Cell-Autonomous Cxcl1 Sustains Tolerogenic Circuitries and Stromal Inflammation via Neutrophil-Derived TNF in Pancreatic Cancer.

We have shown that KRAS-TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC. SIGNIFICANCE: By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell-excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer. This article is highlighted in the In This Issue feature, p. 1275.

Correction to: Angiosarcoma patients treated with immune checkpoint inhibitors: a case series of seven patients from a single institution.

Following publication of the original article [1], the authors have reported that the following sentence "While of the same IgG1 class as ipilimumab, preclinical data suggests this molecule may have enhanced activity against T regulatory cells".

Angiosarcoma patients treated with immune checkpoint inhibitors: a case series of seven patients from a single institution.

BACKGROUND: Angiosarcoma is an uncommon endothelial malignancy and a highly aggressive soft tissue sarcoma. Due to its infiltrative nature, successful management of localized angiosarcoma is often challenging. Systemic chemotherapy is used in the metastatic setting and occasionally in patients with high-risk localized disease in neoadjuvant or adjuvant settings. However, responses tend to be short-lived and most patients succumb to metastatic disease. Novel therapies are needed for patients with angiosarcomas. METHODS: We performed a retrospective analysis of patients with locally advanced or metastatic angiosarcoma, who were treated with checkpoint inhibitors at our institution. We collected their clinical information and outcome measurements. In one patient with achieved complete response, we analyzed circulating and infiltrating T cells within peripheral blood and tumor tissue. RESULTS: We have treated seven angiosarcoma (AS) patients with checkpoint inhibitors either in the context of clinical trials or off label [Pembrolizumab + Axitinib (NCT02636725; n = 1), AGEN1884, a CTLA-4 inhibitor (NCT02694822; n = 2), Pembrolizumab (n = 4)]. Five patients had cutaneous angiosarcoma, one primary breast angiosarcoma and one radiation-associated breast angiosarcoma. At 12 weeks, 5/7 patients (71%) had partial response of their lesions either on imaging and/or clinical exam and two (29%) had progressive disease. 6/7 patients are alive to date and, thus far, 3/7 patients (43%) have progressed (median 3.4 months)- one achieved partial response after pembrolizumab was switched to ongoing Nivolumab/Ipilimumab, one died of progressive disease at 31 weeks (primary breast angiosarcoma) and one was placed on pazopanib. One patient had a complete response (CR) following extended treatment with monotherapy AGEN1884. No patient experienced any ≥ grade 2 toxicities. CONCLUSIONS: This case series underscores the value of targeted immunotherapy in treating angiosarcoma. It also identifies genetic heterogeneity of cutaneous angiosarcomas and discusses specific genetic findings that may explain reported benefits from immunotherapy.

Axitinib plus pembrolizumab in patients with advanced sarcomas including alveolar soft-part sarcoma: a single-centre, single-arm, phase 2 trial.

BACKGROUND: VEGF promotes an immunosuppressive microenvironment and contributes to immune checkpoint inhibitor resistance in cancer. We aimed to assess the activity of the VEGF receptor tyrosine-kinase inhibitor axitinib plus the anti-PD-1 immune checkpoint inhibitor pembrolizumab in patients with sarcoma. METHODS: This single-centre, single-arm, phase 2 trial was undertaken at a tertiary care academic medical centre in Miami, FL, USA, and participants were recruited from all over the USA and internationally. Patients were eligible if they were aged 16 years or older, and had histologically confirmed advanced or metastatic sarcomas, including alveolar soft-part sarcoma (ASPS); measurable disease with one site amenable to repeated biopsies; an ECOG performance status of 0-1; and progressive disease after previous treatment with at least one line of systemic therapy (unless no standard treatment existed or the patient declined therapy). The first five patients were enrolled in a lead-in cohort and were given axitinib 5 mg orally twice daily and pembrolizumab 200 mg intravenously for 30 min on day 8 and every 3 weeks for cycles of 6 weeks for up to 2 years. Thereafter, patients received escalating doses of axitinib (2-10 mg) plus flat dose pembrolizumab according to the schedule above. The primary endpoint was 3-month progression-free survival. All patients were evaluable for survival and safety analyses. This study is registered with ClinicalTrials.gov, number NCT02636725, and is closed to accrual. FINDINGS: Between April 19, 2016, and Feb 7, 2018, of 36 patients assessed for eligibility, 33 (92%) were enrolled and given study treatment (intention-to-treat population and safety population), 12 (36%) of whom had ASPS. With a median follow-up of 14·7 months (IQR 10·1-19·1), 3-month progression-free survival for all evaluable patients was 65·6% (95% CI 46·6-79·3). For patients with ASPS, 3-month progression-free survival was 72·7% (95% CI 37·1-90·3). The most common grade 3 or 4 treatment-related adverse events included hypertension (five [15%] of 33 patients), autoimmune toxicities (five [15%]), nausea or vomiting (two [6%]), and seizures (two [6%]). Serious treatment-related adverse events occurred in seven (21%) patients, including autoimmune colitis, transaminitis, pneumothorax, haemoptysis, seizures, and hypertriglyceridemia. There were no treatment-related deaths. INTERPRETATION: Axitinib plus pembrolizumab has manageable toxicity and preliminary activity in patients with advanced sarcomas, particularly patients with ASPS, warranting further investigation in randomised controlled trials. FUNDING: Merck, Pfizer, American Cancer Society, and Sylvester Comprehensive Cancer Center.

