cancer and B16F10 melanoma, and even enabled the growth of allogeneic CT26 Balb/c tumors. Adoptive transfer of IRF4-GFP transduced Pmel-1 T cells was significantly more potent than GFP-control transuded Pmel-1 cells in inhibiting B16F10 melanoma growth. In the GFP-control cell-transfer group, most Pmel-1 and endogenous CD8 T cells in tumors remained naïve-like or transitory states (expressed TCF1 but not effector molecules). In the IRF4-GFP cell-transfer group, more than half of Pmel-1 and endogenous CD8 T cells in tumors successfully developed into effector T cells that expressed multiple effector molecules.

Conclusions

IRF4 in T cells governs anti-tumor immunity. IRF4 overexpression in therapeutic T cells improves their anti-tumor potency.
Blockade of CD80 interaction with PD-L1 between T and tumor cells augments effector T expansion and tumor immunity in a PD-1-dependent manner
Authors: ZHANG YUANKUN , Cassady Kaniel , Lee Michael , Nasri Ubaydah , Riggs Arthur D. , Feng Ru , Zeng Defu
Published: January 2023

PD-L1 interactions with PD-1 and CD80 play a critical role in regulating tumor immunity. Blockade of PD-L1 interaction with PD-1 between tumor and T cells augments tumor immunity. Cis-PD-L1/CD80 interactions reduce PD-L1 interaction with PD-1 on T cells and augment tumor immunity. However, whether trans-PD-L1 interaction with CD80 between tumor and T cells play a role in regulating tumor immunity remains unknown. In the current studies, using B16 and MC38 tumor lines in C57BL/6 mice with or without PD-1 or PD-L1 deficiency, we show that administration of anti-PD-L1 (43H12), a monoclonal antibody that specifically blocks PD-L1 interaction with CD80, augments tumor immunity. The effect is tumor PD-L1 interaction with PD-1 on T cells-dependent, because although 43H12 blockade does not augment tumor immunity in PD-1−/− mice or in PD-L1−/− mice with PD-L1 KO tumor lines, 43H12 blockade does augment tumor immunity in PD-L1−/− mice with wild-type tumor lines. Furthermore, consistent with CD28 requirement for augmenting anti-tumor immunity during blocking anti-PD-1 treatment, 43H12 blockade of PD-L1/CD80 interaction does not augment tumor immunity in CD28−/− mice. Finally, 43H12 blockade augments CD8+ T effector expansion in the tumor draining lymph nodes, with increased production of IFN-g and down-regulation of ps21-EZH2. These results indicate that blockade of PD-L1 interaction with CD80 can augment tumor immunity via down-regulating the effect of PD-L1 interaction with PD-1. (This work is supported by DMRI institutional funds and NIH 1R01CA228465-01 to Zeng).
DNA Hypomethylation of PDCD1 Gene in Korean Colorectal Cancer Patients
Authors: Lee YeongUn , Lee Ji-Yeon , Dho So-Hee , Cho Min-Jeong , Kim Jeong-Min , Kim Lark-Kyun
Published: January 2023

Colorectal cancer (CRC) is one of the most common and critical cancers, hence considerable effort has been focused on the identification of prognostic, diagnostic markers and therapeutic targets for CRC. Meanwhile, CRC is extensively known to be related to DNA methylation.

PD-1, also known as CD279, is a cell surface protein encoded by PDCD1 gene which acts as the immune checkpoint to terminate pro-inflammatory response of T cells at the right time. Recently, PD-1 has been reported to be aberrantly overexpressed in multiple cancers, hampering the anti-tumor function of T cells. Monoclonal antibodies (mAb) which target PD-1 have been used as cancer therapy, but there has been limited success with the use of immune checkpoint inhibitors.

