Effects of immune checkpoint blockade on antigen-specific CD8+ T cells for use in adoptive cellular therapy.

Adoptive T-cell therapies have been successfully used as prophylaxis or treatment for immunocompromised patients at risk of viral infections or advanced cancers. Unfortunately, for some refractory cancers, they have failed. To overcome this, checkpoint inhibitors are used to rescue immune antitumor responses. We hypothesized that in vitro checkpoint blockade during T-cell stimulation and expansion with messenger RNA (mRNA)-pulsed DCs may enhance the activity of antigen-specific T cells and improve the efficacy of adoptive cellular therapy platforms. Human peripheral blood mononuclear cells were isolated from cytomegalovirus (CMV)-seropositive donors to generate DCs. These were pulsed with CMV matrix phosphoprotein 65 (CMVpp65)-mRNA to educate T cells in coculture for 15 days. Three checkpoint blockade conditions were evaluated (anti-PD1, anti-Tim3, and anti-PD1 + Tim3). IL-2 and antibodies blockades were added every 3 days. Immunophenotyping was performed on Day 0 and Day 15. Polyfunctional antigen-specific responses were evaluated upon rechallenge with CMVpp65 peptides. CMVpp65-activated CD8+ T cells upregulate Lag3 and Tim3 (P ≤ 0.0001). Tim3 antibody blockade alone or in combination led to a significant upregulation of Lag3 expression on CD8+ pp65Tetramer+ central memory, effector memory, and terminal effector memory cells re-expressing RA (TEMRA) T cells. This latter T-cell subset uniquely maintains double-positive Tim3/Lag3 expression after checkpoint blockade. By contrast, PD1 blockade had minimal effects on Tim3 or Lag3 expression. In addition, IFN-γ secretion was reduced in T cells treated with Tim3 blockade in a dose-dependent manner (P = 0.004). In this study, we have identified a potential activating component of Tim3 and linkage between Tim3 and Lag3 signaling upon blocking the Tim3 axis during T-cell-antigen-presenting cell interactions that should be considered when targeting immune checkpoints for clinical use.

RNA-Modified T Cells Mediate Effective Delivery of Immunomodulatory Cytokines to Brain Tumors.

With the presence of the blood-brain barrier (BBB), successful immunotherapeutic drug delivery to CNS malignancies remains a challenge. Immunomodulatory agents, such as cytokines, can reprogram the intratumoral microenvironment; however, systemic cytokine delivery has limited access to the CNS. To bypass the limitations of systemically administered cytokines, we investigated if RNA-modified T cells could deliver macromolecules directly to brain tumors. The abilities of T cells to cross the BBB and mediate direct cytotoxic killing of intracranial tumors make them an attractive tool as biological carriers. Using T cell mRNA electroporation, we demonstrated that activated T cells can be modified to secrete granulocyte macrophage colony-stimulating factor (GM-CSF) protein while retaining their inherent effector functions in vitro. GM-CSF RNA-modified T cells effectively delivered GM-CSF to intracranial tumors in vivo and significantly extended overall survival in an orthotopic treatment model. Importantly, GM-CSF RNA-modified T cells demonstrated superior anti-tumor efficacy as compared to unmodified T cells alone or in combination with systemic administration of recombinant GM-CSF. Anti-tumor effects were associated with increased IFN-γ secretion locally within the tumor microenvironment and systemic antigen-specific T cell expansion. These findings demonstrate that RNA-modified T cells may serve as a versatile platform for the effective delivery of biological agents to CNS tumors.

Lin-CCR2+ hematopoietic stem and progenitor cells overcome resistance to PD-1 blockade.

