Loss of MTAP Expression by Immunohistochemistry Is a Surrogate Marker for Homozygous 9p21.3 Deletion in Urothelial Carcinoma.

Homozygous deletion of the chromosomal region 9p21.3 is common in urothelial carcinoma (UC) and leads to loss of several genes, including CDKN2A and MTAP, resulting in loss of MTAP protein expression. Here, we aimed to explore the diagnostic potential of MTAP immunohistochemistry (IHC) as a surrogate marker for homozygous 9p21.3 deletion (9p21 homozygous deletion [HD]) in UC. MTAP status was determined by IHC on 27 UC tissue specimens with known 9p21.3 status as defined by fluorescence in situ hybridization in matched cytological specimens, by IHC and fluorescence in situ hybridization on a tissue microarray (TMA) containing 359 UC at different stages, and by IHC on 729 consecutive UC from routine practice. Moreover, we analyzed a longitudinal series of matched specimens from 38 patients with MTAP-negative recurrent UC. MTAP loss by IHC was found in all 17 patients with 9p21 HD and in 2/8 cases without 9p21 HD. In the TMA, MTAP loss was more common in metastases (53%) than in muscle-invasive (33%) and non-muscle-invasive UC (29%) (P = .03). In the consecutive series, 164/729 (22%) cases showed loss of MTAP expression. In 41 of these 164 cases (25%), loss of MTAP expression was heterogenous. We also discovered loss of MTAP expression in flat urothelium adjacent to MTAP-negative low-grade UC, suggesting true flat low-grade neoplasia that could not be diagnosed by morphology alone. Longitudinal analysis of recurrences showed persistent negative MTAP status over time in 37/38 (97%) patients. MTAP IHC can serve as a surrogate marker for 9p21 HD in UC and as a diagnostic tool to differentiate reactive urothelium from urothelial neoplasia. It also provides a unique opportunity to study clinicopathological associations and the heterogeneity of 9p21 HD across the whole spectrum of UC manifestations.

Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma.

Around 30-50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

Infraorbital Subcutaneous Intravascular Lobular Capillary Hemangioma After Rhinoplasty.

BACKGROUND: Lobular capillary hemangioma (LCH; also referred to as pyogenic granuloma) is a common benign vascular tumor that is characterized by proliferation of capillaries with a lobular architecture. Lobular capillary hemangioma can involve superficial cutaneous, mucosal, or subcutaneous structures; the subcutaneous and intravascular variant is very rare. METHODS: A 26-year-old female patient presented with a small infraorbital mass that was slowly growing within the last 6 months. She reported no pain but an uncomfortable feeling of pressure in this area. Six months before symptom onset, the patient had undergone a closed rhinoplasty with osteotomies without any reported complication. RESULTS: An excisional biopsy was performed via a transconjunctival approach, and the histopathological findings were characteristic for a subcutaneous intravascular LCH. At the 3-month follow-up, the patient was asymptomatic with no evidence of a recurrent lesion. CONCLUSION: To our knowledge, this is the first report of a subcutaneous intravascular LCH after rhinoplasty. We would like to draw the attention of stakeholders to this rare condition and raise awareness among clinicians to what seems to be a late finding after rhinosurgery.

EBV persistence without its EBNA3A and 3C oncogenes in vivo.

The oncogenic Epstein Barr virus (EBV) infects the majority of the human population and usually persists within its host for life without symptoms. The EBV oncoproteins nuclear antigen 3A (EBNA3A) and 3C (EBNA3C) are required for B cell transformation in vitro and are expressed in EBV associated immunoblastic lymphomas in vivo. In order to address the necessity of EBNA3A and EBNA3C for persistent EBV infection in vivo, we infected NOD-scid γcnull mice with reconstituted human immune system components (huNSG mice) with recombinant EBV mutants devoid of EBNA3A or EBNA3C expression. These EBV mutants established latent infection in secondary lymphoid organs of infected huNSG mice for at least 3 months, but did not cause tumor formation. Low level viral persistence in the absence of EBNA3A or EBNA3C seemed to be supported primarily by proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression without elevated lytic replication. In vitro, EBNA3A and EBNA3C deficient EBV infected B cells could be rescued from apoptosis through CD40 stimulation, mimicking T cell help in secondary lymphoid tissues. Thus, even in the absence of the oncogenes EBNA3A and 3C, EBV can access a latent gene expression pattern that is reminiscent of EBV persistence in healthy virus carriers without prior expression of its whole growth transforming program.

