Chimeric antigen receptor (CAR)–T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)-a subtype of Hodgkin lymphoma (HL)-is characterized by a low content of tumor cells, the lymphocyte predominant (LP) cells. Transformation into diffuse large B-cell lymphoma (DLBCL) occurs in about 10% of patients. We performed whole-genome mutation analysis of the DLBCL components from two composite lymphomas consisting of clonally related NLPHL and DLBCL as a means to identify candidate tumor suppressor genes and oncogenes in NLPHL. The analysis of LP cells for selected mutations of the DLBCL revealed that most mutations are also present in the LP cells, indicating a close relationship between the two components. The analysis of 62 selected genes in NLPHL by targeted ultra-deep sequencing revealed three novel highly recurrently mutated genes (each mutated in ~50% of cases), that is, DUSP2, SGK1 and JUNB. SGK1 was expressed in the LP cells of primary NLPHL cases and in the NLPHL cell line DEV. Administration of an SGK1 inhibitor induced apoptosis in the NLPHL cell line DEV and the DLBCL cell line Farage, suggesting a pathogenetic role of SGK1 in the LP and DLBCL cells. In summary, the present study identifies SGK1, DUSP2 and JUNB as novel key players in the pathogenesis of NLPHL.

Multinucleated Reed-Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well.

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cellderived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity ϳ10-to ϳ40-fold.Like many retroviruses, human immunodeficiency virus type 1 (HIV-1) has a very limited host range; spreading replication is seen only in Homo sapiens and by artificial inoculation in the close relative the chimpanzee (Pan troglodytes) (2, 16), preventing the setup of an efficient small animal model for HIV-1 research. Many reasons argue against the widespread use of chimpanzees in research, including the ethical problems involved in the use of an endangered species, budgetary problems, and the very low induction of simian AIDS either from HIV-1 or its ancestor simian immunodeficiency virus cpz (SIVcpz) in infected chimpanzees (25,26,30,57,58,60,63).In general, the tropism of HIV-1 in human tissue is determined by the expression of its receptor protein CD4 together with CCR5 or CXCR4 chemokine receptors. Simian CD4 but not murine CD4 supports entry of HIV-1 (15, 33). HIV-1 entry through receptor-mediated membrane fusion is required for reverse transcription of the viral genomic RNA into a doublestranded DNA molecule. Murine T cells show early postentry restriction of HIV-1 at reverse transcription (3). In simian cells, a related restriction of HIV-1, but not of SIVs (5, 10, 53), involves the simian TRIM5␣ protein, which leads to increased viral uncoating and thereby suppresses reverse transcription (73). Since HIV-1 does not show spreading replication in nonhuman cells, cell type-and t...

In contrast to the commonly indolent clinical behavior of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), T cell/histiocyte rich large B cell lymphoma (THRLBCL) is frequently diagnosed in advanced clinical stages and has a poor prognosis. Besides the different clinical presentations of these lymphoma entities, there are variants of NLPHL with considerable histopathologic overlap compared to THRLBCL. Especially THRLBCL-like NLPHL, a diffuse form of NLPHL, often presents a histopathologic pattern similar to THRLBCL, suggesting a close relationship between both lymphoma entities. To corroborate this hypothesis, we performed gene expression profiling of microdissected tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. In unsupervised analyses, the lymphomas did not cluster according to their entity. Moreover, even in supervised analyses, very few consistently differentially expressed transcripts were found, and for these genes the extent of differential expression was only moderate. Hence, there are no clear and consistent differences in the gene expression of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. Based on the gene expression studies, we identified BAT3/ BAG6, HIGD1A, and FAT10/UBD as immunohistochemical markers expressed in the tumor cells of all three lymphomas. Characterization of the tumor microenvironment for infiltrating T cells and histiocytes revealed significant differences in the cellular composition between typical NLPHL and THRLBCL cases. However, THRLBCL-like NLPHL presented a histopathologic pattern more related to THRLBCL than NLPHL. In conclusion, NLPHL and THRLBCL may represent a spectrum of the same disease. The different clinical behavior of these lymphomas may be strongly influenced by differences in the lymphoma microenvironment, possibly related to the immune status of the patient at the timepoint of diagnosis.