Immune checkpoint blockade using anti-PD-1 monoclonal antibodies has shown considerable promise in the treatment of solid tumors, but brain tumors remain notoriously refractory to treatment. In CNS malignancies that are completely resistant to PD-1 blockade, we found that bone marrow-derived, lineage-negative hematopoietic stem and progenitor cells (HSCs) that express C-C chemokine receptor type 2 (CCR2+) reverses treatment resistance and sensitizes mice to curative immunotherapy. HSC transfer with PD-1 blockade increases T-cell frequency and activation within tumors in preclinical models of glioblastoma and medulloblastoma. CCR2+HSCs preferentially migrate to intracranial brain tumors and differentiate into antigen-presenting cells within the tumor microenvironment and cross-present tumor-derived antigens to CD8+ T cells. HSC transfer also rescues tumor resistance to adoptive cellular therapy in medulloblastoma and glioblastoma. Our studies demonstrate a novel role for CCR2+HSCs in overcoming brain tumor resistance to PD-1 checkpoint blockade and adoptive cellular therapy in multiple invasive brain tumor models.

ß-Catenin-SOX2 signaling regulates the fate of developing airway epithelium.

Wnt-ß-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. To address this question, we used a Cre recombinase (Cre)-LoxP approach to activate canonical Wnt signaling ectopically in developing lung endoderm. We found that persistent activation of canonical Wnt signaling within distal lung endoderm was permissive for normal development of alveolar epithelium, yet led to the loss of developing bronchiolar epithelium and ectasis of distal conducting airways. Activation of canonical Wnt led to ectopic expression of a lymphoid-enhancing factor and a T-cell factor (LEF and TCF, respectively) and absence of SRY (sex-determining region Y)-box 2 (SOX2) and tumor protein p63 (p63) expression in proximal derivatives. Conditional loss of SOX2 in airways phenocopied epithelial differentiation defects observed with ectopic activation of canonical Wnt. Our data suggest that Wnt negatively regulates a SOX2-dependent signaling program required for developmental progression of the bronchiolar lineage.

Molecular staging of epithelial maturation using secretory cell-specific genes as markers.

Bronchiolar Clara cells undergo phenotypic changes during development and in disease. These changes are poorly described due to a paucity of molecular markers. We used chemical and transgenic approaches to ablate Clara cells, allowing identification of their unique gene expression profile. Flavin monooxygenase 3 (Fmo3), paraoxonase 1 (Pon1), aldehyde oxidase 3 (Aox3), and claudin 10 (Cldn10) were identified as novel Clara cell markers. New and existing Clara cell marker genes were categorized into three classes based on their unique developmental expression pattern. Cldn10 was uniformly expressed in the epithelium at Embryonic Day (E)14.5 and became restricted to secretory cells at E18.5. This transition was defined by induction of CCSP. Maturation of secretory cells was associated with progressive increases in the expression of Fmo3, Pon1, Aox3, and Cyp2f2 between late embryonic and postnatal periods. Messenger RNA abundance of all categories of genes was dramatically decreased after naphthalene-induced airway injury, and displayed a sequence of temporal induction during repair that suggested sequential secretory cell maturation. We have defined a broader repertoire of Clara cell-specific genes that allows staging of epithelial maturation during development and repair.

Conditional stabilization of beta-catenin expands the pool of lung stem cells.

Maintenance of classic stem cell hierarchies is dependent upon stem cell self-renewal mediated in part by Wnt/beta-catenin regulation of the cell cycle. This function is critical in rapidly renewing tissues due to the obligate role played by the tissue stem cell. However, the stem cell hierarchy responsible for maintenance of the conducting airway epithelium is distinct from classic stem cell hierarchies. The epithelium of conducting airways is maintained by transit-amplifying cells in the steady state; rare bronchiolar stem cells are activated to participate in epithelial repair only following depletion of transit-amplifying cells. Here, we investigate how signaling through beta-catenin affects establishment and maintenance of the stem cell hierarchy within the slowly renewing epithelium of the lung. Conditional potentiation of beta-catenin signaling in the embryonic lung results in amplification of airway stem cells through attenuated differentiation rather than augmented proliferation. Our data demonstrate that the differentiation-modulating activities of stabilized beta-catenin account for expansion of tissue stem cells.

