Figure 14 Analysis of peripheral CDS T cells of chimeric mice.
3.3 Peripheral CD8 T cells from NP47F5 are heterogeneous with respect to antigen reactivity.
3.3.3 Phenotypic and functional analysis of peripheral CD8 T cells after transfer into NP47 Ag expressing hosts.
Having analysed the response to the antigen upon transfer of naïve F5 Tg T cells to NP47 Ag expressing hosts, the phenotype and the function of the cells in the stable stage 20 days after transfer, was studied. Peripheral cells from spleen and lymph nodes from naïve F5, as well as NP47F5 were isolated and analysed for the expression of activation markers and compared to F5 T cells anergised after transfer to NP47 mice for at least 20 days.
Twenty days after transfer into NP47 mice peripheral F5 CDS T cells display an activated phenotype CD44 high, CD 62-L low, but in com parison to NP47F5 CDS peripheral T cells, the transferred cells form a homogeneous population where all the cells have high levels of CD44 expression. Also 20 days after transfer F5 CDS T cells have downregulated the expression of CD69 early activation marker (F ig.l9). This indicates that the cells have been exposed for long time to antigenic stimulation.
Characteristically F5 peripheral CDS T cells after transfer into NP47F5 mice express low levels of CD25 (IL-2 R) surface marker similarly to peripheral NP47F5 CDS T cells. F5 peripheral CDS T cells after transfer into NP47 mice were also analysed for the expression of CD5 surface marker. CD5 is a monomeric glycoprotein, m em ber o f the cystein rich receptor family, constitutively expressed on mature T lym phocytes. This surface molecule has been reported to regulate negatively naïve CD4 T cell responses to peptide MHC ligand (Smith et al., 2001) and to regulate negatively Ig receptor signalling in anergic B cells (Hippen et al., 2000). Interestingly after transfer to NP47 mice F5 CDS T cells express higher levels of C D 5 surface m arker com pared to naïve F5 CDS T cells (F ig.l9).
When the F5 CDS T cells after transfer into NP47 mice were compared with naïve F5 peripheral CDS T cells for the expression of TCR and CDS molecules, the surface levels of expression were comparable, with only a few (< 5%) transferred cells to have lower surface expression of TCR molecules. (Fig.20 A, B).
To characterise the function of peripheral CDS T cells after transfer into NP47 mice, total spleen or lymph node cells from NP47 host mice were isolated at different time points after injection of F5 CDS T cells and cultured in vitro for 4S hrs with H-2^ syngeneic DCs and different concentrations of NP6S peptide. Upon in vitro antigenic stimulation, the
recovered F5 T cells after transfer into NP47 mice, showed the characteristic unresponsive condition of strongly im paired proliferation, IL-2, IFN-y cytokines production, when compared to naïve F5 CDS T cells (Fig.20 C, D, E).
To further investigate the functional status of the peripheral CDS upon transfer into NP47 mice, cells were assessed for their capacity to produce IFN-y upon in vitro antigenic stimulation. As control the same experiment was performed on peripheral CDS cells from NP47F5 mice or on control naïve F5 and on memory F5 peripheral CDS T cells.
Analysis of the ability of peripheral NP47F5 CDS T cells to express IFN-y cytokine per cell basis by intracellular staining, confirmed that there was a small population (9.93% IFN-y positive cells) (Fig. 21 C) of NP47F5 CDS T cells able to express IFN-y cytokine in response to a 19 hours NP6S in vitro antigenic stim ulation In com parison to naïve or memory cells that are able to respond to such an antigenic stimuli (32% IFN-y positive cells) (Fig. 21 A, B) the percentage of NP47F5 CDS T cells, able to respond under these conditions by expression of IFN-y was greatly reduced.
In vitro antigenic stimulation, of F5 CDS T cells recovered from NP47 hosts, showed that these cells had impaired capacity (3% IFN-y positive) (Fig.21 D) to express IFN-y in comparison to naïve or memory F5 cells, as judged by intracellular staining. The slightly increased capacity o f NP47F5 CDS T cells to express IFN-y upon in vitro antigenic stimulation, in comparison to F5 CDS T cells recovered from NP47 hosts, may be due to peripheral NP47F5 CDS T cells that appear as naive and are not tolerised yet. These data indicate that in the adoptive transfer system there are no cells able to respond to in vitro
antigenic re-stimulation and that all cells are in the same functional status. That further supports that chronic exposure to antigen in the absence of thymic export generates homogeneous T cell population that appears anergic.
3.3.4 Summary
NP47F5 peripheral CDS T cells were shown to be heterogeneous and contain a subpopulation of cells, that are able to respond to antigenic stimulation in vivo, possibly recent thymic emigrants that were not tolerised yet.
An adoptive transfer approach of peripheral F5 CDS T cells into NP47 Ag-expressing host without endogenous T cells was applied in order to generate a homogenous anergic peripheral CDS T cell population. Peripheral CDS T cells in that system, display an activated phenotype and have all the functional characteristics of being unresponsive to antigenic stimulation in vitro, and therefore are characterised as being anergic.
Analysis of the response of naïve F5 CDS T cells after adoptive transfer to NP47 hosts show that upon antigenic stimulation in vivo cells pass through an effector expansion phase, followed by a short deletion phase after which a population of cells that has escaped deletion remains in an unresponsive state.