We investigated the DNA methylation status of normal and tumor tissues of Korean CRC patients by performing the targeted bisulfite sequencing and identified that the promoter CpG island of PDCD1 is significantly hypomethylated in CRC tissue compared to its paired adjacent normal tissue. We have hypothesized the hypomethylation of promoter CpG island of PDCD1 is associated with aberrant expression of PD-1 and promotes immune evasion, eventually, development of CRC. Additionally, we are currently checking the possibility that hypomethylation of promoter CpG island of PDCD1 hinders the therapeutic effects of PD-1 monoclonal antibodies in CRC patients.
Detailing the mechanism of CDK5-mediated PD-L1 regulation in Medulloblastoma
Authors: abiff muta , Kingsley Daniel , Rauhe Peter , Benson Bryan , Petrosiute Agne , Huang Alex Yee-Chen
Published: January 2023

Novel therapies for the pediatric brain tumor medulloblastoma (MB) are hindered by limited knowledge of the roles played by host-derived cells at the local tumor site. Immune resistance in a murine model of MB (MM1) is associated with enhanced tumor expression of programmed death ligand-1 (PD-L1) in response to anti-tumor cytokine interferon-gamma (IFNγ) signaling. Disrupting this pathway via knockdown of cyclin dependent kinase 5 (CDK5), an essential transducer of the IFNγ signal, leads to an inflammatory tumor microenvironment (TME) and enhanced tumor rejection in vivo. In this study, we sought to detail the mechanism of CDK5-mediated PD-L1 regulation.

In response to IFNγ, PD-L1 expression is thought to be regulated at the promoter level by competition between the transcriptional activator IRF1 and repressor IRF2. We hypothesized that CDK5-IFNγ signaling inhibits IRF2 activity, resulting in unleashed PD-L1 promoter activity. We assessed IRF1/IRF2-DNA interactions using chromatin immunoprecipitation (ChIP) and gene reporter assays, with complementary analysis of PD-L1 transcription and surface expression with IRF1/IRF2 depletion.

ChIP-qPCR revealed a similar, IFNγ-induced enrichment of both IRF1 and IRF2 at the PD-L1 promoter. This is a surprising result, given the assumption that they compete for the same binding site and exert opposing effects. Additionally, inducible PD-L1 promoter activity was found to be decreased in tumors with IRF2 depletion. Together, these results suggest that IRF2 may be necessary for full activity of the PD-L1 promoter in MB. IRF1/IRF2 also bind to other cis-regulatory sites at genes that contribute to tumor immunogenicity, necessitating genome-wide characterization of their interactions.
Targeting TNFR2 to overcome acquired adaptive resistance to immune checkpoint blockade
Authors: Mayer Lena Sophie , Orlowski Robert J. , Giles Josephine , Benci Joseph L. , Ellis Gavin , Deng Guoping , Attanasio John , Chen Zeyu , Bengsch Bertram , Kahn Omar , Manne Sasikanth , Herati Ramin S. , Ngiow Shin , George Sangeeth M. , Faustman Denise L. , Gilliland Gary , Mick Rosemarie , Xu Wei , McGettigan Suzanne , Xu Xiaowei , Amaravadi Ravi K. , Karakousis Giorgos C. , Schuchter Lynn M. , Mitchell Tara C. , Riley James L. , Huang Alexander C. , Minn Andy , Tomov Vesselin , Wherry E. John
Published: January 2023

Overcoming acquired adaptive immune resistance to anti-PD-1 therapy is imperative for enhancing the efficacy of immune checkpoint blockade (ICB) in solid tumors. Regulatory T cells (Tregs) play a prominent role in the suppressive tumor microenvironment (TME) and are major contributors to adaptive immune resistance. Tregs limit CD8+ T cell reinvigoration and are a promising target for combination therapy. While the clinical efficacy of anti-CTLA4 may be partially explained by restriction of Tregs, its co-administration with anti-PD1 causes significant toxicity. Thus, safer approaches to limit Treg activity are needed. To elucidate the dynamic changes in immuno-regulatory circuits within the TME during ICB, we performed deep immune profiling of peripheral blood and tumors from patients with advanced melanoma prior to (n=7) and after 1 cycle of anti-PD-1 therapy with pembrolizumab (n=9). Tregs were abundant in the TME and retained their immunosuppressive phenotype and functionality following anti-PD-1. Epigenetic, transcriptomic, and proteomic analysis of Tregs after ICB identified tumor necrosis factor receptor 2 (TNFR2) signaling as a possible driver of CD8+ T cell suppression. TNFR2 was preferentially expressed by Tregs in the TME (mean 18.03 %, SD +/− 10.13 %) relative to CD8+ T cells (mean 0.64 %, SD +/− 0.82 %) and peripheral Tregs (mean 3.16 %, SD +/− 3.21 %), suggesting it might be a safe and effective target for combination therapy. Indeed, dual blockade of TNFR2 and PD-1 led to potent CD8+ T cell expansion in two mouse tumor models, and restored sensitivity to ICB in a resistant murine model of melanoma. Our data suggest that anti-TNFR2 might synergize with current ICB by countering the development of adaptive immune resistance.
Tumor-specific CD8 T cell activation in draining lymph nodes supports the anti-tumor CD8 T cell response
Authors: Prokhnevska Nataliya , Cardenas Maria , Jansen Caroline , Valanparambil Rajesh , Master Viraj , Sanda Martin , Kissick Haydn
Published: January 2023