Breast cancer molecular signatures as determined by SAGE: correlation with lymph node status.

Global gene expression measured by DNA microarray platforms have been extensively used to classify breast carcinomas correlating with clinical characteristics, including outcome. We generated a breast cancer Serial Analysis of Gene Expression (SAGE) high-resolution database of approximately 2.7 million tags to perform unsupervised statistical analyses to obtain the molecular classification of breast-invasive ductal carcinomas in correlation with clinicopathologic features. Unsupervised statistical analysis by means of a random forest approach identified two main clusters of breast carcinomas, which differed in their lymph node status (P=0.01); this suggested that lymph node status leads to globally distinct expression profiles. A total of 245 (55 up-modulated and 190 down-modulated) transcripts were differentially expressed between lymph node (+) and lymph node (-) primary breast tumors (fold change, >or=2; P<0.05). Various lymph node (+) up-modulated transcripts were validated in independent sets of human breast tumors by means of real-time reverse transcription-PCR (RT-PCR). We validated significant overexpression of transcripts for HOXC10 (P=0.001), TPD52L1 (P=0.007), ZFP36L1 (P=0.011), PLINP1 (P=0.013), DCTN3 (P=0.025), DEK (P=0.031), and CSNK1D (P=0.04) in lymph node (+) breast carcinomas. Moreover, the DCTN3 (P=0.022) and RHBDD2 (P=0.002) transcripts were confirmed to be overexpressed in tumors that recurred within 6 years of follow-up by real-time RT-PCR. In addition, meta-analysis was used to compare SAGE data associated with lymph node (+) status with publicly available breast cancer DNA microarray data sets. We have generated evidence indicating that the pattern of gene expression in primary breast cancers at the time of surgical removal could discriminate those tumors with lymph node metastatic involvement using SAGE to identify specific transcripts that behave as predictors of recurrence as well.

Gene expression signature of estrogen receptor alpha status in breast cancer.

BACKGROUND: Estrogens are known to regulate the proliferation of breast cancer cells and to modify their phenotypic properties. Identification of estrogen-regulated genes in human breast tumors is an essential step toward understanding the molecular mechanisms of estrogen action in cancer. To this end we generated and compared the Serial Analysis of Gene Expression (SAGE) profiles of 26 human breast carcinomas based on their estrogen receptor alpha (ER) status. Thus, producing a breast cancer SAGE database of almost 2.5 million tags, representing over 50,000 transcripts. RESULTS: We identified 520 transcripts differentially expressed between ERalpha-positive (+) and ERalpha-negative (-) primary breast tumors (Fold change >or= 2; p < 0.05). Furthermore, we identified 220 high-affinity Estrogen Responsive Elements (EREs) distributed on the promoter regions of 163 out of the 473 up-modulated genes in ERalpha (+) breast tumors. In brief, we observed predominantly up-regulation of cell growth related genes, DNA binding and transcription factor activity related genes based on Gene Ontology (GO) biological functional annotation. GO terms over-representation analysis showed a statistically significant enrichment of various transcript families including: metal ion binding related transcripts (p = 0.011), calcium ion binding related transcripts (p = 0.033) and steroid hormone receptor activity related transcripts (p = 0.031). SAGE data associated with ERalpha status was compared with reported information from breast cancer DNA microarrays studies. A significant proportion of ERalpha associated gene expression changes was validated by this cross-platform comparison. However, our SAGE study also identified novel sets of genes as highly expressed in ERalpha (+) invasive breast tumors not previously reported. These observations were further validated in an independent set of human breast tumors by means of real time RT-PCR. CONCLUSION: The integration of the breast cancer comparative transcriptome analysis based on ERalpha status coupled to the genome-wide identification of high-affinity EREs and GO over-representation analysis, provide useful information for validation and discovery of signaling networks related to estrogen response in this malignancy.

From mice to humans: identification of commonly deregulated genes in mammary cancer via comparative SAGE studies.

