Evaluation of single domain antibodies as nuclear tracers for imaging of the immune checkpoint receptor human lymphocyte activation gene-3 in cancer.

Recent advancements in the field of immune-oncology have led to a significant increase in life expectancy of patients with diverse forms of cancer, such as hematologic malignancies, melanoma and lung cancer. Unfortunately, these encouraging results are not observed in the majority of patients, who remain unresponsive and/or encounter adverse events. Currently, researchers are collecting more insight into the cellular and molecular mechanisms that underlie these variable responses. As an example, the human lymphocyte activation gene-3 (huLAG-3), an inhibitory immune checkpoint receptor, is increasingly studied as a therapeutic target in immune-oncology. Noninvasive molecular imaging of the immune checkpoint programmed death protein-1 (PD-1) or its ligand PD-L1 has shown its value as a strategy to guide and monitor PD-1/PD-L1-targeted immune checkpoint therapy. Yet, radiotracers that allow dynamic, whole body imaging of huLAG-3 expression are not yet described. We here developed single-domain antibodies (sdAbs) that bind huLAG-3 and showed that these sdAbs can image huLAG-3 in tumors, therefore representing promising tools for further development into clinically applicable radiotracers.

Antigen-presenting cell-targeted lentiviral vectors do not support the development of productive T-cell effector responses: implications for in vivo targeted vaccine delivery.

Targeting transgene expression specifically to antigen-presenting cells (APCs) has been put forward as a promising strategy to direct the immune system towards immunity. We developed the nanobody-display technology to restrict the tropism of lentiviral vectors (LVs) to APCs. However, we observed that immunization with APC-targeted LVs (DC2.1-LVs) did not evoke strong antigen-specific T-cell immunity when compared to immunization with broad tropism LVs (VSV.G-LVs). In this study, we report that VSV.G-LVs are more immunogenic than DC2.1-LVs because they transduce stromal cells, which has a role in activating antigen-specific T cells. Moreover, VSV.G-LVs trigger a pro-inflammatory innate immune response through transduction of APCs and stromal cells, while DC2.1-LVs trigger a type I interferon response with anti-viral capacity. These findings question the rationale of targeting LVs to APCs and argue for the development of VSV.G-LVs with an improved safety profile.

Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells.

Lentiviral vectors (LVs) provide unique opportunities for the development of immunotherapeutic strategies, as they transduce a variety of cells in situ, including antigen-presenting cells (APCs). Engineering LVs to specifically transduce APCs is required to promote their translation towards the clinic. We report on the Nanobody (Nb) display technology to target LVs to dendritic cells (DCs) and macrophages. This innovative approach exploits the budding mechanism of LVs to incorporate an APC-specific Nb and a binding-defective, fusion-competent form of VSV.G in the viral envelope. In addition to production of high titer LVs, we demonstrated selective, Nb-dependent transduction of mouse DCs and macrophages both in vitro and in situ. Moreover, this strategy was translated to a human model in which selective transduction of in vitro generated or lymph node (LN)-derived DCs and macrophages, was demonstrated. In conclusion, the Nb display technology is an attractive approach to generate LVs targeted to specific cell types.

Claudin-1, claudin-2 and claudin-11 genes differentially associate with distinct types of anti-inflammatory macrophages in vitro and with parasite- and tumour-elicited macrophages in vivo.

Macrophages altered by various Th2-associated and anti-inflammatory mediators--including IL-4 and IL-13 [inducing alternatively activated macrophages (AAMs)], IL-10 and TGF-ß--were generically termed M2. However, markers that discriminate between AAMs and other M2 remain scarce. We previously described E-cadherin as a marker for AAMs, permitting these macrophages to fuse upon IL-4 stimulation. To identify novel potential contributors to macrophage fusion, we assessed the effect of IL-4 on other adherens and tight junction-associated components. We observed an induction of claudin-1 (Cldn1), Cldn2 and Cldn11 genes by IL-4 in different mouse macrophage populations. Extending our findings to other stimuli revealed Cldn1 as a mainly TGF-ß-induced gene and showed that Cldn11 is predominantly associated with IL-4-induced AAMs. Cldn2 is upregulated by diverse stimuli and is not associated with a specific macrophage activation state in vitro. Interestingly, different claudin genes preferentially associate with M2 from distinct diseases. While Cldn11 is predominantly expressed in AAMs from helminth-infected mice, Cldn1 is the major macrophage claudin during chronic trypanosomiasis and Cldn2 dominates in tumour-associated macrophages. Overall, we identified Cldn1, Cldn2 and Cldn11 as genes that discriminate between diverse types of M2.