CD8 T cells are a critical part of the immune response to tumors, with CD8 T cell infiltration predicting disease progression in many types of cancer. Recent work in CD8 T cell immunology described how CD8 T cells respond to chronic diseases, finding two subsets of CD8 T cells within tumors. One is a stem-like CD8 T cell and the other is a terminally differentiated CD8 T cell with cytotoxic capabilities. Determining how tumor-specific CD8 T cells activate and differentiate is critical to understanding why some tumors are highly infiltrated. To study how tumor-specific CD8 T cells are activated, I have made a prostate cancer model that expresses the viral LCMV glycoprotein (GP) which acts as a tumor-specific antigen. We have used this model to study tumor-specific CD8 T cell activation by adoptively transferring LCMV GP specific TCR transgenic P14 CD8 T cells into TRAMPC1-LCMV-GP bearing mice. We have found when tumor-specific CD8 T cells are activated in the tumor-draining lymph node they acquire an undifferentiated but activated program, upregulating CD44, PD1 but retaining high TCF1 and CD62L expression. These undifferentiated activated CD8 T cells do not acquire a typical effector program that is seen in an acute viral infection such as LCMV Armstrong, yet they are able to migrate to the tumor to establish the anti-tumor response. In conclusion, tumor-specific CD8 T cells do not acquire an effector program after activation and instead gain an undifferentiated activated phenotype. These data suggest that tumor-specific CD8 T cells are activated in the TDLN and differentiate to become the stem-like CD8 T cells within the tumor, establishing the anti-tumor CD8 response.
Effect of ontogeny and vaccination on plasma adenosine deaminase (ADA)-1 and -2 levels across the first week of human life
Authors: Smolen Kinga , Plotkin Alec , Pak Jensen , Odumade Oludare , Idoko Olubukola , Pettengill Matthew , Ozonoff Al , Kollmann Tobias , Kampmann Beate , Levy Ofer
Published: January 2023

Introduction

Adenosine is a purine metabolite that acts on its leukocyte receptors to inhibit inflammatory and Th1-polarizing cytokine production. Newborns exhibit lower plasma levels of adenosine deaminase (ADA), an enzyme that metabolizes extracellular adenosine, resulting in elevated plasma adenosine levels and a Th2/anti-inflammatory bias. Humans express two ADA isoforms: ADA1 and ADA2. Characterizing the impact of ontogeny and vaccination on levels of ADA1 and ADA2 may provide insight into mechanisms regulating early life immune development.

Methods

Newborns were recruited at the MRC Unit-Gambia and randomized to either Hepatitis B (HepB), BCG, or the combination of both vaccines. Plasma was collected at birth (day of life (DOL) 0) and at DOL 1, 3, or 7. Plasma levels of ADA1 and ADA2 were measured by a chromogenic assay. Log-transformed ADA concentrations were compared using ANOVA.

Results

In this interim analysis, we measured n = 51–53 participants per vaccine group per timepoint. ADA2 activity ex vivo in plasma derived from Gambian newborns increased by median fold changes of 1.6 (control), 1.6 (HepB), 1.5 (BCG), and 1.9 (HepB+BCG) over the first week of life. The changes in ADA2 for all groups were statistically significant over DOL 0. HepB and BCG immunizations were both associated with statistically significant 1.5-fold decrease in ADA1 across the first week of life.

