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Maternal immune activation (MIA), related to autoimmune/inflammatory diseases or acute infections, during the two first trimesters of pregnancy is a risk factor for autism spectrum disorders (ASD) in offspring. In mice, MIA has a long-term impact on offspring's immune equilibrium resulting in a pro-inflammatory phenotype. We therefore hypothesized that children with ASD and a history of MIA could display a similar phenotype specifically assessed by a higher neutrophil to lymphocyte ratio (NLR). In this study, we used a retrospective sample of 231 dyads involving children with ASD and their mothers. Among ASD patients, 12% had a history of MIA. The multivariate analysis revealed a significant association between NLR in children with ASD and maternal history of MIA (F = 2.27, p = 0.03). Using a categorical approach, we observed an abnormal NLR (over 3) in 7.4% of children with ASD MIA+ compared to 1.9% for MIA-. Our study supports the hypothesis suggesting an impact of MIA on the risk of ASD. Further studies could contribute to the development of biomarkers in MIA+ ASD and enable the development of targeted immunomodulatory therapies., (© 2023. The Author(s).)

Autism spectrum disorder (ASD) are neurodevelopmental conditions characterised by deficits in social communication and interaction and repetitive behaviours. Maternal immune activation (MIA) during the mid-pregnancy is a known risk factor for ASD. Although reported in 15% of affected individuals, little is known about the specificity of their clinical profiles. Adaptive skills represent a holistic approach to a person's competencies and reflect specifically in ASD, their strengths and difficulties. In this study, we hypothesised that ASD individual with a history of MIA (MIA + ) could be more severely socio-adaptively impaired than those without MIA during pregnancy (MIA - ). To answer this question, we considered two independent cohorts of individuals with ASD (PARIS study and FACE ASD) screened for pregnancy history, and used supervised and unsupervised machine learning algorithms. We included 295 mother-child dyads with 14% of them with MIA + . We found that ASD-MIA + individuals displayed more severe maladaptive behaviors, specifically in their socialization abilities. MIA + directly influenced individual's socio-adaptive skills, independent of other covariates, including ASD severity. Interestingly, MIA + affect persistently the socio-adaptive behavioral trajectories of individuals with ASD. The current study has a retrospective design with possible recall bias regarding the MIA event and, even if pooled from two cohorts, has a relatively small population. In addition, we were limited by the number of covariables available potentially impacted socio-adaptive behaviors. Larger prospective study with additional dimensions related to ASD is needed to confirm our results. Specific pathophysiological pathways may explain these clinical peculiarities of ASD- MIA + individuals, and may open the way to new perspectives in deciphering the phenotypic complexity of ASD and for the development of specific immunomodulatory strategies., (© 2023. Springer Nature Limited.)

The mechanisms regulating inflammation in large vessels vasculitis (LVV) are poorly understood. Interleukin 33 (IL-33) has been shown to license innate and adaptive immunity by enhancing Th2 cytokines production. We aimed to examine the role of IL-33 in the immunomodulation of T cell activation in LVV. T cell homeostasis and cytokines production were determined in peripheral blood from 52 patients with giant cell arteritis (GCA) and 50 healthy donors (HD), using Luminex assay, flow cytometry, quantitative RT-PCR and by immunofluorescence analysis in inflammatory aorta lesions. We found increased level of IL-33 and its receptor ST2/IL-1R4 in the serum of patient with LVV. Endothelial cells were the main source of IL-33, whereas Th2 cells, Tregs and mast cells (MC) express ST2 in LVV vessels. IL-33 had a direct immunomodulatory impact by increasing Th2 and Tregs. IL-33 and MC further enhanced Th2 and regulatory responses by inducing a 6.1 fold increased proportion of Tregs (p = 0.008). Stimulation of MC by IL-33 increased indoleamine 2 3-dioxygenase (IDO) activity and IL-2 secretion. IL-33 mRNA expression was significantly correlated with the expression of IL-10 and TGF-β within aorta inflammatory lesions. To conclude, our findings suggest that IL-33 may exert a critical immunoregulatory role in promoting Tregs and Th2 cells in LVV.

The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4(+) T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.