B7-1, IFN gamma and anti-CTLA-4 co-operate to prevent T-cell tolerization during immunotherapy against a murine T-lymphoma.

We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8(+) CTL response capable of mediating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, resulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing animals, with or without B7-mediated costimulation, even increased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN gamma, together with B7-1, could have beneficial effects on immunotherapy. Although immunotherapy with IFN gamma and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN gamma double transfectants resulted in a more beneficial outcome. This phenomenon correlated with an increased CTL-inducing potential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regulation of the anti-tumor response. Indeed, when immunotherapy was provided along with anti-CTLA-4, the protection by B71/IFN gamma double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN gamma and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth.

Cellular expression of the cytolytic factor in earthworms Eisenia foetida.

Coelomic fluid of earthworms contains a 42 kDa protein designated CCF-1 (coelomic cytolytic factor 1), which accounts for approximately 40% of cytolytic activity of the entire coelomic fluid. CCF-1 was documented to be present on cells of the mesenchymal lining of the coelomic cavity as well as on free coelomocytes. Both cellular and humoral levels of CCF-1 were significantly increased after parenteral injection of endotoxin. Moreover, CCF-1 seems to be involved in cell mediated cytotoxicity, because cytotoxic activity is blocked in the presence of anti-CCF-1 monoclonal antibody (mAb).

Active antitumor immunotherapy, with or without B7-mediated costimulation, increases tumor progression in an immunogenic murine T cell lymphoma model.

BW-Sp3 is a BW-5147-derived T cell lymphoma with limited immunogenicity since, despite regression of the majority of subcutaneous tumors, an important fraction of the animals will die from metastases. In the present study, the BW-Sp3 cells were transfected with genes encoding B7-1 or B7-2, known to be involved in the induction of T cell responses. The resulting transfectants exhibited a reduced tumorigenicity and did not cause mortality in the syngeneic recipients. Furthermore, immunization with the B7-1 or B7-2 transfectants resulted in an increased generation of cytotoxic T lymphocytes (CTL) that lysed both the transfectants and the wild-type BW-Sp3 cells. Since the B7 transfectants were completely rejected in syngeneic recipients and induced potent CTL recognizing the wild-type BW-Sp3 cells, these engineered cells were considered as candidates for immunotherapy. Vaccinations with the B7-1 or B7-2 transfectants could completely protect the animals from metastatic disease when subsequently challenged with wild-type BW-Sp3 cells. Furthermore, immunization with the B7 transfectants could prolong the survival time of mice that had been challenged intravenously with BW-Sp3 cells. Surprisingly, however, when these transfectants, as well as the wild-type BW-Sp3 cells, were used for vaccination of tumor-bearing animals, the presence of the subcutaneous BW-Sp3 tumors clearly interfered with the outcome of immunotherapy, resulting in increased malignancy, as reflected by a higher incidence of progressing tumors and a reduced survival rate. Possible implications for immunotherapy in humans are discussed.

Multiple effects of transfection with interleukin 2 and/or interferon gamma on the behavior of mouse T lymphoma cells.

We have previously reported that transfection of mouse interferon gamma (IFN-gamma) in H-2Kk-positive BW variants (BW-Sp3) abolishes tumorigenicity and reduces metastatic capacity. To further increase the immunogenicity of BW-Sp3 cells, the gene for human interleukin 2 (huIL-2) was transfected in these cancer cells. Single BW-Sp3(huIL-2) and double BW-Sp3(huIL-2+IFN-gamma) transfectants were generated and their behavior was investigated as compared to parental and IFN-gamma-transfected BW-Sp3. Although expression of huIL-2 was equally effective as IFN-gamma in preventing tumor formation and reducing experimental metastasis, it did not confer protection to spontaneous metastases and even reversed the anti-metastatic activity of IFN-gamma. Inoculation of the BW variants in immunocompromised mice revealed that expression of IL-2 activates both T cells and aspecific immune effectors. However, in immunocompromised mice a clear pro-metastatic effect of IL-2 was recorded. Analysis of membrane antigens on the different variants showed a selective effect of huIL-2 on the expression of two surface antigens, i.e. L-selectin and metastatic T cell hybridoma antigen (MTH), which may contribute to metastasis. Hence upon expression of huIL-2 in T lymphoma variants, tumor outcome will depend on the balanced effects of the transfected cytokines on the immune response and the redirected effect on tumor progression.