Conclusions

Neonatal plasma ADA2 activity rises across the first week of life, suggestive of ontogeny-specific changes that promote Th2/anti-inflammatory immune status. Immunization at birth with HepB or BCG may be associated with lower ADA1 across the first week of life. ADA isoforms may be useful biomarkers of immune development and vaccine responses.
Programming effective respiratory immunity to influenza with combination microparticle adjuvants
Authors: Bakke Brock , Marinaik Chandranaik , Toy Randall , Larsen Autumn , Lee Woojong , Kawaoka Yoshihiro , Roy Krishnendu , Suresh Marulasiddappa
Published: January 2023

T cell-mediated immunity (CMI) can provide long-lived protection following parenteral immunization, but eliciting durable and protective CMI in the mucosa remains a significant challenge. Here, using polylactic-co-glycolic acid (PLGA)-based cationic pathogen-like particles (PLPs) for delivery of TLR agonists, we have investigated novel ways of presenting vaccine components to the immune system to simulate the potent innate immune stimulating effects of pathogens. We generated micro particle sized PLPs loaded with TLR4 (glucopyranosyl lipid adjuvant, GLA) and/or TLR9 (CpG) agonists, and formulated them with and without a mucosal delivery enhancing carbomer-based nano-emulsion adjuvant (ADJ). These adjuvants delivered intranasally to mice safely elicited robust and durable influenza nucleoprotein (NP)-specific CD8+/CD4+ effector and tissue-resident memory responses in lungs and airways. PLPs delivering TLR4 versus TLR9 agonists drove phenotypically and functionally distinct populations of effector and memory T cells. Following influenza virus challenge, functional and phenotypic attributes of effector T cells unique to the individual PLP formulation were associated with disparate levels of viral control in the respiratory tract. These studies demonstrate: (1) how adjuvant formulations affect effector/memory T cell differentiation in the lungs; (2) feasibility of using PLGA-based PLPs in vaccine formulations to elicit protective T cell immunity in the respiratory tract.
Development of alternative vaccination strategies to overcome Tc1 vaccine failure caused by chronic schistosomiasis
Authors: Shollenberger Lisa M , Norwood Stephanie K , Bement Zachary J
Published: January 2023

It is a global public health imperative to develop vaccine strategies that produce effective cell-mediated immunity in schistosome-endemic areas, such as sub-Saharan Africa, where HIV infection is common. Vaccine delivery techniques effective in immune-suppressed populations are critical to combating infectious diseases in these already compromised groups. It is well-known that immune suppression, such as by chronic schistosome infection, can cause Tc1/Th1 type vaccines to fail. Listeria, a strong inducer of cell-mediated immunity, can overcome the Th2 biasing and immune suppression caused by schistosome infection. However, safety concerns cause reluctance among the vaccine community and the public to incorporate this vector into immunization programs. Since neither DNA nor the Listeria vaccine vector are ideal for clinical translation, and Gag is likely not the ideal HIV vaccine antigen, we are using this system as a model for enumeration of Tc1 responses (IFNγ+ CD8+ cytotoxic T lymphocytes, CTL); CTLs are likely necessary components of functional responses for next-generation vaccines targeting diseases that currently lack effective vaccines (HIV, TB, malaria). Here, we investigate why DNA vaccine vectors fail during chronic schistosomiasis when Listeria vaccine vectors can generate robust CTL responses to HIV Gag. Specifically, we are examining live bacteria as adjuvants for an episomally expressed or concurrently administered DNA vaccine for HIV Gag. This project is one step toward our overall goal, which is to generate cell-mediated vaccine immunity in schistosome-infected, immune-suppressed patients.
An Ebola virus vaccine that leverages helper T cells induced by prior BCG immunization
Authors: Ng Tony W , Wirchnianski Ariel A , Chan John , Jacobs William R , Chandran Kartik , Marzi Andrea , Porcelli Steven A
Published: January 2023