The observation that depletion or inhibition of regulatory T cells (Tregs) unleashes efficient antitumor effector immune responses that can lead to tumor eradication in mice has opened new perspectives for the development of cancer immunotherapy. The quality and overall efficiency of the effector immune responses induced in the absence of Tregs seem to depend on multiple factors that determine the result of a battle involving effector T cells (Teffs), Tregs and tumor cells. In this study, we investigated the quality of tumor-associated antigens (TAAs) as one such factor. We show that the presence of a strong dominant antigen is required for the induction of effector responses capable of tumor eradication in the absence of Tregs. The sole addition of a dominant antigen on tumor cells does not change tumor growth in unmanipulated mice, but improves tumor eradication rate from a few to almost 100% in the absence of Tregs. This eradication can be shown to result from the recruitment and activation of specific Teffs recognizing this antigen. We also show that the presence of such dominant antigens has the side effect of restricting the breadth of the immune response to other TAAs, which could favor the generation of escape mutant by tumor editing. Taken together, our results highlight the potential, and some requirements for cancer immunotherapy based on Treg depletion. They also show that, ultimately, tumor fate depends on multiple factors that should all be taken into consideration for the design of more efficient immunotherapy.

In humans, no efficient treatment exists not only against multifocal liver metastases (LM) but also against recurrent microscopic liver metastases within the liver remnant following curative liver resection. Furthermore, in nonmultifocal LM, partial liver resection could be performed, but in more than 50% of the patients, tumor recurrence within liver remnant is observed, partly due to the growth of dormant cancer cells in the setting of postoperative host immune dysfunction. We investigated the therapeutic potential of interleukin-12 (IL-12) immuno-gene therapies in these experimental models under total vascular exclusion (TVE) of the liver. In rats with multiple LM of DHDK12 colon cancer cells, we observed a significant reduction in tumor volume after retroviral-mediated gene transfer of either herpes simplex virus thymidine kinase (HSV1-TK) and ganciclovir (GCV) administration, or IL-12. Combined treatment with HSV1-TK/GCV and IL-12 resulted in improved tumor volume reduction and even survival. In rats with recurrent microscopic DHDK12 LM established after partial liver resection, we observed significantly decreased recurrent tumor volumes and increased survival after retroviral-mediated IL-12 gene transfer. In both settings, immunohistological analysis revealed that IL-12 immuno-gene therapy was accompanied by an infiltration of CD8+ T lymphocytes within the tumors. Altogether, our results suggest that IL-12 adjuvant gene therapy could improve the management of patients with either resectable or unresectable LM.

Poor efficiency of gene transfer into cancer cells constitutes the major bottleneck of current cancer gene therapy. We reasoned that because tumors are masses of rapidly dividing cells, they would be most efficiently transduced with vector systems allowing transgene propagation. We thus designed two replicative retrovirus-derived vector systems: one inherently replicative vector, and one defective vector propagated by a helper retrovirus. In vitro, both systems achieved very efficient transgene propagation. In immunocompetent mice, replicative vectors transduced >85% tumor cells, whereas defective vectors transduced <1% under similar conditions. It is noteworthy that viral propagation could be efficiently blocked by azido-thymidine, in vitro and in vivo. In a model of established brain tumors treated with suicide genes, replicative retroviral vectors (RRVs) were approximately 1000 times more efficient than defective adenoviral vectors. These results demonstrate the advantage and potential of RRVs and strongly support their development for cancer gene therapy.

The immunosuppressive drug cyclosporin A (CsA) represents the standard preventive treatment of graft-versus-host disease (GVHD), the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, its efficacy is only partial and many patients develop lethal GVHD despite CsA. A strategy of genetic immunosuppression based on conditional elimination of donor T cells expressing the Herpes simplex type 1 thymidine kinase (TK) suicide gene was recently developed. In this system, ganciclovir (GCV) selectively kills dividing but not quiescent TK T cells. Since CsA is known to have a cytostatic effect on T cells, it could negatively interfere with the division-dependent TK gene therapy. We thus tested whether administration of CsA would antagonize elimination of alloreactive donor TK T cells mediated by GCV in a murine model of GVHD. In vivo experiments revealed that, contrary to GCV, CsA only transiently controlled alloactivation-induced T cell proliferation, and likewise could not prevent lethal GVHD. When T cells resumed proliferation under CsA, they were however still sensitive to GCV. Survival, as well as immune reconstitution, was excellent in mice treated with GCV alone or in combination with CsA. These observations should help to design improved suicide gene therapy trials in the field of allogeneic HSCT.

