IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR-engineered T cells.

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

Boosting Interleukin-12 Antitumor Activity and Synergism with Immunotherapy by Targeted Delivery with isoDGR-Tagged Nanogold.

The clinical use of interleukin-12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor-homing peptide containing isoDGR, an αvß3-integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head-to-tail cyclized-peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvß3 and IL12-receptor recognition. Low-dose Iso1/Au/IL12, equivalent to 18-75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low-dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T-cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR-tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T-cell therapy.

Invariant NKT cells contribute to chronic lymphocytic leukemia surveillance and prognosis.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant CD5+ B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance. We investigated the impact of iNKT cells in the natural history of the disease in the Eµ-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients. We found that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression.

miR-17∼92 family clusters control iNKT cell ontogenesis via modulation of TGF-ß signaling.

Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-ß receptor II (TGF-ßRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-ß signaling. miRNA members of the miR-17∼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17∼92 family clusters (miR-17∼92, miR-106a∼363, miR-106b∼25) phenocopied both increased TGF-ßRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-ß signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.

Of self-lipids, CD1-restricted T cells, and contact sensitization.

Contact hypersensitivity (CHS) in rodents and contact dermatitis in humans are long-known pathological conditions caused by MHC-restricted T-cell responses. These responses are triggered upon T-cell recognition of neo-antigenic determinants, which are generated by a variety of environmental contact sensitizer (CS) chemicals associating with self-proteins to comprise these neo-antigens. In this issue of the European Journal of Immunology, Betts et al. [Eur. J. Immunol. 2017. 47: 1171-1180] provide intriguing data implying that common small molecule CSs such as dinitrochlorobenzene can also recruit and activate autoreactive CD1-restricted T cells specific for cell-endogenous lipids, which are enriched in human skin. The effects of dinitrochlorobenzene on CD1 T-cell recruitment and function were dependent on newly synthesized CD1 molecules and the presence of endogenous lipids. These findings shed new light on unanticipated mechanisms that have potential clinical relevance on a common and highly distressing disease state.

T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes.

OBJECTIVE: Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies. DESIGN: We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions. RESULTS: Several unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+ and CD4+ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+ T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo. CONCLUSIONS: These results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

The circulating microRNome demonstrates distinct lymphocyte subset-dependent signatures.

Upon activation, lymphocytes release vesicles containing microRNAs (miRNAs). However, little is known as to whether this release results in modulation of circulating miRNAs (the miRNome) in the serum. The present work aims to identify lymphocyte subset-specific signatures of miRNAs within the serum circulating miRNome. We therefore assessed serum miRNA expression profiles in wild-type mice; in mice lacking either CD4(+) T cells, CD8(+) T cells, invariant natural killer T (iNKT) cells, or B cells; and, as a control, in mice in which Dicer has been ablated in T lymphocytes. We found that specific serum miRNAs are differentially modulated when different lymphocyte subsets are lacking. In particular, the serum level of miR-181b-5p, previously demonstrated to be fundamental for the development of iNKT cells, is specifically reduced in mice in which iNKT cells are absent. Interestingly, our results indicate a direct link between the biological role of a single miRNA in lymphocyte development and its serum level, and prove that even a population composed of relatively few cells in vivo, such as iNKT lymphocytes, has a measurable effect on the serum circulating miRNome.

A Subset of CD8αß+ Invariant NKT Cells in a Humanized Mouse Model.

Invariant NKT (iNKT) cells are unconventional innate-like T cells demonstrating potent antitumor function in conventional mouse models. However, the iNKT cell ligands have had limited efficacy in human antitumor clinical trials, mostly due to the profound differences in the properties and compositions of iNKT cells between the two species, including the presence of a CD8(+) subset of iNKT cells only in humans. To build reliable in vivo models for studying human iNKT cells, we recently developed the first humanized mouse model (hCD1d-KI) with human CD1d knocked in. To further humanize the mouse model, we now introduced the human invariant NKT TCRα-chain (Vα24Jα18) into the hCD1d-knockin mice. Similar to humans, this humanized mouse model developed a subset of CD8αß(+) iNKT cells among other human-like iNKT subsets. The presence of the CD8αß(+) iNKT cells in the thymus suggests that these cells developed in the thymus. In the periphery, these NKT cells showed a strong Th1-biased cytokine response and potent cytotoxicity for syngeneic tumor cells upon activation, as do human CD8αß(+) iNKT cells. The low binding avidity of iNKT TCRs to the human CD1d/lipid complex and high prevalence of Vß7 TCRß among the CD8(+) iNKT cells strongly point to a low avidity-based developmental program for these iNKT cells, which included the suppression of Th-POK and upregulation of eomesodermin transcriptional factors. Our establishment of this extensively humanized mouse model phenotypically and functionally reflecting the human CD1d/iNKT TCR system will greatly facilitate the future design and optimization of iNKT cell-based immunotherapies.