Protective antibody responses against the Ebola virus (EBOV) are dependent on the quality of CD4+ helper T (Th) cells. We propose that BCG specific Th cells may provide a source of Th cells that can be recruited to drive antibody responses against EBOV, given that the majority of individuals residing in EBOV endemic regions have been previously vaccinated with BCG. To take advantage of pre-existing BCG Th cells, a vaccine was developed that consists of the EBOV GP fused to the mycobacteria Th cell epitope (P25) of Ag85B (P25-EBOV GP). Presentation of the P25 epitope by B cells recruited cognate interactions with P25 Th cells to promote antibody responses against the EBOV GP. Experiments in mice showed BCG priming induced a broad range of Th cells including Th1, Th2, and Tfh subsets. These P25 Th subsets provided a significant dose sparing effect on the P25-EBOV GP vaccine in the induction of EBOV GP antibodies and resulted in increased titers of high affinity neutralizing IgG1 antibodies. The induction of IgG2c antibodies only occurred in BCG primed mice, where BCG vaccination is known to be a strong inducer of Th1 responses that drive class-switching to IgG2c. Microscopy of sections from secondary lymphoid organs of BCG primed mice showed that the majority of P25 Th cells remained in extrafollicular regions outside germinal centers and were most responsible for the induction of the IgG2c class-switched antibodies that were weakly neutralizing but capable of mediating antibody-dependent cellular toxicity (ADCC). BCG priming followed by vaccination with P25-EBOV GP protected mice against lethal EBOV infection, thus suggesting a potentially useful strategy for vaccination against EBOV in populations with high prevalence of previous BCG vaccination.
Structural and functional analysis of cooperativity in a potent human antibody cocktail against Ebola virus
Authors: Gilchuk Pavlo , Murin Charles D , Milligan Jacob C , Cross Robert W , Mire Chad E , Ilinykh Philipp A , Huang Kai , Kuzmina Natalia , Altman Pilar X , Hui Sean , Gunn Bronwyn M , Bryan Aubrey L , Davidson Edgar , Doranz Benjamin J , Turner Hannah L , Alkutkar Tanwee , Flinko Robin , Orlandi Chiara , Carnahan Robert , Nargi Rachel , Bombardi Robin G , Vodzak Megan E , Li Sheng , Okoli Adaora , Ibeawuchi Morris , Ohiaeri Benjamin , Lewis George K , Alter Galit , Bukreyev Alexander , Saphire Erica Ollmann , Geisbert Thomas W , Ward Andrew B , Crowe James E
Published: January 2023

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus (EBOV). Systematic analysis of antibody repertoire in the human survivors identified a pair of potently neutralizing mAbs that cooperatively bind to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb/GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. MAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated high levels of effectiveness against EBOV in nonhuman primates. These data illuminate structural principles of antibody cooperativity with implications for the design and development of antiviral immunotherapeutics.
Dendritic cell targeting anti-CD40 antibody-CD40L-HIV-1 vaccines are adjuvant intrinsic
Authors: Ceglia Valentina , Zurawski Sandy , Montes Monica , Bouteau Aurelie , Wang Zhiqing , Ellis Jerome , Kroll Mitchell , Igyarto Botond Z , Levy Yves , Zurawski Gerard
Published: January 2023