Immunogenization of a murine T-cell lymphoma via transfection with interferon-gamma.

Tumor cell variants were derived from the BW5147 T-cell lymphoma that differ in major histocompatibility complex (MHC) class I antigen expression, tumorigenicity and metastatic potential. In general, increased H-2Kk expression was found to be correlated with a reduced tumorigenicity and spontaneous metastasis. CD8+ T cells were identified in the immune recognition of such variants, implicating a role for H-2Kk in the presentation of tumor-associated antigens. In the present study, H-2Kk+ BW variants were transfected with a gene encoding interferon-gamma (IFN-gamma), a potent inducer of MHC class I expression. The resulting transfectants exhibited an increased expression of H-2Kk and concomitantly an inability to generate visible tumors and a reduced metastatic capacity. Furthermore, immunization with the IFN-gamma transfectants resulted in an increased generation of cytotoxic T lymphocytes (CTLs) that lysed both the transfectants and the parental tumor cells. Based on these results, vaccinations with the IFN-gamma transfectants were performed against the parental tumor cells. The results clearly demonstrated that such vaccinations reduced significantly the tumorigenicity and metastatic capacity of the parental tumor cells. Hence, in this tumor model, IFN-gamma gene transfection provides a means to immunogenize H-2Kk+ BW tumor cells.

Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity.

The interaction between B7-1 and CD28 provides costimulatory signals not only for T cells but also for natural killer (NK) cells. Highly metastatic mouse T lymphoma cells (BW-Li) can escape from NK cell-mediated killing by expressing H-2Dk molecules that negatively regulate NK lytic activity. We have analyzed whether B7-1:CD28 overrules the MHC class I-mediated inactivation of NK cells by transfecting BW-Li with the gene coding for B7-1. Expression of B7-1 rendered BW-Li cells sensitive toward NK cells. The experimental metastatic capacity of the B7-1 transfectants was drastically reduced in both syngeneic AKR and SCID mice but could be restored in SCID-bg mice. These results provide direct evidence that B7-1 expression leads to NK-mediated elimination of metastasizing, NK-resistant tumor cells.

Linkage analysis of bipolar illness with X-chromosome DNA markers: a susceptibility gene in Xq27-q28 cannot be excluded.

Transmission studies have supported the presence of a susceptibility gene for bipolar (BP) illness on the X-chromosome. Initial linkage studies with color blindness (CB), glucose-6-phosphate dehydrogenase (G6PD) deficiency, and the blood coagulation factor IX (F9) have suggested that a gene for BP illness is located in the Xq27-q28 region. We tested linkage with several DNA markers located in Xq27-q28 in 2 families, MAD3 and MAD4, that previously were linked to F9 and 7 newly ascertained families of BP probands. Linkage was also examined with the gene encoding the alpha 3 subunit of the gamma-amino butyric acid receptor (GABRA3), a candidate gene for BP illness located in this region. The genetic data were analyzed with the LOD score method using age-dependent penetrance of an autosomal dominant disease gene and narrow and broad clinical models. In MAD3 and MAD4 the multipoint LOD score data suggested a localization of a BPI gene again near F9. In the 7 new families the overall linkage data excluded the Xq27-q28 region. However, if the families were grouped according to their proband's phenotype BPI or BPII, a susceptibility gene for BPI disorder at the DXS52-F8 cluster could not be excluded.

[Extrauterine pregnancies in patients with intrauterine devices (apropos of 9 cases)]. / Grossesses extra-utérines sur dispositifs intra-utérins (à propos de neuf observations).

PIP: The published literature reports an increase of ectopic pregnancies in patients wearing an IUD. Over the total number of pregnancies with IUD in situ, ectopic pregnancies account for about 10-17%. The incidence is lower for the first 6 months after insertion, and higher after that. The mechanism responsible for extrauterine pregnancies can be the appearance of infectious lesions in the genital tract, or the slow and continuous secretion of prostaglandin promoted by the presence of the IUD. Diagnosis of ectopic pregnancy is not always easy, and it usually follows episodes of pelvic pain and metrorrhagia.^ieng