David Laurin

Phenotype Frequencies of Autosomal Minor Histocompatibility Antigens Display Significant Differences among Populations

Minor histocompatibility (H) antigens are allogeneic target molecules having significant roles in alloimmune responses after human leukocyte antigen–matched solid organ and stem cell transplantation (SCT). Minor H antigens are instrumental in the processes of transplant rejection, graft-versus-host disease, and in the curative graft-versus-tumor effect of SCT. The latter characteristic enabled the current application of selected minor H antigens in clinical immunotherapeutic SCT protocols. No information exists on the global phenotypic distribution of the currently identified minor H antigens. Therefore, an estimation of their overall impact in human leukocyte antigen–matched solid organ and SCT in the major ethnic populations is still lacking. For the first time, a worldwide phenotype frequency analysis of ten autosomal minor H antigens was executed by 31 laboratories and comprised 2,685 randomly selected individuals from six major ethnic populations. Significant differences in minor H antigen frequencies were observed between the ethnic populations, some of which appeared to be geographically correlated.

Photochemotherapy Induces the Apoptosis of Monocytes Without Impairing Their Function

Background. Extracorporeal photopheresis (ECP) is a powerful therapy currently used to treat various hematological disorders as in graft versus host disease. Clinical data clearly demonstrate its efficacy and immunomodulation toward the pathogenic T cells. However, ECP mechanism of action is still poorly understood. Monocytes represent up to 30% of the total amount of treated cells and are known to play an important role in adaptive immunity. However, data from previous reports analyzing the effect of psoralen and UV-A irradiation (PUVA) on their functions are heterogeneous. In this study, we focused on the effect of PUVA on human monocytes functions in adaptive immunity. Design and Methods. Purified human monocytes were treated in vitro by PUVA. We measured their kinetic of apoptosis after the treatment. We also determine whether their phenotype and functionalities were modified. Finally, we assessed the functionalities of PUVA-treated monocytes-derived dendritic cells (DC). Results. PUVA treatment sentenced purified monocytes to die in 6 days and immediately altered their migratory capacities without impairing their ability of endocytosis. It also up-regulated co-stimulatory molecules and production of inflammatory cytokines on activation and consequently stimulated allogeneic or autologous T cells as efficiently as untreated monocytes. Moreover, PUVA-treated monocytes retained their ability to differentiate into fully functional DC that maturated and stimulated T cells as well as normal DC. Conclusions. Our data demonstrate that monocytes undergo apoptosis and loose a part of their migratory capacity after ECP and the surviving cell functionalities are not impaired, suggesting that monocytes have a minor effect on ECP-mediated immunomodulation.

Immunomonitoring of graft-versus-host minor histocompatibility antigen correlates with graft-versus-host disease and absence of relapse after graft

BACKGROUND: After HLA‐identical hematopoietic stem cell transplantation, minor histocompatibility (mH) antigen alloreactivity plays a dominant role in the development of graft‐versus‐host disease (GVHD) and graft versus leukemia (GVL).

Poly-functional and long-lasting anticancer immune response elicited by a safe attenuated Pseudomonas aeruginosa vector for antigens delivery

Live-attenuated bacterial vectors for antigens delivery have aroused growing interest in the field of cancer immunotherapy. Their potency to stimulate innate immunity and to promote intracellular antigen delivery into antigen-presenting cells could be exploited to elicit a strong and specific cellular immune response against tumor cells. We previously described genetically-modified and attenuated Pseudomonas aeruginosa vectors able to deliver in vivo protein antigens into antigen-presenting cells, through Type 3 secretion system of the bacteria. Using this approach, we managed to protect immunized mice against aggressive B16 melanoma development in both a prophylactic and therapeutic setting. In this study, we further investigated the antigen-specific CD8+ T cell response, in terms of phenotypic and functional aspects, obtained after immunizations with a killed but metabolically active P. aeruginosa attenuated vector. We demonstrated that P. aeruginosa vaccine induces a highly functional pool of antigen-specific CD8+ T cell able to infiltrate the tumor. Furthermore, multiple immunizations allowed the development of a long-lasting immune response, represented by a pool of predominantly effector memory cells which protected mice against late tumor challenge. Overall, killed but metabolically active P. aeruginosa vector is a safe and promising approach for active and specific antitumor immunotherapy.

