Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells.

Despite the impressive rates of clinical response to programmed death 1 (PD-1) blockade in multiple cancers, the majority of patients still fail to respond to this therapy. The CT26 tumor in mice showed similar heterogeneity, with most tumors unaffected by anti-PD-1. As in humans, response of CT26 to anti-PD-1 correlated with increased T- and B-cell infiltration and IFN expression. We show that intratumoral injection of a highly interferogenic TLR9 agonist, SD-101, in anti-PD-1 nonresponders led to a complete, durable rejection of essentially all injected tumors and a majority of uninjected, distant-site tumors. Therapeutic efficacy of the combination was also observed with the TSA mammary adenocarcinoma and MCA38 colon carcinoma tumor models that show little response to PD-1 blockade alone. Intratumoral SD-101 substantially increased leukocyte infiltration and IFN-regulated gene expression, and its activity was dependent on CD8+ T cells and type I IFN signaling. Anti-PD-1 plus intratumoral SD-101 promoted infiltration of activated, proliferating CD8+ T cells and led to a synergistic increase in total and tumor antigen-specific CD8+ T cells expressing both IFN-γ and TNF-α. Additionally, PD-1 blockade could alter the CpG-mediated differentiation of tumor-specific CD8+ T cells into CD127lowKLRG1high short-lived effector cells, preferentially expanding the CD127highKLRG1low long-lived memory precursors. Tumor control and intratumoral T-cell proliferation in response to the combined treatment is independent of T-cell trafficking from secondary lymphoid organs. These findings suggest that a CpG oligonucleotide given intratumorally may increase the response of cancer patients to PD-1 blockade, increasing the quantity and the quality of tumor-specific CD8+ T cells.

Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses.

BACKGROUND: Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization. OBJECTIVE: Small-molecule imidazoquinoline Toll-like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age- and species-specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs. METHODS: Chemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist-encapsulating polymersome formulations were then synthesized. Novel 96-well in vitro assays using neonatal human monocyte-derived DCs and humanized TLR8 mouse bone marrow-derived DCs enabled benchmarking of the TLR8 agonist-encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25-loaded BCG-mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice. RESULTS: Although alum-adjuvanted vaccines induced modest costimulatory molecule expression, limited TH-polarizing cytokine production, and significant cell death, BCG induced a robust adult-like maturation profile of neonatal DCs. Remarkably, TLR8 agonist polymersomes induced not only newborn DC maturation profiles similar to those induced by BCG but also stronger IL-12p70 production. On subcutaneous injection to neonatal mice, the TLR8 agonist-adjuvanted Ag85B peptide 25 formulation was comparable with BCG in inducing Ag85B-specific CD4+ T-cell numbers. CONCLUSION: TLR8 agonist-encapsulating polymersomes hold substantial potential for early-life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early-life vaccines.

TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus.

Glucocorticoids are widely used to treat patients with autoimmune diseases such as systemic lupus erythematosus (SLE). However, regimens used to treat many such conditions cannot maintain disease control in the majority of SLE patients and more aggressive approaches such as high-dose methylprednisolone pulse therapy are used to provide transient reductions in disease activity. The primary anti-inflammatory mechanism of glucocorticoids is thought to be NF-kappaB inhibition. Recognition of self nucleic acids by toll-like receptors TLR7 and TLR9 on B cells and plasmacytoid dendritic cells (PDCs) is an important step in the pathogenesis of SLE, promoting anti-nuclear antibodies and the production of type I interferon (IFN), both correlated with the severity of disease. Following their activation by self-nucleic acid-associated immune complexes, PDCs migrate to the tissues. We demonstrate, in vitro and in vivo, that stimulation of PDCs through TLR7 and 9 can account for the reduced activity of glucocorticoids to inhibit the IFN pathway in SLE patients and in two lupus-prone mouse strains. The triggering of PDCs through TLR7 and 9 by nucleic acid-containing immune complexes or by synthetic ligands activates the NF-kappaB pathway essential for PDC survival. Glucocorticoids do not affect NF-kappaB activation in PDCs, preventing glucocorticoid induction of PDC death and the consequent reduction of systemic IFN-alpha levels. These findings unveil a new role for self nucleic acid recognition by TLRs and indicate that inhibitors of TLR7 and 9 signalling could prove to be effective corticosteroid-sparing drugs.