Deep functional immunophenotyping predicts risk of cytomegalovirus reactivation after hematopoietic cell transplantation.

Cytomegalovirus (CMV) is the most common viral infection in hematopoietic cell transplantation (HCT) recipients. We performed deep phenotyping of CMV-specific T cells to predict CMV outcomes following allogeneic HCT. By using 13-color flow cytometry, we studied ex vivo CD8+ T-cell cytokine production in response to CMV-pp65 peptides in 3 clinically distinct subgroups of CMV-seropositive HCT patients: (1) Elite Controllers (n = 19): did not have evidence of CMV DNAemia on surveillance testing; (2) Spontaneous Controllers (n = 16): spontaneously resolved low-grade CMV DNAemia without antiviral therapy; and (3) Noncontrollers (NC; n = 21): experienced clinically significant CMV. Two CMV-specific CD8+ T-cell functional subsets were strongly associated with risk of CMV: (i) the nonprotective signature (NPS; IL-2-IFN-γ+TNF-α-MIP-1ß+), found at increased levels among NC; and (ii) the protective signature (PS; IL-2+IFN-γ+TNF-α+MIP-1ß+) found at low levels among NC. High levels of the NPS and low levels of PS were associated with an increased 100-day cumulative incidence of clinically significant CMV infection (35% vs 5%; P = .02; and 40% vs 12%; P = .05, respectively). The highest predictive value was observed when these signatures were combined into a composite biomarker consisting of low levels of the PS and high levels of the NPS (67% vs 10%; P < .001). After adjusting for steroid use or donor type, this composite biomarker remained associated with a fivefold increase in the risk of clinically significant CMV infection. CMV-specific CD8+ T-cell cytokine signatures with robust predictive value for risk of CMV reactivation should prove useful in guiding clinical decision making in HCT recipients.

CD28 aptamers as powerful immune response modulators.

CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.Molecular Therapy - Nucleic Acids (2013) 2, e98; doi:10.1038/mtna.2013.26; published online 11 June 2013.

Targeting 4-1BB costimulation to disseminated tumor lesions with bi-specific oligonucleotide aptamers.

The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. Here, we show that bi-specific oligonucleotide aptamer conjugates can deliver costimulatory ligands to tumor cells in situ and enhance antitumor immunity. In poorly immunogenic subcutaneously implanted tumor and lung metastasis models, systemic delivery of an agonistic 4-1BB aptamer ligand conjugated to a prostate specific membrane antigen (PSMA)-binding tumor-targeting aptamer led to inhibition of tumor growth, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared to costimulation with 4-1BB antibodies. Tumor inhibition was dependent on homing to PSMA-expressing tumor cells and 4-1BB costimulation. Aptamer targeted costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions. This study suggests that potentiating naturally occurring antitumor immunity via tumor-targeted costimulation could be an effective approach to elicit protective immunity to control tumor progression in cancer patients.

Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay.

The main reason why tumours are not controlled by the immune system is that, unlike pathogens, they do not express potent tumour rejection antigens (TRAs). Tumour vaccination aims at stimulating a systemic immune response targeted to, mostly weak, antigens expressed in the disseminated tumour lesions. Main challenges in developing effective vaccination protocols are the identification of potent and broadly expressed TRAs and effective adjuvants to stimulate a robust and durable immune response. Here we describe an alternative approach in which the expression of new, and thereby potent, antigens are induced in tumour cells by inhibiting nonsense-mediated messenger RNA decay (NMD). Small interfering RNA (siRNA)-mediated inhibition of NMD in tumour cells led to the expression of new antigenic determinants and their immune-mediated rejection. In subcutaneous and metastatic tumour models, tumour-targeted delivery of NMD factor-specific siRNAs conjugated to oligonucleotide aptamer ligands led to significant inhibition of tumour growth that was superior to that of vaccination with granulocyte-macrophage colony-stimulating factor (GM-CSF)-expressing irradiated tumour cells, and could be further enhanced by co-stimulation. Tumour-targeted NMD inhibition forms the basis of a simple, broadly useful, and clinically feasible approach to enhance the antigenicity of disseminated tumours leading to their immune recognition and rejection. The cell-free chemically synthesized oligonucleotide backbone of aptamer-siRNAs reduces the risk of immunogenicity and enhances the feasibility of generating reagents suitable for clinical use.

Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice.

4-1BB is a major costimulatory receptor that promotes the survival and expansion of activated T cells. Administration of agonistic anti-4-1BB Abs has been previously shown to enhance tumor immunity in mice. Abs are cell-based products posing significant cost, manufacturing, and regulatory challenges. Aptamers are oligonucleotide-based ligands that exhibit specificity and avidity comparable to, or exceeding, that of Abs. To date, various aptamers have been shown to inhibit the function of their cognate target. Here, we have described the development of an aptamer that binds 4-1BB expressed on the surface of activated mouse T cells and shown that multivalent configurations of the aptamer costimulated T cell activation in vitro and mediated tumor rejection in mice. Because aptamers can be chemically synthesized, manufacturing and the regulatory approval process should be substantially simpler and less costly than for Abs. Agonistic aptamers could therefore represent a superior alternative to Abs for the therapeutic manipulation of the immune system.

Modulation of dendritic cell differentiation and function by YopJ of Yersinia pestis.

Yersinia pestis evades immune responses in part by injecting into host immune cells several effector proteins called Yersinia outer proteins (Yops) that impair cellular function. This has been best characterized in the innate effector cells, but much less so for cells involved in adaptive immune responses. Dendritic cells (DC) sit at the crossroads between innate and adaptive immunity, and can function to initiate or inhibit adaptive immune responses. Although Y. pestis can target and inactivate DC, the mechanism responsible for this remains unclear. We have found that injection of Y. pestis YopJ into DC progenitors disrupts key signal transduction pathways and interferes with DC differentiation and subsequent function. YopJ injection prevents up-regulation of the NF-kappaB transcription factor Rel B and inhibits MAPK/ERK activation--both having key roles in DC differentiation. Furthermore, YopJ injection prevents costimulatory ligand up-regulation, LPS-induced cytokine expression, and yields differentiated DC with diminished capability to induce T cell proliferation and IFN-gamma induction. By modulating DC function through YopJ-mediated disruption of signaling pathways during progenitor to DC differentiation, Yersinia may interfere with the adaptive responses necessary to clear the infection as well as establish a tolerant immune environment that leads to chronic infection/carrier state in the surviving host.

CD28-mediated regulation of multiple myeloma cell proliferation and survival.

Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown, due in large part to the complexity of the bone marrow microenvironment itself. The T-cell costimulatory receptor CD28 is also expressed on normal and malignant plasma cells, and CD28 expression in MM correlates significantly with poor prognosis and disease progression. In contrast to T cells, activation and function of CD28 in myeloma cells is largely undefined. We have found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induces activation of PI3K and NFkappaB, suppresses MM cell proliferation, and protects against serum starvation and dexamethasone (dex)-induced cell death. Coculture with dendritic cells (DCs) expressing the CD28 ligands CD80 and CD86 also elicits CD28-mediated effects on MM survival and proliferation, and DCs appear to preferentially localize within myeloma infiltrates in primary patient samples. Our findings suggest a previously undescribed myeloma/DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also point to CD28 as a potential therapeutic target in the treatment of MM.

Regulation of RelB expression during the initiation of dendritic cell differentiation.

The transcription factor RelB is required for proper development and function of dendritic cells (DCs), and its expression is upregulated early during differentiation from a variety of progenitors. We explored this mechanism of upregulation in the KG1 cell line model of a DC progenitor and in the differentiation-resistant KG1a subline. RelB expression is relatively higher in untreated KG1a cells but is upregulated only during differentiation of KG1 by an early enhancement of transcriptional elongation, followed by an increase in transcription initiation. Restoration of protein kinase CbetaII (PKCbetaII) expression in KG1a cells allows them to differentiate into DCs. We show that PKCbetaII also downregulated constitutive expression of NF-kappaB in KG1a-transfected cells and restores the upregulation of RelB during differentiation by increased transcriptional initiation and elongation. The two mechanisms are independent and sensitive to PKC signaling levels. Conversely, RelB upregulation was inhibited in primary human monocytes where PKCbetaII expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKCbetaII-mediated regulation of transcriptional initiation and elongation.

Protein kinase C betaII plays an essential role in dendritic cell differentiation and autoregulates its own expression.