Natural killer cell-based adoptive immunotherapy eradicates and drives differentiation of chemoresistant bladder cancer stem-like cells.

BACKGROUND: High-grade non-muscle invasive bladder cancer (NMIBC) has a high risk of recurrence and progression to muscle-invasive forms, which seems to be largely related to the presence of tumorigenic stem-like cell populations that are refractory to conventional therapies. Here, we evaluated the therapeutic potential of Natural Killer (NK) cell-based adoptive immunotherapy against chemoresistant bladder cancer stem-like cells (CSCs) in a pre-clinical relevant model, using NK cells from healthy donors and NMIBC patients. METHODS: Cytokine-activated NK cells from healthy donors and from high-grade NMIBC patients were phenotypically characterized and assayed in vitro against stem-like and bulk differentiated bladder cancer cells. Stem-like cells were isolated from two bladder cancer cell lines using the sphere-forming assay. The in vivo therapeutic efficacy was evaluated in mice bearing a CSC-induced orthotopic bladder cancer. Animals were treated by intravesical instillation of interleukin-activated NK cells. Tumor response was evaluated longitudinally by non-invasive bioluminescence imaging. RESULTS: NK cells from healthy donors upon activation with IL-2 and IL-15 kills indiscriminately both stem-like and differentiated tumor cells via stress ligand recognition. In addition to cell killing, NK cells shifted CSCs towards a more differentiated phenotype, rendering them more susceptible to cisplatin, highlighting the benefits of a possible combined therapy. On the contrary, NK cells from NMIBC patients displayed a low density on NK cytotoxicity receptors, adhesion molecules and a more immature phenotype, losing their ability to kill and drive differentiation of CSCs. The local administration, via the transurethral route, of activated NK cells from healthy donors provides an efficient tumor infiltration and a subsequent robust tumoricidal activity against bladder cancer with high selective cytolytic activity against CSCs, leading to a dramatic reduction in tumor burden from 80 % to complete remission. CONCLUSION: Although pre-clinical, our results strongly suggest that an immunotherapeutic strategy using allogeneic activated NK cells from healthy donors is effective and should be exploited as a complementary therapeutic strategy in high-risk NMIBC patients to prevent tumor recurrence and progression.

Adoptive transfer of EBV specific CD8+ T cell clones can transiently control EBV infection in humanized mice.

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.

Role for early-differentiated natural killer cells in infectious mononucleosis.

A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially during viral infections in humans. However, both phenotype and role of NK cells during acute symptomatic Epstein-Barr virus (EBV) infection, termed infectious mononucleosis (IM), remain unclear. Here, we longitudinally assessed the kinetics, the differentiation, and the proliferation of subsets of NK cells in pediatric IM patients. Our results indicate that acute IM is characterized by the preferential proliferation of early-differentiated CD56(dim) NKG2A(+) immunoglobulin-like receptor(-) NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on exposure to EBV-infected B cells expressing lytic antigens. Thus, early-differentiated NK cells might play a key role in the immune control of primary infection with this persistent tumor-associated virus.

Role of the 2B4 Receptor in CD8+ T-Cell-Dependent Immune Control of Epstein-Barr Virus Infection in Mice With Reconstituted Human Immune System Components.

Patients with X-linked lymphoproliferative (XLP) disease due to deficiency in the adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) are highly susceptible to one specific viral pathogen, the Epstein-Barr virus (EBV). This susceptibility might result from impaired CD8(+) T-cell and natural killer cell responses to EBV infection in these patients. We demonstrate that antibody blocking of the SAP-dependent 2B4 receptor is sufficient to induce XLP-like aggravation of EBV disease in mice with reconstituted human immune system components. CD8(+) T cells require 2B4 for EBV-specific immune control, because 2B4 blockade after CD8(+) T-cell depletion did not further aggravate symptoms of EBV infection.

Infectious diseases in humanized mice.