Blocking TLR7- and TLR9-mediated IFN-α production by plasmacytoid dendritic cells does not diminish immune activation in early SIV infection.

Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.

Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward ANCA induction and associated autoimmunity.

Antineutrophil cytoplasmic antibodies (ANCAs) target proteins normally retained within neutrophils, indicating that cell death is involved in the autoimmunity process. Still, ANCA pathogenesis remains obscure. ANCAs activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NETs), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with antimicrobial activity. NETs have been consistently detected in ANCA-associated small-vessel vasculitis, and this association prompted us to test whether the peculiar structure of NET favors neutrophil proteins uploading into myeloid dendritic cells and the induction of ANCAs and associated autoimmunity. Here we show that myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with myeloid dendritic cells also positive for neutrophil myeloperoxidase in myeloperoxidase-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity.

Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation.

Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-alpha and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-alpha production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-alpha production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1-positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-alpha from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1-positive endosomes accompanied by the loss of IFN-alpha production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-alpha and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-alpha response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs.

Triggering CD40 on endothelial cells contributes to tumor growth.

Inflammatory cells can either promote or inhibit tumor growth. Here we studied whether CD40, a key molecule for adaptive immune response, has any role in mammary carcinogenesis of BALB/NeuT transgenic tumor-prone mice. We transferred the HER2/neu oncogene into CD40-null background to obtain the CD40-KO/NeuT strain. CD40-KO/NeuT mice showed delayed tumor onset and reduced tumor multiplicity. BM (BM) transplantation experiments excluded a role of BM-derived cells in the reduced tumorigenicity associated with CD40 deficiency. Rather, CD40 expressed by endothelial cells (ECs) takes part to the angiogenic process. Accordingly, large vessels, well organized around the tumor lobular structures, characterize BALB/NeuT tumors, whereas tiny numerous vessels with scarce extracellular matrix are dispersed in the parenchyma of poorly organized CD40-KO/NeuT tumors. Activated platelets, which may interact with and activate ECs, are a possible source of CD40L. Their localization within tumor vessels prompted the idea of treating BALB/NeuT and CD40-KO/NeuT mice chronically with the anti-platelet drug clopidogrel, known to inhibit platelet CD40L expression. Treatment of BALB/NeuT mice reduced tumor growth to a level similar to CD40-deficient mice, whereas CD40-KO/NeuT mice treated or not showed the same attenuated tumor outgrowth, indicating that activated platelets are the likely source of CD40L in this model. Collectively, these data point to a participation of CD40/CD40L in the angiogenic processes associated with mammary carcinogenesis of BALB/NeuT mice.

Intralesional SD-101 in Combination with Pembrolizumab in Anti-PD-1 Treatment-Naïve Head and Neck Squamous Cell Carcinoma: Results from a Multicenter, Phase II Trial.

PURPOSE: To determine whether SD-101, a Toll-like receptor 9 agonist, potentiates the antitumor activity of anti-PD-1 antibodies in patients with anti-PD-1/PD-L1 naïve, recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: Patients with PD-1 Ab-naïve HNSCC received either 2 mg SD-101 injected in one to four lesions or 8 mg SD-101 injected into a single lesion weekly × 4 doses then every 3 weeks × 7 doses. Pembrolizumab was administered at 200 mg every 3 weeks. RESULTS: A total of 28 patients received 2 mg and 23 received 8 mg per injection, respectively. A total of 76% of patients had received prior systemic therapy. Combined positive score was ≥1 to < 20 in 35 patients (70%) and ≥ 20 in 15 patients (30%) of 50 patients with available data. There were 12 patients with grade ≥3 treatment-related adverse events (24%), and no treatment-related deaths. The objective response rate was 24% including 2 complete and 10 partial responses. The median duration of response was 7.0 [95% confidence interval (CI): 2.1-11.1] months. The response rate was higher in human papillomavirus-positive (HPV+) patients (44%, N = 16). Responses were not associated with PD-L1 expression levels or IFNγ-related gene expression at baseline. Responses were observed both in injected (32%) and in noninjected lesions (29%). Progression-free and overall survival at 9 months were 19.0% (95% CI: 9.1-31.7) and 64.7% (95% CI: 45.3-78.7), respectively. CONCLUSIONS: SD-101 combined with pembrolizumab induced objective responses, especially in HPV+ tumors, which were frequently associated with increased intratumoral inflammation and effector immune cell activity.

Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma.

Secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is a Ca2+-binding matricellular glycoprotein involved in development, wound healing, and neoplasia. However, the role of SPARC in tumors is ill defined mostly because it is expressed by both tumor and stromal cells, especially inflammatory cells. We analyzed the respective roles of host- and tumor-derived SPARC in wild-type and congenic SPARC knockout (SPARC-/-) mice on a BALB/c genetic background injected into the mammary fat pad with SPARC-producing mammary carcinoma cells derived from c-erB2 transgenic BALB/c mice. Reduced tumor growth but massive parenchyma infiltration, with large areas of necrosis and impaired vascularization were observed in SPARC-/- mice. Immunohistochemical analysis showed a defect in collagen type IV deposition in the stroma of lobular tumors from SPARC-/- mice. Chimeric mice expressing SPARC only in bone marrow-derived cells were able to organize peritumoral and perilobular stroma, whereas reciprocal chimeras transplanted with bone marrow from SPARC-/- mice developed tumors with less defined lobular structures, lacking assembled collagen type IV and with a parenchyma heavily infiltrated by leukocytes. Together, the data indicate that SPARC produced by host leukocytes, rather than the tumor, determines the assembly and function of tumor-associated stroma through the organization of collagen type IV.

Low-dose metronomic cyclophosphamide complements the actions of an intratumoral C-class CpG TLR9 agonist to potentiate innate immunity and drive potent T cell-mediated anti-tumor responses.

The synthetic oligonucleotide SD-101 is a potent and specific agonist for toll-like receptor 9. Intratumoral injection of SD-101 induces significant anti-tumor immunity in preclinical and clinical studies, especially when combined with PD-1 blockade. To build upon this strategy, we studied the enhancement of SD-101 activities by combination with low-dose cyclophosphamide, a well-characterized agent with potentially complementary activities. In multiple mouse tumor models, we demonstrate substantial anti-tumor activity of the combination, compared to each single agent. Combination therapy generated CD8+ T cell dependent immunity leading to rejection of both non-injected and injected tumors and long-term survival, even in very large tumors. Mechanistic studies encompassing global gene expression changes and characterization of immune cell infiltrates show the rapid, sequential induction of innate and adaptive responses and identify discrete contributions of SD-101 and cyclophosphamide. Importantly, these changes were prominent in tumors not injected directly with SD-101. Combination treatment resulted in creation of a permissive environment for a systemic anti-tumor immune response, including a reduction of intratumoral regulatory T cells (Tregs) and an increase in "M1" versus "M2" tumor-associated macrophage (TAM) phenotypes. Additionally, we observed increased immunogenic cell death as well as antigen processing in response to combination treatment.

Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25- lymphocytes is thymus and proliferation independent.

The CD25- and CD25+ CD4 T-lymphocyte compartments are tightly regulated. We show here that tumors break such balance, increasing the number of CD4+CD25+ T cells in draining lymph node and spleen but not contralateral node of tumor-bearing mice. Tumor injection in thymectomized and CD25-depleted mice shows that CD4+CD25+ T-cell expansion occurs even in the absence of the thymus and independently from proliferation of preexisting CD25+ T cells. These newly generated cells are bona fide regulatory T cells (T reg) in terms of Foxp3 expression and suppression of CD3-stimulated or allogeneic effector cell proliferation. Transfer of congenic Thy1.1 CD4+CD25- T cells, from mice treated or not with vinblastine, into tumor-bearing or tumor-free mice and analysis of recovered donor lymphocytes indicate that conversion is the main mechanism for acquiring the expression of CD25 and Foxp3 through a process that does not require proliferation. Although conversion of CD4+CD25- T cells for generation of T regs has been described as a natural process that maintains peripheral T-reg population, this process is used by the tumor for immune escape. The prompt recovery of T regs from monoclonal antibody-mediated CD25 depletion in tumor-bearing mice suggests attempts able to inactivate rather than deplete them when treating existing tumors.

Low surface expression of B7-1 (CD80) is an immunoescape mechanism of colon carcinoma.