Soluble CD40L and agonistic anti-CD40 monoclonal antibody are adjuvants used in vaccination settings. Vaccines based on anti-CD40 antibodies fused to HIV-1 antigens are in clinical development. Studies with current anti-CD40-based dendritic cell (DC) targeting vaccines show that co-administration of an adjuvant is needed for maximal immune responses. We show that by fusing CD40L to CD40-targeting antibodies, activation of DCs concomitant with antigen uptake and processing is maximized, and this provides a context CD40-targeting vaccines with intrinsic adjuvant activity. Direct fusion of CD40L to L or H chain C-termini results in CD40 agonists with ‘superagonist’ properties. Especially on DCs, both potency and efficacy for induction of cytokine secretion and activation markers is greatly enhanced compared to known strong agonists like Pfizer’s anti-CD40 CP-870-89 antibody. This potency was maintained by anti-CD40-CD40L constructs fused to HIV-1 antigens from Gag, Nef, and Pol regions (HIV5pep). Anti-CD40-CD40L-HIV5pep preferentially expanded CD8+ T cells from HIV-1+ donor PBMCs compared to the same antibody-antigen fusions without attached CD40L. Anti-CD40-CD40L-TEα and anti-CD40-TEα both evoked robust proliferation of TEα-specific CD4+ T cells in human CD40 transgenic mice, but only anti-CD40-CD40L-TEα vaccine elicited TEα-specific CD4+ T cells producing IFNγ. Also, anti-CD40-CD40L-Env gp140 vaccine without adjuvant in human CD40 transgenic mice elicited stronger anti-Env gp140 antibody responses than anti-CD40-Env gp140 vaccine. Thus superagonist anti-CD40 antibodies directly fused to the natural ligand show great advantage in inducing immune responses without the use of an extrinsic adjuvant.
Characterization of a protective antibody against dengue virus non-structural protein 1 (NS1) reveals critical domains required for NS1-triggered pathogenesis
Authors: Wong Marcus P , Biering Scott B , Akey David L , Lo Nicholas TN , Brown W. Clay , de Sousa Francielle Tramontini Gomes , Puerta-Guardo Henry , Wang Chunling , Konwerski Jamie , Espinosa Diego , Li Jeffery , Blanc Sophie , Beatty P. Robert , Smith Janet L , Harris Eva
Published: January 2023

Dengue virus (DENV) is a medically important, mosquito-borne flavivirus that infects up to 390 million people annually. A signature of severe DENV infection is vascular leak, which can lead to shock and organ failure. We and others have shown that secreted DENV non-structural protein 1 (NS1) triggers endothelial cell (EC) hyperpermeability and vascular leak. Antibodies (Abs) targeting NS1 prevent NS1-induced EC hyperpermeability in vitro and are protective against lethal DENV challenge, as shown by vaccination of mice against NS1 and passive transfer of α-NS1 polyclonal serum. How α-NS1 Abs protect against pathogenesis, however, remains obscure. Here we characterize possible mechanisms behind the protective ability of an NS1-specific monoclonal Ab, 2B7. We show that 2B7 abrogates NS1 binding to ECs and EC hyperpermeability in vitro and inhibits vascular leak and mortality in vivo. Guided by crystal structures of NS1 complexed to a 2B7 Fab fragment, we mutated the amino acids on NS1 that comprise the NS1:2B7 interface. Intriguingly, while these contact sites were critical for the interaction between 2B7 and NS1, they were dispensable for NS1:EC binding. The structures and mutagenesis results suggest that 2B7 abrogates NS1 binding indirectly by sterically hindering the binding domain on NS1 from interacting with ECs. The mutagenesis also identified a key residue on NS1 that is necessary for inducing EC hyperpermeability without affecting NS1:EC binding, suggesting that 2B7 may also block an additional step in NS1 pathogenesis. This work provides mechanistic insight into the blockade of DENV NS1-mediated pathogenesis by α-NS1 Abs and identifies critical domains that serve as targets for drugs and vaccine-induced α-NS1 Ab responses.
Adjuvanted bacteriophage vaccine targeting Pseudomonas aeruginosa
Authors: Román-Cruz Valery Cristina , Miller Shannon , Buhl Cassandra , Schooner Roman , Walsh Lois , Secor Patrick , Burkhart David J , Evans Jay T
Published: January 2023

