Human immune-monitoring core

The human immune-monitoring core facility at Toulouse Institute For Infectious and Inflammatory Diseases (INFINITy, Inserm UMR1291 – CNRS UMR5051 – Université Toulouse III) specializes in the conservation and the characterization of human immune cells.

Our team

Head: Raphaëlle Romieu-Mourez (PhD, raphaelle.romieu-mourez@inserm.fr)

Manager of the biobanks: Pierre Emmanuel Paulet (pierre-emmanuel.paulet@inserm.fr)

Scientific Director: Roland Liblau (MD PhD, roland.liblau@inserm.fr)

 
To contact the platform, email imp.infinity@inserm.fr or phone +33 5 62 74 86 66
Steering Committee
Claire Cénac (Team J-C. Guéry)
Nathalie Jonca (Team M. Simon)
Emmanuel Treiner (Team A. Saoudi / R. Liblau)
Xavier Iriart (Team N. Blanchard)
Florence Abravanel (Team J. Izopet / B. Lagane)
Claire Detraves (Team J. van Meerwijk / O. Joffre)
Nicolas Fazilleau (Team N. Fazilleau / S. Guerder)
Loïc Dupré (Team R. Lesourne / L. Dupré)
Laurent Guilleminault (Team L. Reber)

Expertise

BIOBANKING OF PBMC INCLUDING IN CLINICAL SITUATION AND ON FRAGILE SAMPLES

BIOBANKING OF LYMPHOID TISSUES

QUALITY CONTROLS ASSOCIATED WITH BIOBANKING

AUTOMATED COUNTING OF FRESH AND CRYOPRESERVED PBMC (CELLOMETER AUTO2000, BT20, CYTOMETRY)

PHENOTYPIC AND FUNCTIONAL IMMUNOMONITORING ON CLINICAL COHORTS

CYTOMETRY

ELISPOT

DOSAGE OF BIOMARKERS WITH THE MESO SCALE DISCOVERY TECHNOLOGY

LINK WITH INSTITUTIONAL DEPARTMENTS FOR REGULATORY REQUIREMENTS OF RESEARCH ON HUMAN PERSONS

Service request

We provide biobanks of PBMC, services and trainings in biobanking and human immune-monitoring in our facilities or those of the requesting team.

Our technical facilities and procedures allow the manipulation of human samples in diverse clinical settings, including infectious ones.

Our biobanks are available to investigators within INFINITy upon approval by the platform Steering Committee. Please follow instructions on the cryovial request form PBMC request_Infinity.

Services and trainings are accessible to any research team from a public institution or a private sector.

A service is open and not “à la carte”, depending on the needs of the requesting team. Continuity can be established between the team and the platform through trainings and interventions at intervals.

The services offered by the platform are detailed below on this page. Requests for assistance by the platform can be sent by email at imp.infinity@inserm.fr

 

Services: biobanks, immune-monitoring and training

Healthy subject PBMC, plasma, DNA and lymphoid tissue biobanks

Healthy subject PBMC, plasma, DNA biobanking

  • PBMC are isolated from buffy coats provided by the “Établissement Français du Sang” (EFS) at the Purpan hospital in Toulouse, France
  • The PBMC bank is from adult volunteers donating their blood for a therapeutic use and who are recruited in the greater Toulouse – Occitania region. Donors reported feeling well and were evaluated in good general health by a doctor at the EFS. Each blood donation was monitored for the absence of permanent contraindication for blood donation (viral hepatitis, syphilis, HIV or HTLV infection)
  • The core facility provides frozen PBMC vials, each containing a minimum of 15 million PBMC. Plasma and DNA are also conserved for purposes of molecular analyses
  • Between 500 and 2000 ampoules are used each year for research on humans by INFINITy teams. 100 new donors are added annually to the biobank in order to reach a constant availability of at least 350 donors since 2015
  • At least 1 vial from each donor is thawed by the core facility and analyzed for PBMC recovery or viability. Other quality controls include the analysis of the production of interferon by memory CD8+ T cells.
  • PBMC are characterized for donor gender, age, blood type, HLA-A2 and HLA-B7 or -B8 expression, CMV and toxoplasmosis serology

 

Healthy subject lymphoid tissue biobanking

  • One of the scientific collaborations between our institution and the Toulouse University Hospital is focused on the study of pediatric lymphoid tissues, which functions and cellular composition differ from peripheral blood. The tissues analyzed are surgical wastes from pediatric tonsils and thymus obtained with the agreement of families informed of the research.
  • The platform supervises the transfer of samples and their regulatory monitoring as part of studies within INFINITy
Immune-monitoring on patient clinical samples

We perform phenotypic and functional immunomonitoring on samples of patients followed at different University Hospital Centers in France. The samples reach us fresh or cryopreserved. For example, analyzes can include: functional analysis of the Th response, standardized immunophenotyping in collaboration with the INFINITy cytometry platform, standardized ELISPOT analyzes, optimization of biobanking and cryopreservation parameters, etc.

