Recombinant Modified Allergens for Immunotherapy Design and Testing by means of an In Vitro System
Coordinating Institution:
Centre Hospitalier de Luxembourg ,
CRP Sante
From: 01/07/2002
To: 31/12/2005
Budget: 490,000.00€
Contact(s):
Hentges François
Summary
The goals of this project were to study the mechanisms of immune response and tolerance in order to acquire the skills and tools to make allergic persons tolerant to the allergens they are sensitised to. The problem addressed was the allergic sensitisation to the major cat allergens Fel d1 (an uteroglobin) and cat serum albumin (CSA). Analysis of the cellular immune response at the T cell level, the crossroad of all cognate immune responses, was performed by an in vitro assay where T cells were stimulated by matured allergen–pulsed myeloid dendritic cells (DCs). This allowed the definition of T cell epitopes (the allergen parts recognised by T lymphocytes).
During the first year, the human T cell response to 12 recombinant fragments of CSA was studied for 35 persons, according to their different HLA-class II specificities. During the second year, the in vitro proliferation of splenocytes from naïve and immunised BALB/c mice was studied for Fel d1 (by overlapping peptides) and CSA (by 12 recombinant fragments) presented by myeloid DCs. First data on natural regulatory T cells was obtained.
In 2005, focus was given on the regulation of immune responses to CSA and Fel d1. The role of natural T regulatory cells (nTregs) was primarily addressed and that of inducible T regulatory cells (iTregs) secondarily.
The cellular proliferation of unseparated splenocytes, separated T helper cells, nTregs, in chosen compositions, was tested. The tested cells originated from naive or immunised mice. Tests were performed with DCs pulsed with the major CSA epitopes (fragments IV, V, VI), and with Fel d1 and its major epitope F1.4. Culture supernatants were screened on day 1, 2, 3, and 4 for cytokine secretion (IFN-gamma, IL-10, IL-2, IL-4, IL-12, and IL-5). nTregs were able to suppress proliferation of T helper cells in a quantitative manner, proliferation inhibition being complete for a ½ ratio (nTregs/T helper). Cytokine secretion was independent of T cell proliferation inhibition and differed depending on the cytokines tested.
For the investigation of iTregs in mice, a hybrid MSA-CSA (CSA fragment V on a mouse SA backbone) was constructed and tested. To make further progress in nTreg analysis, the in vitro amplification of nTregs of desired specificity in quantities allowing to test their regulatory effect in vivo is a major goal.