Pseudomonas aeruginosa (Pa) is a Gram-negative bacterium which is associated with chronic diabetic wounds as well as potentially deadly lung infections in cystic fibrosis patients. Approximately 51,000 cases of Pa infections occur annually; of these cases, 13% are resistant to antibiotics which results in the deaths of ~400 individuals every year. Diversity between Pa strains presents a challenge in developing a vaccine or therapeutic capable of resolving infections caused by different strains. Pf phage (a filamentous bacteriophage) has been found in Pa from chronic diabetic wound isolates and has been show to increase virulence of Pa. Recent findings by our team indicate that a Pf phage-targeted vaccine or monoclonal antibody provide protection from the establishment of Pa infection in mice. To further improve this innovative new vaccine targeting Pa, adjuvant systems were used to enhance humoral response to the vaccine. A consensus peptide from Pf Phage coat protein was conjugated to the carrier protein CRM197 and combined with novel adjuvants and delivery systems. Selected adjuvants significantly enhanced humoral immunity to the Pf Phage peptide in a dose-dependent manner. Combination adjuvant systems were also used to further enhance antigen-specific immunity to Pf Phage and cell-mediated immunity to the carrier protein. The development of an adjuvanted Pf phage vaccine to protect against Pa infections is a new and innovative strategy with the potential to protect patients at risk for opportunistic bacterial infections.
Vaccine-Elicited T cells are Important for Intravenous BCG Mediated Protection in a Rhesus macaque Model of Tuberculosis
Authors: Zeppa Joseph John , Darrah Patricia A , Sutton Matthew , Pokkali Supriya , Scanga Chuck A , Maiello Pauline , Lin Philana L , Roederer Mario , Seder Robert A , Flynn JoAnne L
Published: January 2023

Our laboratories have shown that intravenous (IV) vaccination with Bacille Calmette-Guerin (BCG; 5×107 CFU) provides robust protection against low-dose Mycobacterium tuberculosis (Mtb) infection in rhesus macaques (9/10 protected [<50 thoracic Mtb CFU]; 100,000-fold reduction compared to intradermal [ID] BCG). IV BCG also resulted in a 100-fold increase in mycobacterium--specific T cells in the airways of animals compared to ID or aerosol administration. To investigate the role of T cells in this protective model, we employed two methodologies: 1. Vaccination with decreasing doses of BCG to stimulate lower-magnitude T cell responses and; 2. Antibody-mediated CD4 and CD8a T cell depletion post-vaccination but pre-infection. Animals were vaccinated for 6 months followed by a 2–3 month low-dose Mtb challenge. Surprisingly, even at low doses of BCG (0.4–2 × 105), a subset of animals was protected, with a range of outcomes across the doses. In the second study, depletion was successful in blood, airways and tissue. Interestingly, depletion of either T cell subset resulted in an intermediate total thoracic CFU phenotype. In both studies, correlates of immunity will be assessed comparing protection (CFU) to BAL and tissue resident immune responses. Our data supports that sterilizing protection is observed across the BCG IV dose spectrum and that T cells are important, but are likely not the only factors involved in this protection. Our findings are critical for identifying correlates of protection and generating a safe and effective vaccine against TB.
Identifying mediators of STING-dependent TB vaccine immunity
Authors: Jong Robyn MY , Van Dis Erik , Baltodano Alexander , Nguyenla Xammy , Blackshire Bianca , Magee Kiran , Stanley Sarah
Published: January 2023

Tuberculosis (TB) is the top infectious killer in the world. The only current licensed vaccine for TB, Mycobacterium bovis Bacille Calmette-Guérin (BCG), has not proven reliably effective against adult pulmonary TB in all studies. It furthermore cannot be administered safely to immunocompromised individuals as it is a live attenuated vaccine. Protein subunit vaccines offer increased safety over live attenuated vaccines. Many recently developed protein subunit TB vaccines use toll-like receptor-stimulating adjuvants that induce potent inflammatory responses, but it is uncertain whether they can induce long-lasting memory protection against an intracellular bacterium such as TB.

Cyclic-di-nucleotides are an alternative adjuvant that target the cytosolic immunosurveillance pathway mediated by STING. Recently, a STING-activating cyclic-di-nucleotide (CDN)-adjuvanted TB protein subunit vaccine was shown to elicit an effective memory T cell response against TB challenge in mice. Intriguingly, intranasal delivery of the vaccine provided superior protection compared to subcutaneous administration of the CDN-adjuvanted vaccine or BCG. Protection induced by mucosal administration correlates with a potent Th17 response rather than the Th1-dominated CD4+ T cell response induced by subcutaneous vaccination. Protection is partially dependent on IL-17 and type I interferon, while the transcription factor STAT6 is dispensable. The Th17 response and protection both precedes Th1 influx into the lungs and also persists after the Th1 response wanes. We are currently determining whether the transcription factor IRF3 and cytokine IFNγ are also mediators of Th17 memory induction and protection against TB infection.