Despite many theoretical incompatibilities between mouse and human cells, mice with reconstituted human immune system components contain nearly all human leukocyte populations. Accordingly, several human-tropic pathogens have been investigated in these in vivo models of the human immune system, including viruses such as human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV), as well as bacteria such as Mycobacterium tuberculosis and Salmonella enterica Typhi. While these studies initially aimed to establish similarities in the pathogenesis of infections between these models and the pathobiology in patients, recent investigations have provided new and interesting functional insights into the protective value of certain immune compartments and altered pathology upon mutant pathogen infections. As more tools and methodologies are developed to make these models more versatile to study human immune responses in vivo, such improvements build toward small animal models with human immune components, which could predict immune responses to therapies and vaccination in human patients.

CAR T cells outperform CAR NK cells in CAR-mediated effector functions in head-to-head comparison.

BACKGROUND: CAR NK cells as vehicles for engineered "off-the-shelf" cellular cancer immunotherapy have attracted significant interest. Nonetheless, a comprehensive comparative assessment of the anticancer activity of CAR T cells and CAR NK cells carrying approved benchmark anti-CD19 CAR constructs is missing. Here, we report a direct head-to-head comparison of CD19-directed human T and NK cells. METHODS: We generated CAR T and CAR NK cells derived from healthy donor PBMC by retroviral transduction with the same benchmark second-generation anti-CD19 CAR construct, FMC63.28z. We investigated IFN-γ secretion and direct cytotoxicity in vitro against various CD19+ cancer cell lines as well as in autologous versus allogeneic settings. Furthermore, we have assessed anticancer activity of CAR T and CAR NK cells in vivo using a xenograft lymphoma model in an autologous versus allogeneic setting and a leukemia model. RESULTS: Our main findings are a drastically reduced capacity for CAR-mediated IFN-γ production and lower CAR-mediated cytotoxicity of CAR NK cells relative to CAR T cells in vitro. Consistent with these in vitro findings, we report superior anticancer activity of autologous CAR T cells compared with allogeneic CAR NK cells in vivo. CONCLUSIONS: CAR T cells had significantly higher CAR-mediated effector functions than CAR NK cells in vitro against several cancer cell lines and autologous CAR T cells outperformed allogeneic CAR NK cells both in vitro and in vivo. CAR NK cells will likely benefit from further engineering to enhance anticancer activity to ultimately fulfill the promise of an effective off-the-shelf product.

Mutation-specific CAR T cells as precision therapy for IGLV3-21R110 expressing high-risk chronic lymphocytic leukemia.

The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21R110) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells.

CD4+ T cells in cancer.

Cancer immunology and immunotherapy are driving forces of research and development in oncology, mostly focusing on CD8+ T cells and the tumor microenvironment. Recent progress highlights the importance of CD4+ T cells, corresponding to the long-known fact that CD4+ T cells are central players and coordinators of innate and antigen-specific immune responses. Moreover, they have now been recognized as anti-tumor effector cells in their own right. Here we review the current status of CD4+ T cells in cancer, which hold great promise for improving knowledge and therapies in cancer.

The NK cell checkpoint NKG2A maintains expansion capacity of human NK cells.

Human natural killer (NK) cells are cytotoxic effector cells that are increasingly harnessed in cancer immunotherapy. NKG2A/CD94 is an inhibitory receptor on NK cells that has established regulatory functions in the direct interaction with target cells when engaged with its ligand, the non-classical HLA class I molecule HLA-E. Here, we confirmed NKG2A as a checkpoint molecule in primary human NK cells and identified a novel role for NKG2A in maintaining NK cell expansion capacity by dampening both proliferative activity and excessive activation-induced cell death. Maintenance of NK cell expansion capacity might contribute to the preferential accumulation of human NKG2A+ NK cells after hematopoietic cell transplantation and enrichment of functionally impaired NK cells in human cancers. Functional silencing of NKG2A for cancer immunotherapy is highly attractive but will need to consider that this might also lead to a reduced survival by driving activation-induced cell death in targeted NK cells.

Targeted adenovirus-mediated transduction of human T cells in vitro and in vivo.