Cancer-initiating cells from colorectal cancer patients escape from T cell-mediated immunosurveillance in vitro through membrane-bound IL-4.

Cancer-initiating cells (CICs) that are responsible for tumor initiation, propagation, and resistance to standard therapies have been isolated from human solid tumors, including colorectal cancer (CRC). The aim of this study was to obtain an immunological profile of CRC-derived CICs and to identify CIC-associated target molecules for T cell immunotherapy. We have isolated cells with CIC properties along with their putative non-CIC autologous counterparts from human primary CRC tissues. These CICs have been shown to display "tumor-initiating/stemness" properties, including the expression of CIC-associated markers (e.g., CD44, CD24, ALDH-1, EpCAM, Lgr5), multipotency, and tumorigenicity following injection in immunodeficient mice. The immune profile of these cells was assessed by phenotype analysis and by in vitro stimulation of PBMCs with CICs as a source of Ags. CICs, compared with non-CIC counterparts, showed weak immunogenicity. This feature correlated with the expression of high levels of immunomodulatory molecules, such as IL-4, and with CIC-mediated inhibitory activity for anti-tumor T cell responses. CIC-associated IL-4 was found to be responsible for this negative function, which requires cell-to-cell contact with T lymphocytes and which is impaired by blocking IL-4 signaling. In addition, the CRC-associated Ag COA-1 was found to be expressed by CICs and to represent, in an autologous setting, a target molecule for anti-tumor T cells. Our study provides relevant information that may contribute to designing new immunotherapy protocols to target CICs in CRC patients.

iNKT-cell help to B cells: a cooperative job between innate and adaptive immune responses.

T-cell help to B lymphocytes is one of the most important events in adaptive immune responses in health and disease. It is generally delivered by cognate CD4(+) T follicular helper (T(FH)) cells via both cell-to-cell contacts and soluble mediators, and it is essential for both the clonal expansion of antibody (Ab)-secreting B cells and memory B-cell formation. CD1d-restricted invariant natural killer T (iNKT) cells are a subset of innate-like T lymphocytes that rapidly respond to stimulation with specific lipid antigens (Ags) that are derived from infectious pathogens or stressed host cells. Activated iNKT cells produce a wide range of cytokines and upregulate costimulatory molecules that can promote activation of dendritic cells (DCs), natural killer (NK) cells, and T cells. A decade ago, we discovered that iNKT cells can help B cells to proliferate and to produce IgG Abs in vitro and in vivo. This adjuvant-like function of Ag-activated iNKT cells provides a flexible set of helper mechanisms that expand the current paradigm of T-cell-B-cell interaction and highlights the potential of iNKT-cell targeting vaccine formulations.

Somatically mutated tumor antigens in the quest for a more efficacious patient-oriented immunotherapy of cancer.

Although cancer immunotherapy shows efficacy with adoptive T cell therapy (ACT) and antibody-based immune checkpoint blockade, efficacious therapeutic vaccination of cancer patients with tumor-associated antigens (TAAs) remains largely unmet. Current cancer vaccines utilize nonmutated shared TAAs that may have suboptimal immunogenicity. Experimental evidence underscores the strong immunogenicity of unique TAAs derived from somatically mutated cancer proteins, whose massive characterization has been precluded until recently by technical limitations. The development of cost-effective, high-throughput DNA sequencing approaches makes now possible the rapid identification of all the somatic mutations contained in a cancer cell genome. This method, combined with robust bioinformatics platforms for T cell epitope prediction and established reverse immunology approaches, provides us with an integrated strategy to identify patient-specific unique TAAs in a relatively short time, compatible with their potential use in the clinic. Hence, it is now for the first time possible to quantitatively define the patient's unique tumor antigenome and exploit it for vaccination, possibly in combination with ACT and/or immune checkpoint blockade to further increase immunotherapy efficacy.

Alliance Against Cancer, the network of Italian cancer centers bridging research and care.

Alliance Against Cancer (ACC) was established in Rome in 2002 as a consortium of six Italian comprehensive cancer centers (Founders). The aims of ACC were to promote a network among Italian oncologic institutions in order to develop specific, advanced projects in clinical and translational research. During the following years, many additional full and associate members joined ACC, that presently includes the National Institute of Health, 17 research-oriented hospitals, scientific and patient organizations. Furthermore, in the last three years ACC underwent a reorganization process that redesigned the structure, governance and major activities. The present goal of ACC is to achieve high standards of care across Italy, to implement and harmonize principles of modern personalized and precision medicine, by developing cost effective processes and to provide tailored information to cancer patients. We herein summarize some of the major initiatives that ACC is currently developing to reach its goal, including tumor genetic screening programs, establishment of clinical trial programs for cancer patients treated in Italian cancer centers, facilitate their access to innovative drugs under development, improve quality through an European accreditation process (European Organization of Cancer Institutes), and develop international partnerships. In conclusion, ACC is a growing organization, trying to respond to the need of networking in Italy and may contribute significantly to improve the way we face cancer in Europe.