Artificially enforced expression of CD80 (B7-1) and CD86 (B7-2) on tumor cells renders them more immunogenic by triggering the CD28 receptor on T cells. The enigma is that such B7s interact with much higher affinity with CTLA-4 (CD152), an inhibitory receptor expressed by activated T cells. We show that unmutated CD80 is spontaneously expressed at low levels by mouse colon carcinoma cell lines and other transplantable tumor cell lines of various tissue origins. Silencing of CD80 by interfering RNA led to loss of tumorigenicity of CT26 colon carcinoma in immunocompetent mice, but not in immunodeficient Rag-/- mice. CT26 tumor cells bind CTLA-4Ig, but much more faintly with a similar CD28Ig chimeric protein, thus providing an explanation for the dominant inhibitory effects on tumor immunity displayed by CD80 at that expression level. Interestingly, CD80-negative tumor cell lines such as MC38 colon carcinoma and B16 melanoma express CD80 at dim levels during in vivo growth in syngeneic mice. Therefore, low CD80 surface expression seems to give an advantage to cancer cells against the immune system. Our findings are similar with the inhibitory role described for the dim CD80 expression on immature dendritic cells, providing an explanation for the low levels of CD80 expression described in various human malignancies.

Inhaled TLR9 Agonist Renders Lung Tumors Permissive to PD-1 Blockade by Promoting Optimal CD4+ and CD8+ T-cell Interplay.

Currently approved inhibitors of the PD-1/PD-L1 pathway represent a major advance for the treatment of lung cancers, yet they are ineffective in a majority of patients due to lack of preexisting T-cell reactivity. Here, we show that a TLR9 agonist delivered by inhalation is able to prime T-cell responses against poorly immunogenic lung tumors and to complement the effects of PD-1 blockade. Inhaled TLR9 agonist causes profound remodeling in tumor-bearing lungs, leading to the formation of tertiary lymphoid structures adjacent to the tumors, CD8+ T-cell infiltration into the tumors, dendritic cell expansion, and antibody production. Inhalation of TLR9 agonist also increased the pool of functional PD-1lowT-bethigh effector CD8+ T cells in tumor-bearing lungs. Effector CD8+ T cells generated by inhaled TLR9 agonist treatment were licensed by PD-1 blockade to become highly functional CTLs, leading to a durable rejection of both lung tumors and tumor lesions outside the lungs. CD4+ T cells activated in response to inhaled TLR9 play a critical role in this process by controlling the proliferation, preventing exhaustion, and guiding the differentiation of optimally functional CTLs. This study characterizes a strategy to apply localized TLR9 stimulation to a tumor type not accessible for direct injection, a strategy that may expand the therapeutic potential of PD-1 blockade in non-small cell lung cancer.Significance: These findings demonstrate that local delivery of a toll-like receptor 9 agonist can change the immune content of an entire organ and enhance the efficacy of immune checkpoint inhibition.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4943/F1.large.jpg Cancer Res; 78(17); 4943-56. ©2018 AACR.

Redirecting in vivo elicited tumor infiltrating macrophages and dendritic cells towards tumor rejection.

A hostile tumor microenvironment interferes with the development and function of the adaptive immune response. Here we report the mechanisms by which large numbers of tumor-infiltrating macrophages and dendritic cells (DC) can be redirected to become potent effectors and activators of the innate and adaptive immunity, respectively. We use adenoviral delivery of the CCL16 chemokine to promote accumulation of macrophages and DC at the site of preestablished tumor nodules, combined with the Toll-like receptor 9 ligand CpG and with anti-interleukin-10 receptor antibody. CpG plus anti-interleukin-10 receptor antibody promptly switched infiltrating macrophages infiltrate from M2 to M1 and triggered innate response debulking large tumors within 16 hours. Tumor-infiltrating DC matured and migrated in parallel with the onset of the innate response, allowing the triggering of adaptive immunity before the diffuse hemorrhagic necrosis halted the communication between tumor and draining lymph nodes. Treatment of B6>CXB6 chimeras implanted with BALB/c tumors with the above combination induced an efficient innate response but not CTL-mediated tumor lysis. In these mice, tumor rejection did not exceed 25%, similarly to that observed in CCR7-null mice that have DC unable to prime an adaptive response. The requirement of CD4 help was shown in CD40-KO, as well as in mice depleted of CD4 T cells, during the priming rather than the effector phase. Our data describe the critical requirements for the immunologic rejection of large tumors: a hemorrhagic necrosis initiated by activated M1 macrophages and a concomitant DC migration to draining lymph nodes for subsequent CTL priming and clearing of any tumor remnants.

Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence: evidence against immortality as a constitutive trait of tumor cells.

Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.

Combination immunotherapy with TLR agonists and checkpoint inhibitors suppresses head and neck cancer.

Checkpoint inhibitors have demonstrated efficacy in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, the majority of patients do not benefit from these agents. To improve the efficacy of checkpoint inhibitors, intratumoral (i.t.) injection with innate immune activators, TLR7 and TLR9 agonists, were tested along with programmed death-1 receptor (PD-1) blockade. The combination therapy suppressed tumor growth at the primary injected and distant sites in human papillomavirus-negative (HPV-negative) SCC7 and MOC1, and HPV-positive MEER syngeneic mouse models. Abscopal effects and suppression of secondary challenged tumor suggest that local treatment with TLR agonists in combination with anti-PD-1 provided systemic adaptive immunity. I.t. treatment with a TLR7 agonist increased the ratio of M1 to M2 tumor-associated macrophages (TAMs) and promoted the infiltration of tumor-specific IFNγ-producing CD8+ T cells. Anti-PD-1 treatment increased T cell receptor (TCR) clonality of CD8+ T cells in tumors and spleens of treated mice. Collectively, these experiments demonstrate that combination therapy with i.t. delivery of TLR agonists and PD-1 blockade activates TAMs and induces tumor-specific adaptive immune responses, leading to suppression of primary tumor growth and prevention of metastasis in HNSCC models.

Enhanced efficacy of tumor cell vaccines transfected with secretable hsp70.

Tumor immunotherapy has exploited the ability of heat shock proteins to chaperone precursors of antigenic peptides to antigen-presenting cells and to activate efficiently an immune response against tumor-associated antigens. The most common strategy is based on the purification of heat shock protein-peptide complexes from tumor cell lines or from tumor surgical samples for in vivo administration. In this article, we have modified the murine-inducible hsp70 into a secreted protein and engineered tumor cells to secrete constitutively their antigenic repertoire associated with the hsp70 protein. In vitro studies showed that the relocalization of hsp70 from the cytoplasm to the secretory pathway did not modify the ability of hsp70 to interact with peptides derived either from natural tumor-associated antigens or model antigens, and that antigen-presenting cells specifically took up the secreted hsp70 and presented the chaperoned epitopes to T cells. In vivo studies showed that tumors secreting hsp70 displayed increased immunogenicity, with induction of a strong and specific CTL response. Mice injected with hsp70-secreting tumors showed increased survival and impaired tumor take compared with mice bearing parental tumors. More than 70% of mice rejected tumor cells secreting hsp70 through mechanisms that involve T lymphocytes and natural killer cells, with the induction of a memory response in the case of T lymphocytes. Moreover, hsp70 secretion increased the immunogenic potential of tumor cell vaccines.

Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus.

Autoantibodies against nucleic acids and excessive type I interferon (IFN) are hallmarks of human systemic lupus erythematosus (SLE). We previously reported that SLE neutrophils exposed to TLR7 agonist autoantibodies release interferogenic DNA, which we now demonstrate to be of mitochondrial origin. We further show that healthy human neutrophils do not complete mitophagy upon induction of mitochondrial damage. Rather, they extrude mitochondrial components, including DNA (mtDNA), devoid of oxidized (Ox) residues. When mtDNA undergoes oxidation, it is directly routed to lysosomes for degradation. This rerouting requires dissociation from the transcription factor A mitochondria (TFAM), a dual high-mobility group (HMG) protein involved in maintenance and compaction of the mitochondrial genome into nucleoids. Exposure of SLE neutrophils, or healthy IFN-primed neutrophils, to antiribonucleotide protein autoantibodies blocks TFAM phosphorylation, a necessary step for nucleoid dissociation. Consequently, Ox nucleoids accumulate within mitochondria and are eventually extruded as potent interferogenic complexes. In support of the in vivo relevance of this phenomenon, mitochondrial retention of Ox nucleoids is a feature of SLE blood neutrophils, and autoantibodies against Ox mtDNA are present in a fraction of patients. This pathway represents a novel therapeutic target in human SLE.