Targeted release of transcription factors for cell reprogramming by a natural micro-syringe

Ectopic expression of defined transcription factors (TFs) for cell fate handling has proven high potential interest in reprogramming differentiated cells, in particular for regenerative medicine, ontogenesis study and cell based modelling. Pluripotency or transdifferentiation induction as TF mediated differentiation is commonly produced by transfer of genetic information with safety concerns. The direct delivery of proteins could represent a safer alternative but still needs significant advances to be efficient. We have successfully developed the direct delivery of proteins by an attenuated bacterium with a type 3 secretion system that does not require challenging and laborious steps for production and purification of recombinant molecules. Here we show that this natural micro-syringe is able to inject TFs to primary human fibroblasts and cord blood CD34+ hematopoietic stem cells. The signal sequence for vectorization of the TF Oct4 has no effect on DNA binding to its nucleic target. As soon as one hour after injection, vectorized TFs are detectable in the nucleus. The injection process is not associated with toxicity and the bacteria can be completely removed from cell cultures. A three days targeted release of Oct4 or Sox2 embryonic TFs results in the induction of the core pluripotency genes expression in fibroblasts and CD34+ hematopoietic stem cells. This micro-syringe vectorization represents a new strategy for TF delivery and has potential applications for cell fate reprogramming.

Targeted release of transcription factors for human cell reprogramming by ZEBRA cell-penetrating peptide

Transcription factors (TFs) are key actors of the control of gene expression and consequently of every major process within cells, ranging from cell fate determination, cell cycle control and response to environment. Their ectopic expression has proven high potential in reprogramming cells for regenerative medicine; ontogenesis studies and cell based modelling. Direct delivery of proteins could represent an alternative to current reprogramming methods using gene transfer but still needs technological improvements. Herein, we set-up an efficient cellular penetration of recombinant TFs fused to the minimal transduction domain (MD) from the ZEBRA protein. We show that ZEBRA MD-fused TFs applied on primary human fibroblasts and cord blood CD34+ hematopoietic stem cells route through the cytoplasm to the nucleus. The delivery of Oct4, Sox2 and Nanog by MD leads to the activation of mRNA transcripts from genes regulated by these TFs. Moreover, the expression of genes involved in the pluripotency network but not directly bound by these TFs, is also induced. Overall, the repeated application of MD-Oct4, MD-Sox2, MD-Nanog TFs and the post-transcriptional regulator RNA-binding protein MD-Lin28a, triggers the rejuvenation of human fibroblasts and CD34+ cells. This study provides powerful tools for cell fate reprogramming without genetic interferences.

Extracorporeal Photopheresis: Scientific and Technical Considerations for Improving Clinical Management of Patients

Extracorporeal photopheresis or chemophototherapy (ECP) is a current therapy and concept that has demonstrated clinical efficacy in the treatment of acute and chronic graft versus host disease (GVHD), Sezary syndrome or cutaneous T-cell lymphoma (CTCL) and organ transplant rejection. Patient inclusion in ECP protocols increases due to the diffusion of technical progress but has to be explored regarding various schedules and processes. ECP mechanisms of action are still unclear despite evident implication of a complex cascade beginning from the apoptosis of the various cell types collected and exposed to the process that is followed by an immunomodulation via antigen presentation. Exploration of the mechanisms in animal models has many limits that weakness human clinical link. Beside major indications, the barrier to ECP treatment extension is the absence of randomized trials in comparison to alternative therapies. The cost of ECP treatment is also a limitation and has to be evaluated relative to other strategies. Nevertheless, the efficacy and the absence of major side effects deserve attention, mainly for the extension of the ECP treatment in clinical trials as new therapeutic line.

Gender influences the birth order effect in HLA-identical stem cell transplantation

To the editor: An earlier report by Bucher et al[1][1] indicated a better stem cell transplant (SCT) outcome with younger sibling donors (YSD). Transmaternal cell flow during pregnancy[2][2] might explain this observation. Notably, cord blood comprises minor H antigen experienced T cells that are

Killed but metabolically active Pseudomonas aeruginosa -based vaccine induces protective humoral- and cell-mediated immunity against Pseudomonas aeruginosa pulmonary infections

Pseudomonas aeruginosa (Pa) is a significant cause of morbidity and mortality, especially in cystic fibrosis patients. Its eradication is difficult due to a wide phenotypic adaptability and an increase of its resistance to antibiotics. After the failure of several recombinant vaccines which mainly triggered humoral response, live-attenuated vaccines received attention thanks to their ability to elicit a broad immunity with both humoral- and cell-mediated responses, essential to fight this pathogen. In this study, we developed an innovative and safer live-attenuated Pa vaccine based on a Killed But Metabolically Active (KBMA) attenuation method. KBMA Pa has been further rationally designed to overexpress beneficial effectors like the type 3 secretion system apparatus. We demonstrated that KBMA Pa elicits a high and broad humoral response in mice against several antigens of particular interest such as OprF and PcrV proteins. Moreover, we assessed cytokines in the serum of immunized mice and showed that KBMA Pa elicits Th1, Th2 and especially Th17 pathways of cell-mediated immune responses. Th17 pathway involvement was also confirmed after specific stimulation of helper T cells in immunized mice. Finally, we showed that this vaccine is safe and has a protective effect in a murine acute pulmonary infectious challenge. In conclusion, KBMA Pa is a new platform with high potential for the development of a vaccine against Pa.

Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry

A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45) and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.