The ability to control T cell reactivity using suicide genes opens new perspectives for the treatment of T cell-mediated diseases. The therapeutic effect is achieved by the selective killing of thymidine kinase gene-modified activated T cells by ganciclovir (GCV). This strategy has been shown to control T cell alloreactivity efficiently after bone marrow or solid organ transplantation. Here, we aimed to determine whether an immunopathological process induced by a viral infection could be controlled by GCV when T cells express a thymidine kinase transgene. When transgenic mice were infected with the lymphocytic choriomeningitis virus, administration of GCV resulted in an efficient, but only transient, control of the immunopathological immune response. Further analysis revealed the existence of a minute population of GCV-insensitive T cells. These cells expand in response to the virus despite the presence of GCV and cause immunopathology before viral elimination is finally obtained. Thus, when confronted with a replicative virus, the efficacy of this genetic immunosuppression strategy is highly dependent on the presence of even small numbers of GCV-insensitive cells. These results emphasize the need for sufficient preclinical investigations with regard to the pathology and the nature of the immune response if suicide gene transfer is envisioned for new therapeutic indications.

Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1 alpha (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro, as well as transgene amplification and persistence in vivo. Retrovirus titers of >10(5) infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10- to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin.

Herpes simplex virus type 1-thymidine kinase (HSV1-TK) in combination with ganciclovir is an efficient and widely used strategy in brain tumour gene therapy. Recently, we have shown effective inhibition of glioma growth in a syngeneic rat model using recombinant adenoviruses expressing the full-length HSV1-TK and an N-terminus truncated variant, HSV1-DeltaTK in the presence of ganciclovir. We also showed active chronic brain inflammation in the long-term survivors (3 months) treated with HSV1-TK plus GCV. Furthermore, our results indicated loss of myelinated fibres, oedema and indices of ongoing axonal degeneration. In this study, we assessed the cytotoxicity of both HSV1-TK variants in the presence or absence of ganciclovir, in primary cultures of neurones and glia, and in the rat brain in vivo. Our results indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotoxicity for either neurones or glial cells grown in primary cultures, (2) on its own the full-length HSV1-TK is more cytotoxic than its truncated version HSV1-DeltaTK for a population of non-neuronal and non-glial cells within neocortical primary cultures, and (3) in vivo, when delivered into the striatum, RAds encoding HSV1-TK are more cytotoxic than RAds encoding HSV1-DeltaTK, after administration of ganciclovir. The effectiveness of HSV1-DeltaTK in preventing brain tumour growth in vivo, combined with its reduced cytotoxicity, both in vivo and in primary cultures of CNS cells, could represent an advantage for treatment of brain tumours using gene therapy.

The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain. In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity. When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR). The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK. TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.

We recently described a novel nonviral/viral vector for gene transfer, the plasmovirus (Noguiez-Hellin P, Robert-le Meur M, Laune S, et al. C R Acad Sci Paris, Sciences de la Vie. 1996;319:45-50; Noguiez-Hellin P, Robert-le Meur M, Salzmann J-L, et al. Proc Natl Acad Sci USA. 1996;93:4175-4180). Plasmoviruses are plasmids capable of expressing all the viral genes required for generating infectious particles and packaging a defective genome containing a transgene. Transfected as plasmids, plasmoviruses transform the transduced cells into packaging cells that release infectious replication-defective retrovirus vectors (RV) containing a transgene, which are capable of infecting nearby cells. We previously showed that such a vector can efficiently "propagate" the transgene after transfection. Here we examine in greater detail the different steps of plasmovirus replication in vitro in human (143 B TK-) and murine (NIH 3T3 TK-) cells. Molecular-biological analysis revealed plasmovirus-coded protein expression starting from 24 hours post-transfection, followed by the detection of infectious RV 48 hours post-transfection. The gag proteins were correctly processed in the released particles. Electron microscopic analysis revealed typical type C particles. Nonintegrated plasmovirus DNA was not toxic for the cells and could be detected for at least 14 days post-transfection. While the transfected gag gene and the transgene could also be detected throughout this period, we observed that env-coded proteins decreased after 72 hours post-transfection. Nevertheless, the production of RV resulted in the propagation of the transgene in the culture, with stable integration of plasmovirus proviral DNA into the host genome of infected cells. We show that this propagation results in a major improvement in therapeutic efficacy using an HSV1-TK transgene and ganciclovir treatment, when compared to that of plasmovirus constructs that cannot propagate. Altogether, these results demonstrate the functionality of this gene transfer method and suggest that improvements in the vector design enhance its efficacy.