Studies are typically carried out in 2 phases:

  • 1st phase of the project: development and validation of protocols, often based on biobanks of healthy subjects
  • 2nd phase of the project: routine implementation of protocols on targeted clinical cohort

Samples from asthmatic children, patients with an infection with the hepatitis E virus, patients hospitalized following an infection with the Sars-CoV-2 virus, and patients with narcolepsy were thus studied by the plateau.

The duration of these analyzes is inherently variable, ranging from a few weeks to several months with, if necessary, the transmission of protocols and know-how procedures to the requesting teams.

Development and standardization of immune assays on human cells

We teach on our biobanking or immunomonitoring approaches to support research on clinical cohorts.

We participate in the study of new techniques and in the evaluation of intra- and inter-experimental variations, namely between experimenters and over time.

Examples include:

  • Quality controls of potentially fragile PBMC biobanks (for instance samples transported from other regions of France, patients with certain conditions that may affect blood cells)
  • Isolation of PBMCs from small volume blood samples for pediatric studies
  • Testing of PBMC isolation kits in comparison with conventional methodologies by Ficoll
  • Comparison of automated counting methods for fresh or cryopreserved PBMCs
  • Standardization of ELISPOT analyzes
  • ELISPOT ex vivo on fresh PBMC in comparison on cryopreserved PBMC
  • Development of multiparameric cytometric profiles in collaboration with the INFINITy cytometry platform
Single and dual ELISPOT - Single and dual FLUOROSPOT

ELISPOT assay on PBMC from a patient hospitalized at the Hospital of Toulouse

The ELISPOT technology and reader are routinely used on the platform as part of fundamental or clinical studies, particularly in coordination with the Toulouse University Hospital. ELISPOT analyzes were included to the monitor effector memory responses specific for the hepatitis E virus, to evaluate the potency of RNA vaccines targeting Sars-CoV-2 in immune-suppressed individuals, to screen potential antigenic peptides, and to analyze antibody production.

We have an ELISPOT S6 Ultra-V analyzer from Cellular Technology Ltd:

  • Allows the reading of single or dual ELISPOT. Substrates are classically BCIP/NBT (blue) and/or AEC (red): spot forming cells appear blue, red, or dark purple depending on single or dual staining
  • Allows reading of single or dual Fluorospot. Filters are FITC (green) and/or PE (red): spot forming cells appear green, red, or yellow depending on single or dual staining
  • Fits all 96-well ELISPOT plates
  • Accessible without reservation

The design and realization of ELISPOTs and the use of the reader are offered by the platform in service and in training.

Quick Plex SQ120 for biomarker quantification (Meso Scale Discovery)

 

The Quick Plex SQ120 allows the assay of biomarkers with a technology based on electrochemiluminescence developed by the company Meso Scale Discovery.

 

 

The site https://www.mesoscale.com/ lists the hundreds of quantification possible in humans, primates and rodents, all of which are suitable for the Quick Plex SQ120:

  • The analyzes use several types of kits (R-plex, U-plex, T-plex, V-plex, S-plex) which all offer excellent linearity (5-log) and some the possibility of multiplexing (ex U- plex up to 10-plex)
  • Lower detection limits for U-plexes are generally around the picogram/mL. S-plexes, measuring 1 single analyte at a time, offer the gain of increased sensitivity with lower limits of detection commonly on the order of femtogram/mL
  • The analyzes can be carried out on various biological substrates such as plasma, serum, cell supernatant, tissue homgenates, cerebrospinal fluid….
  • The assays are carried out on complete or partial 96-well plates with less than 25microL of substrate by simple or duplicate assay

The use of the Quick Plex SQ120 reader is offered by the platform in service and in training. Assistance during the first dosages is not invoiced and this service is carried out by the platform and the engineers at Meso Scale Discovery (contact cchaise@meso-scale.come)

Network with hospitals and human research regulatory affairs

We act as a liaison with several institutions’ departments of regulatory affairs to ensure that the center complies with the ethical and regulatory requirements for human research. Examples: writing of patient information sheets, informed consent forms, or administrative documents related to collections of human biological samples.