Clinical success in T cell therapy has stimulated widespread efforts to increase safety and potency and to extend this technology to solid tumors. Yet progress in cell therapy remains restricted by the limited payload capacity, specificity of target cell transduction, and transgenic gene expression efficiency of applied viral vectors. This renders complex reprogramming or direct in vivo applications difficult. Here, we developed a synergistic combination of trimeric adapter constructs enabling T cell-directed transduction by the human adenoviral vector serotype C5 in vitro and in vivo. Rationally chosen binding partners showed receptor-specific transduction of otherwise non-susceptible human T cells by exploiting activation stimuli. This platform remains compatible with high-capacity vectors for up to 37 kb DNA delivery, increasing payload capacity and safety because of the removal of all viral genes. Together, these findings provide a tool for targeted delivery of large payloads in T cells as a potential avenue to overcome current limitations of T cell therapy.

Human NK cells of mice with reconstituted human immune system components require preactivation to acquire functional competence.

To investigate human natural killer (NK)-cell reactivity in vivo we have reconstituted human immune system components by transplantation of human hematopoietic progenitor cells into NOD-scid IL2Rγ(null) mice. We demonstrate here that this model allows the development of all NK-cell subsets that are also found in human adult peripheral and cord blood, including NKp46(+)CD56(-) NK cells. Similar to human cord blood, NK cells from these reconstituted mice require preactivation by interleukin-15 to reach the functional competence of human adult NK cells. Mainly the terminally differentiated CD16(+) NK cells demonstrate lower reactivity without this stimulation. After preactivation, both CD16(+) and CD16(-) NK cells efficiently produce interferon-γ and degranulate in response to stimulation with NK cell-susceptible targets, including K562 erythroleukemia cells. NK-cell lines, established from reconstituted mice, demonstrate cytotoxicity against this tumor cell line. Importantly, preactivation can as well be achieved by bystander cell maturation via poly I:C stimulation in vitro and injection of this maturation stimulus in vivo. Preactivation in vivo enhances killing of human leukocyte antigen class I negative tumor cells after their adoptive transfer. These data suggest that a functional, but resting, NK-cell compartment can be established in immune-compromised mice after human hematopoietic progenitor cell transfer.

Mice with human immune system components as in vivo models for infections with human pathogens.

Many pathogens relevant to human disease do not infect other animal species. Therefore, animal models that reconstitute or harbor human tissues are explored as hosts for these. In this review, we will summarize recent advances to utilize mice with human immune system components, reconstituted from hematopoietic progenitor cells in vivo. Such mice can be used to study human pathogens that replicate in leukocytes. In addition to studying the replication of these pathogens, the reconstituted human immune system components can also be analyzed for initiating immune responses and control against these infections. Moreover, these new animal models of human infectious disease should replicate the reactivity of the human immune system to vaccine candidates and, especially, the adjuvants contained in them, more faithfully.

Interactions of human myeloid cells with natural killer cell subsets in vitro and in vivo.

In both human and mouse it has been recently realized that natural killer (NK) cells do not emerge from the bone marrow with full functional competence but rather acquire functions in interaction with antigen-presenting cells (APCs), primarily dendritic cells (DCs). Here we review the mechanisms and the consequences of this NK-cell preactivation, as well as discuss new experimental models that now allow investigating these interactions for human NK cells and their response to human pathogens in vivo. These investigations will allow harnessing NK cells during vaccination for improved innate and adaptive immunity.

KSHV infection drives poorly cytotoxic CD56-negative natural killer cell differentiation in vivo upon KSHV/EBV dual infection.

Herpesvirus infections shape the human natural killer (NK) cell compartment. While Epstein-Barr virus (EBV) expands immature NKG2A+ NK cells, human cytomegalovirus (CMV) drives accumulation of adaptive NKG2C+ NK cells. Kaposi sarcoma-associated herpesvirus (KSHV) is a close relative of EBV, and both are associated with lymphomas, including primary effusion lymphoma (PEL), which nearly always harbors both viruses. In this study, KSHV dual infection of mice with reconstituted human immune system components leads to the accumulation of CD56-CD16+CD38+CXCR6+ NK cells. CD56-CD16+ NK cells were also more frequently found in KSHV-seropositive Kenyan children. This NK cell subset is poorly cytotoxic against otherwise-NK-cell-susceptible and antibody-opsonized targets. Accordingly, NK cell depletion does not significantly alter KSHV infection in humanized mice. These data suggest that KSHV might escape NK-cell-mediated immune control by driving CD56-CD16+ NK cell differentiation.