Functional education of invariant NKT cells by dendritic cell tuning of SHP-1.

Invariant NKT (iNKT) cells play key roles in host defense by recognizing lipid Ags presented by CD1d. iNKT cells are activated by bacterial-derived lipids and are also strongly autoreactive toward self-lipids. iNKT cell responsiveness must be regulated to maintain effective host defense while preventing uncontrolled stimulation and potential autoimmunity. CD1d-expressing thymocytes support iNKT cell development, but thymocyte-restricted expression of CD1d gives rise to Ag hyperresponsive iNKT cells. We hypothesized that iNKT cells require functional education by CD1d(+) cells other than thymocytes to set their correct responsiveness. In mice that expressed CD1d only on thymocytes, hyperresponsive iNKT cells in the periphery expressed significantly reduced levels of tyrosine phosphatase SHP-1, a negative regulator of TCR signaling. Accordingly, heterozygous SHP-1 mutant mice displaying reduced SHP-1 expression developed a comparable population of Ag hyperresponsive iNKT cells. Restoring nonthymocyte CD1d expression in transgenic mice normalized SHP-1 expression and iNKT cell reactivity. Radiation chimeras revealed that CD1d(+) dendritic cells supported iNKT cell upregulation of SHP-1 and decreased responsiveness after thymic emigration. Hence, dendritic cells functionally educate iNKT cells by tuning SHP-1 expression to limit reactivity.

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help.

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.

Fine tuning by human CD1e of lipid-specific immune responses.

CD1e is a member of the CD1 family that participates in lipid antigen presentation without interacting with the T-cell receptor. It binds lipids in lysosomes and facilitates processing of complex glycolipids, thus promoting editing of lipid antigens. We find that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect is caused by the capacity of CD1e to facilitate rapid formation of CD1-lipid complexes, as shown for CD1d, and also to accelerate their turnover. Similar results were obtained with antigen-presenting cells from CD1e transgenic mice in which lipid complexes are assembled more efficiently and show faster turnover than in WT antigen-presenting cells. These effects maximize and temporally narrow CD1-restricted responses, as shown by reactivity to Sphingomonas paucimobilis-derived lipid antigens. CD1e is therefore an important modulator of both group 1 and group 2 CD1-restricted responses influencing the lipid antigen availability as well as the generation and persistence of CD1-lipid complexes.

IL-12 reprograms CAR-expressing natural killer T cells to long-lived Th1-polarized cells with potent antitumor activity.

Human natural killer T cells (NKTs) are innate-like T lymphocytes increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintains a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observe that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates a de novo programming of memory NKTs in CD62L-negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy, and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 show enhanced antitumor activity in leukemia and neuroblastoma tumor models, persist long-term in vivo and conserve the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics.

Invariant NKT cell reconstitution in pediatric leukemia patients given HLA-haploidentical stem cell transplantation defines distinct CD4+ and CD4- subset dynamics and correlates with remission state.

Immune reconstitution plays a crucial role on the outcome of patients given T cell-depleted HLA-haploidentical hematopoietic stem cell transplantation (hHSCT) for hematological malignancies. CD1d-restricted invariant NKT (iNKT) cells are innate-like, lipid-reactive T lymphocytes controlling infections, cancer, and autoimmunity. Adult mature iNKT cells are divided in two functionally distinct CD4(+) and CD4(-) subsets that express the NK receptor CD161 and derive from thymic CD4(+)CD161(-) precursors. We investigated iNKT cell reconstitution dynamics in 33 pediatric patients given hHSCT for hematological malignancies, with a follow-up reaching 6 y posttransplantation, and correlated their emergence with disease relapse. iNKT cells fully reconstitute and rapidly convert into IFN-γ-expressing effectors in the 25 patients maintaining remission. CD4(+) cells emerge earlier than the CD4(-) ones, both displaying CD161(-) immature phenotypes. CD4(-) cells expand more slowly than CD4(+) cells, though they mature with significantly faster kinetics, reaching full maturation by 18 mo post-hHSCT. Between 4 and 6 y post-hHSCT, mature CD4(-) iNKT cells undergo a substantial expansion burst, resulting in a CD4(+)