Examples include the setting up (ethics, administration and logistics) of transfers of human surgical wastes for research, the declaration of research patients with multiple sclerosis or narcolepsy.

Useful links to understand the rules of clinical research in France:

Billing

TABLE SUMMARY OF INVOICING FOR EXTERNAL USERS IMP INFINITy Toulouse Purpan Grille tarifaire 2021

INFINITy teams

  • The price for the delivery of PBMC vials (at least 15 million cells) is 11 euros excluding tax per unit
  • Biobanking and immune-monitoring services are invoiced to cover the costs advanced by the platform (in most instances consumable plastics, reagents, cytometry, and hospital services in link with immune-monitoring) with an increase of 10%
  • There is no charge for using the ELISPOT reader and the Quick Plex SQ12 Meso Scale discovery reader
  • The trainings are not invoiced

 

External public research teams

  • Biobanks are not available to external members because of regulatory requirements
  • Reading of ELISPOT plates: 25 euros/plate in autonomy and 50 euros/plate when done by the facility (excluding tax and not including the plate shipping fees)
  • Reading on the Quick Plex SQ12 Meso Scale Discovery: 40 euros/plate (excluding tax). Assistance for the first dosages is not invoiced and this service is carried out by the platform and the engineers at Meso Scale Discovery (contact cchaise@meso-scale.com)
  • A quotation is made for the other services, biobanking and immune-monitoring services, and trainings

 

External private research teams

  • Biobanks are not available to external members because of regulatory requirements
  • Reading of ELISPOT plates: 70 euros/plate in autonomy and 95 euros/plate when done by the facility (excluding tax and not including the plate shipping fees)
  • Reading on the Quick Plex SQ12 Meso Scale Discovery: 50 euros/plate (excluding tax). Assistance for the first dosages is not invoiced and this service is carried out by the platform and the engineers at Meso Scale Discovery (contact cchaise@meso-scale.com)
  • A quotation is made for the other services, biobanking and immune-monitoring services, and trainings

Contact, requests and booking

– To use the biobanks, please email the plateau at least 1 week in advance the cryovial request form PBMC request_Infinity

– To use the Quick Plex SQ120, book online https://www.infinity.inserm.fr/reservations/ (section “Appareils”) and indicate if the thermoshaker will be borrowed.

– The other equipments are accessible without booking.

Other information

Publications

Healthy subject PBMC, plasma, DNA and lymphoid tissue biobanks

– Effector memory CD8 T cell response elicits Hepatitis E Virus genotype 3 pathogenesis in the elderly El Costa H, Gouilly J, Abravanel F, Bahraoui E, Peron J-M, Kamar N, Jabrane-Ferrat N, Izopet J PLoS Pathog. 2021 Feb; 17(2): e1009367

– Circulating follicular helper T cells exhibit reduced ICOS expression and impaired function in narcolepsy type 1 patients. Nguyen XH, Dauvilliers Y, Quériault C, Perals C, Romieu-Mourez R, Paulet PE, Bernard-Valnet R, Fazilleau N, Liblau R. J Autoimmun. 2018 Nov;94:134-142.

 

Immune-Monitoring on clinical cohorts and assay development

– Conventional and innate lymphocytes response at the acute phase of HEV infection in transplanted patients. Abravanel F, Barragué H, Dörr G, Sauné K, Péron JM, Alric L, Kamar N, Izopet J, Champagne E.J Infect. 2016 Jun;72(6):723-730.

Protocols and technical booklets

Documents are available on request imp.infinity@inserm.fr

PBMC isolation and cryopreservation

PBMC thawing

Fresh versus frozen PBMC counting (BT20 counter, Cellometer auto2000 counting, cytometry)

ELISPOT technology to numerate effector memory T cells

Booklet “ELISPOT reader”

Booklet “Introduction to Meso Scale Discovery technology and Quick Plex SQ120 reader for the dosage of biomarkers”

Inserm

Inserm

CNRS

CNRS

UT3

UT3