In contrast to the commonly indolent clinical behavior of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), T cell/histiocyte rich large B cell lymphoma (THRLBCL) is frequently diagnosed in advanced clinical stages and has a poor prognosis. Besides the different clinical presentations of these lymphoma entities, there are variants of NLPHL with considerable histopathologic overlap compared to THRLBCL. Especially THRLBCL-like NLPHL, a diffuse form of NLPHL, often presents a histopathologic pattern similar to THRLBCL, suggesting a close relationship between both lymphoma entities. To corroborate this hypothesis, we performed gene expression profiling of microdissected tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. In unsupervised analyses, the lymphomas did not cluster according to their entity. Moreover, even in supervised analyses, very few consistently differentially expressed transcripts were found, and for these genes the extent of differential expression was only moderate. Hence, there are no clear and consistent differences in the gene expression of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. Based on the gene expression studies, we identified BAT3/BAG6, HIGD1A, and FAT10/UBD as immunohistochemical markers expressed in the tumor cells of all three lymphomas. Characterization of the tumor microenvironment for infiltrating T cells and histiocytes revealed significant differences in the cellular composition between typical NLPHL and THRLBCL cases. However, THRLBCL-like NLPHL presented a histopathologic pattern more related to THRLBCL than NLPHL. In conclusion, NLPHL and THRLBCL may represent a spectrum of the same disease. The different clinical behavior of these lymphomas may be strongly influenced by differences in the lymphoma microenvironment, possibly related to the immune status of the patient at the timepoint of diagnosis.

Combination antiretroviral therapy is highly effective in HIV infection, leading to decreased incidences of AIDSdefining neoplasms. However, HIV patients still have a 10-fold increased risk of developing classical Hodgkin lymphoma compared with the general population. As Hodgkin-and Reed-Sternberg cells represent only a minority in the tumor infiltrate, the aim of the present study was to characterize the microenvironment of HIV-related classical Hodgkin lymphoma and compare it with classical Hodgkin lymphoma cases of immunocompetent individuals. The major morphologic differences were the presence of necrotic foci and the absence of epithelioid cell formation in HIV-related Hodgkin lymphoma. We observed a significantly decreased number of CD4 þ T-cells and a significantly increased number of CD163 þ macrophages in HIVrelated Hodgkin lymphoma. Cases exhibiting a 'sarcomatoid' pattern of the reactive infiltrate exhibited significantly greater numbers of macrophages, associating the 'sarcomatoid' pattern to the presence of spindle-shaped macrophages. Whereas, rosetting of CD4 þ T-cells around Hodgkin-and Reed-Sternberg cells was frequently observed in classical Hodgkin lymphoma in immunocompetent persons; rosetting in a subset of HIV-related Hodgkin lymphoma cases appeared to involve cytoplasmic protrusions of spindle-shaped macrophages. HIV-related Hodgkin lymphoma, therefore, is characterized by unique morphologic features, which should be recognized by pathologists.

Mature T-cell lymphomas (MTCLs) have an extremely poor prognosis and are much less frequent than immature T-cell leukemias. This suggests that malignant outgrowth of mature T lymphocytes is well controlled. Indeed, in a previous study we found that mature T cells are resistant to transformation with known T-cell oncogenes. Here, however, we observed that T-cell receptor (TCR) mono-/oligoclonal mature T cells from TCR transgenic (tg) mice (OT-I, P14) expressing the oncogenes NPM/ALK or DTrkA readily developed MTCLs in T-cell-deficient recipients. Analysis of cell surface markers largely ruled out that TCR tg lymphomas were derived from T-cell precursors. Furthermore, cotransplanted non-modified TCR polyclonal T cells suppressed malignant outgrowth of oncogene expressing TCR tg T lymphocytes. A dominant role of an anti-leukemic immune response or Tregs in the control of MTCLs seems unlikely as naïve T cells derived from oncogene expressing stem cells, which should be tolerant to leukemic antigens, as well as purified CD4 and CD8 were resistant to transformation. However, our results are in line with a model in which homeostatic mechanisms that stabilize the diversity of the normal T-cell repertoire, for example, clonal competition, also control the outgrowth of potentially malignant T-cell clones. This study introduces a new innate mechanism of lymphoma control.