Genetically engineered mouse mammary cancer models have been used over the years as systems to study human breast cancer. However, much controversy exists on the utility of such models as valid equivalents to the human cancer condition. To perform an interspecies gene expression comparative study in breast cancer we used a mouse model that most closely resembles human breast carcinogenesis. This system relies on the transplant of p53 null mouse mammary epithelial cells into the cleared mammary fat pads of syngeneic hosts. Serial analysis of gene expression (SAGE) was used to obtain gene expression profiles of normal and tumor samples from this mouse mammary cancer model (>300,000 mouse mammary-specific tags). The resulting mouse data were compared with 25 of our human breast cancer SAGE libraries (>2.5 million human breast-specific tags). We observed significant similarities in the deregulation of specific genes and gene families when comparing mouse with human breast cancer SAGE data. A total of 72 transcripts were identified as commonly deregulated in both species. We observed a systematic and significant down-regulation in all of the tumors from both species of various cytokines, including CXCL1 (GRO1), LIF, interleukin 6, and CCL2. All of the mouse and most human mammary tumors also displayed decreased expression of genes known to inhibit cell proliferation, including NFKBIA (IKBalpha), GADD45B, and CDKN1A (p21); transcription-related genes such as CEBP, JUN, JUNB, and ELF1; and apoptosis-related transcripts such as IER3 and GADD34/PPP1R15A. Examples of overexpressed transcripts in tumors from both species include proliferation-related genes such as CCND1, CKS1B, and STMN1 (oncoprotein 18); and genes related to other functions such as SEPW1, SDFR1, DNCI2, and SP110. Importantly, abnormal expression of several of these genes has not been associated previously with breast cancer. The consistency of these observations was validated in independent mouse and human mammary cancer sets. This is the first interspecies comparison of mammary cancer gene expression profiles. The comparative analysis of mouse and human SAGE mammary cancer data validates this p53 null mouse tumor model as a useful system closely resembling human breast cancer development and progression. More importantly, these studies are allowing us to identify relevant biomarkers of potential use in human studies while leading to a better understanding of specific mechanisms of human breast carcinogenesis.

Transcriptomic changes in human breast cancer progression as determined by serial analysis of gene expression.

INTRODUCTION: Genomic and transcriptomic alterations affecting key cellular processes such us cell proliferation, differentiation and genomic stability are considered crucial for the development and progression of cancer. Most invasive breast carcinomas are known to derive from precursor in situ lesions. It is proposed that major global expression abnormalities occur in the transition from normal to premalignant stages and further progression to invasive stages. Serial analysis of gene expression (SAGE) was employed to generate a comprehensive global gene expression profile of the major changes occurring during breast cancer malignant evolution. METHODS: In the present study we combined various normal and tumor SAGE libraries available in the public domain with sets of breast cancer SAGE libraries recently generated and sequenced in our laboratory. A recently developed modified t test was used to detect the genes differentially expressed. RESULTS: We accumulated a total of approximately 1.7 million breast tissue-specific SAGE tags and monitored the behavior of more than 25,157 genes during early breast carcinogenesis. We detected 52 transcripts commonly deregulated across the board when comparing normal tissue with ductal carcinoma in situ, and 149 transcripts when comparing ductal carcinoma in situ with invasive ductal carcinoma (P < 0.01). CONCLUSION: A major novelty of our study was the use of a statistical method that correctly accounts for the intra-SAGE and inter-SAGE library sources of variation. The most useful result of applying this modified t statistics beta binomial test is the identification of genes and gene families commonly deregulated across samples within each specific stage in the transition from normal to preinvasive and invasive stages of breast cancer development. Most of the gene expression abnormalities detected at the in situ stage were related to specific genes in charge of regulating the proper homeostasis between cell death and cell proliferation. The comparison of in situ lesions with fully invasive lesions, a much more heterogeneous group, clearly identified as the most importantly deregulated group of transcripts those encoding for various families of proteins in charge of extracellular matrix remodeling, invasion and cell motility functions.