The integration and expression of the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene in localized tumors results in tumor regression after the administration of the specific nucleoside analogue ganciclovir (GCV). Although only 10% to 20% of the tumor cells take up the HSV1-TK gene, the neighboring cells also die, a phenomenon termed "bystander effect.". In the present study, coinjection of the MC26 mouse colon carcinoma cell line and the HSV1-TK expressing retroviral packaging cell line followed after 7 days by the intraperitoneal administration of GCV resulted in almost total tumor regression in the immunocompetent BALB/c mice but not in immunocompromised athymic BALB/c mice. This suggested a strong cell-mediated immune component to the bystander effect.

Somatic transgenesis can be used to confer endogenous production of proteins with therapeutic properties. One such product, recombinant soluble human CD4 (sCD4), has been shown to be an efficient inhibitor of human immunodeficiency virus 1 (HIV-1) in vitro, but its too short half-life in vivo has impaired long-term clinical trials in AIDS patients. Using a retroviral vector, we introduced the cDNA of sCD4 into primary mouse fibroblasts. The cells were enclosed in a lattice of collagen and synthetic fibers coated with basic fibroblast growth factor, and implanted in the peritoneal cavity of syngeneic mice. Implantation of such sCD4-secreting organoids into cyclosporin A-treated C3H mice elicited a strong antibody response against sCD4. Implantation of sCD4-secreting organoids into immunotolerant mice (transgenic for transmembrane human CD4) resulted in continuous sCD4 production, detected during 60 days in animal sera. The serum levels obtained were significant, but too limited as yet for anti-HIV purposes. Nevertheless, this model may be of interest in various fields, as it provides the first demonstration that one potentially therapeutic protein, despite its half-life of a few hours, could remain present in vivo 2 months after a single somatic transgenesis.

Many viruses, including retroviruses, are characterized by their specific cell tropism. Lymphadenopathy-associated virus (LAV) is a human lymphotropic retrovirus isolated from patients with acquired immune deficiency syndrome (AIDS) or related syndromes, that displays selective tropism for a subset of T lymphocytes defined by the expression of a surface glycoprotein of relative molecular mass 62,000 (62K) termed T4 (refs 6-8). This glycoprotein delineates a subset of T lymphocytes with mainly helper/inducer functions, while T lymphocytes of the reciprocal subset express a glycoprotein termed T8, have mainly cytotoxic/suppressor activities, and are unable to replicate LAV. Such a tropism may be controlled at the genomic level by regulatory sequences, as described for the human T-cell leukaemia viruses HTLV-I and -II (refs 2, 3). Alternatively or concomitantly, productive cell infection may be controlled at the membrane level, requiring the interaction of a specific cellular receptor with the virus envelope, as demonstrated recently for Epstein-Barr virus (EBV). Therefore, we have investigated whether the T4 molecule itself is related to the receptor for LAV. We report here that preincubation of T4+ lymphocytes with three individual monoclonal antibodies directed at the T4 glycoprotein blocked cell infection by LAV. This blocking effect was specific, as other monoclonal antibodies--such as antibody to histocompatibility locus antigen (HLA) class II or anti-T-cell natural killer (TNK) target--directed at other surface structures strongly expressed on activated cultured T4+ cells, did not prevent LAV infection. Direct virus neutralization by monoclonal antibodies was also ruled out. These results strongly support the view that a surface molecule directly involved in cellular functions acts as, or is related to, the receptor for a human retrovirus.