Benefits of Using CD45RA and CD28 to Investigate CD8 Subsets in Kidney Transplant Recipients.

The deleterious role of CD8 T cells in kidney graft outcome has regained interest over the years, and memory T cells are considered as one of the main hurdles to achieve transplantation success. Monitoring the CD8 immune response in transplant recipients involved a heterogeneous combination of markers, but the justification of their choice is rarely stated. Whereas the number of parameters is not an issue in phenotypic analysis, functional assays have to accommodate the cell number with the narrowing of the subset. The aim of the study was to investigate the similarities and differences of the subsets identified using three nomenclatures (CD45RA and CCR7/CD27/CD28) in kidney transplant recipients with stable graft function. We found that all three nomenclatures can identify naïve and effector memory (EM) rheumatoid arthritis T cell CD8 with similar features. Whereas CM CD8 could only be documented using CCR7 and CD45RA, the characteristics of EM CD8 will differ according to the nomenclature. We found that the use of the CD45RA and CD28 gives the benefit of examining two EM populations at early and late differentiation states. This systematic comparison provides a cohesive layout of the advantages of using these nomenclature strategies in kidney transplant recipients to guide the choice of their use.

Autologous dendritic cells prolong allograft survival through Tmem176b-dependent antigen cross-presentation.

The administration of autologous (recipient-derived) tolerogenic dendritic cells (ATDCs) is under clinical evaluation. However, the molecular mechanisms by which these cells prolong graft survival in a donor-specific manner is unknown. Here, we tested mouse ATDCs for their therapeutic potential in a skin transplantation model. ATDC injection in combination with anti-CD3 treatment induced the accumulation of CD8(+) CD11c(+) T cells and significantly prolonged allograft survival. TMEM176B is an intracellular protein expressed in ATDCs and initially identified in allograft tolerance. We show that Tmem176b(-/-) ATDCs completely failed to trigger both phenomena but recovered their effect when loaded with donor peptides before injection. These results strongly suggested that ATDCs require TMEM176B to cross-present antigens in a tolerogenic fashion. In agreement with this, Tmem176b(-/-) ATDCs specifically failed to cross-present male antigens or ovalbumin to CD8(+) T cells. Finally, we observed that a Tmem176b-dependent cation current controls phagosomal pH, a critical parameter in cross-presentation. Thus, ATDCs require TMEM176B to cross-present donor antigens to induce donor-specific CD8(+) CD11c(+) T cells with regulatory properties and prolong graft survival.

Cell therapy with autologous tolerogenic dendritic cells induces allograft tolerance through interferon-gamma and epstein-barr virus-induced gene 3.

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)-based therapy is a promising approach for induction of antigen-specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon-gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαß(+) CD3(+) CD4(-) CD8(-) NKRP1(-) cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein-Barr virus-induced gene 3 (EBI3), an IL-12 family member. EBI3 blockade or knock-down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivo led to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC-based therapy in transplantation as well as in the patient immunomonitoring follow-up.

Short communication: Mouse mammary tumor virus driven α-lactalbumin expression effects on lactation and fertility of transgenic mice.

α-Lactalbumin (Alac) is one of the major milk proteins. Its gene expression is restricted to epithelial cells of the lactating mammary gland. The Alac interaction with a uridine 5'-diphosphate-galactosyltransferase induces lactose synthesis, a major osmotic regulator of milk secretion. Other functions attributed to this protein include induction of apoptosis and anti-inflammatory activities. To assess if forced expression of this gene during early gestation or involution could affect mammary physiology, an Alac-encoding minigene was expressed in transgenic mice under the transcriptional regulation of the mouse mammary tumor virus promoter. The mammary expression did not interfere with gestation, resulted in a slight increase in milk yield as indirectly assessed by the 11% increased growth rate of the pups reared by transgenic females compared with that of those reared by control mice, and induced a slight delay in the early involution process, as demonstrated by histological analyses. The use of the mouse mammary tumor virus promoter resulted in Alac expression in several nonmammary tissues, such as the brain, the testis, the ovary, and the uterus. Although it did not affect male reproductive performances, it induced a female subfertile phenotype, characterized by embryonic implantation failure in the transgenic female reproductive tract.

Compliance in the neck structures of the guinea pig spermatozoon, as indicated by rapid freezing and electron microscopy.

Electron microscopy has been used to investigate whether the transversely striated columns of the connecting piece in the neck region of guinea pig spermatozoa, undergo lengthening and shortening as a result of the forces generated during motility. Motile spermatozoa were subjected to near-instantaneous rapid freezing, followed by freeze-substitution fixation and epoxy embedment. Thin sections passing longitudinally through the striated columns revealed that the periodicity was indeed variable. The repeat period, taken to have an unstressed width of 60 nm, could be found extended to 75 nm in some specimens, and reduced to 54 nm in others. The estimates of the coefficients of variation were 6.6% for the width of the 'dense' band and 33.5% for the 'pale' band. The 'pale' band in the extended state showed longitudinal striae. Such variations in length, which - it is suggested - are physiological, and passively induced, would have functional implications for the flagellum - for both bend initiation and bend growth. Also, hypothetically, any mechanism that could increase the degree of compliance in these columns, such as perhaps phosphorylation of the constituent proteins, could permit the flagellum to develop the exaggerated bend angles and asymmetries of the 'hyperactivated' state.

Unexpected high testis-specific transcriptional activity of the cyclin T1 promoter in transgenic mice.

The ubiquitously expressed cyclin T1 gene encodes for a protein involved in human immunodeficiency virus type 1 (HIV-1) transcription activation. The goat gene was recently shown to share an expression pattern similar to that of its endogenous counterpart when incorporated into mice using a BAC insert. To assess if its promoter could target ubiquitous expression of the bovine Prnp in transgenic mice, two constructs carrying either 1 or 30 kb of cyclin T1 5'-flanking sequences were built and microinjected. Both constructs resulted in the unexpected high male germ cell-specific expression of the prion protein. These data re-question the suspected location of the cyclin T1 gene regulatory elements.

Goat PRND expression pattern suggests its involvement in early sex differentiation.

Expression of the goat prion protein gene locus was assessed by reverse transcriptase-polymerase chain reaction on testes and ovaries at various developmental stages. A weak and stochastic expression of the PRNP and PRNT genes was observed. For PRNT, it is consistent with the detected deletions of two single nucleotides within its open reading frame in ruminant genes. PRND was expressed in both tissues at all stages. Whereas its expression is constant in the ovaries, it increases in testes between 36 and 46 days postcoitum (dpc) and remains high thereafter. In testes, Doppel was found in the nucleus of germinal cells and in the cytoplasm of Leydig cells at 44 dpc. It was detected in the cytoplasm of Leydig cells and of some Sertoli and germinal cells at 62 dpc. In the ovaries, it was observed in the nucleus of germinal cells at 44 dpc and mainly in their cytoplasm at 62 dpc. This expression pattern was shown to parallel that of C-kit and suggests Doppel involvement in early testis differentiation.

Efficient and specific down-regulation of prion protein expression by RNAi.

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.