Dendritic cells (DC) arise from a diverse group of hematopoietic progenitors and have marked phenotypic and functional heterogeneity. The signal transduction pathways that regulate the ability of progenitors to undergo DC differentiation, as well as the specific characteristics of the resulting DC, are only beginning to be characterized. We have found previously that activation of protein kinase C (PKC) by cytokines or phorbol esters drives normal human CD34(+) hematopoietic progenitors and myeloid leukemic blasts (KG1, K562 cell lines, and primary patient blasts) to differentiate into DC. We now report that PKC activation is also required for cytokine-driven DC differentiation from monocytes. Of the cPKC isoforms, only PKC-betaII was consistently activated by DC differentiation-inducing stimuli in normal and leukemic progenitors. Transfection of PKC-betaII into the differentiation-resistant KG1a subline restored the ability to undergo DC differentiation in a signal strength-dependent fashion as follows: 1) by development of characteristic morphology; 2) the up-regulation of DC surface markers; 3) the induction of expression of the NFkappaB family member Rel B; and 4) the potent ability to stimulate allo-T cells. Most unexpectedly, the restoration of PKC-betaII signaling in KG1a was not directly due to overexpression of the transfected classical PKC (alpha, betaII, or gamma) but rather through induction of endogenous PKC-beta gene expression by the transfected classical PKC. The mechanism of this positive autoregulation involves up-regulation of PKC-beta promoter activity by constitutive PKC signaling. These findings indicate that the regulation of PKC-betaII expression and signaling play critical roles in mediating progenitor to DC differentiation.

Differentiation of acute and chronic myeloid leukemic blasts into the dendritic cell lineage: analysis of various differentiation-inducing signals.

PURPOSE: Ex vivo differentiation of myeloid leukemic blasts into dendritic cells (DCs) holds significant promise for use as cellular vaccines, as they may present a constellation of endogenously expressed known and unknown leukemia antigens to the immune system. Although variety of stimuli can drive leukemia --> DC differentiation in vitro, these blast-derived DCs typically have aberrant characteristics compared with DCs generated from normal progenitors by the same stimuli. It is not clear whether this is due to underlying leukemogenic mechanisms (e.g., specific oncogenes), genetic defects, stage of maturation arrest, defects in cytokine receptor expression or signal transduction pathways, or whether different stimuli themselves induce qualitatively dissimilar DC differentiation. METHODS: To assess what factors may contribute to aberrant leukemic blast --> DC differentiation, we have examined how the same leukemic blasts (AML and CML) respond to different DC differentiation signals--including extracellular (the cytokine combination GM-CSF + TNF-alpha + IL-4) and intracellular (the protein kinase C agonist PMA, the calcium ionophore A23187, and the combination of PMA plus A23187) stimuli. RESULTS: We have found that the same leukemic blasts will develop qualitatively different sets of DC characteristics in response to differing stimuli, although no stimuli consistently induced all of the characteristic DC features. There were no clear differences in the responses relative to specific oncogene expression or stage of maturation arrest (AML vs CML). Signal transduction agonists that bypassed membrane receptors/proximal signaling (in particular, the combination of PMA and A23187) consistently induced the greatest capability to activate T cells. Interestingly, this ability did not clearly correlate with expression of MHC/costimulatory ligands. CONCLUSIONS: Our findings suggest that signal transduction may play an important role in the aberrant DC differentiation of leukemic blasts, and demonstrate that direct activation of PKC together with intracellular calcium signaling may be an effective method for generating immunostimulatory leukemia-derived DCs.

Induced dendritic cell differentiation of chronic myeloid leukemia blasts is associated with down-regulation of BCR-ABL.

Although differentiation of leukemic blasts to dendritic cells (DC) has promise in vaccine strategies, the mechanisms underlying this differentiation and the differences between leukemia and normal progenitor-derived DC are largely undescribed. In the case of chronic myeloid leukemia (CML), understanding the relationship between the induction of DC differentiation and the expression of the BCR-ABL oncogene has direct relevance to CML biology as well as the development of new therapeutic approaches. We now report that direct activation of protein kinase C (PKC) by the phorbol ester PMA in the BCR-ABL(+) CML cell line K562 and primary CML blasts induced nonterminal differentiation into cells with typical DC morphology (cytoplasmic dendrites), characteristic surface markers (MHC class I, MHC class II, CD86, CD40), chemokine and transcription factor expression, and ability to stimulate T cell proliferation (equivalent to normal monocyte-derived DC). PKC-induced differentiation was associated with down-regulation of BCR-ABL mRNA expression, protein levels, and kinase activity. This down-regulation appeared to be signaled through the mitogen-activated protein kinase pathway. Therefore, PKC-driven differentiation of CML blasts into DC-like cells suggests a potentially novel strategy to down-regulate BCR-ABL activity, yet raises the possibility that CML-derived DC vaccines will be less effective in presenting leukemia-specific Ags.