Th2-polarised PrP-specific transgenic T-cells confer partial protection against murine scrapie

Abstract

Several hurdles must be overcome in order to achieve efficient and safe immunotherapy against conformational neurodegenerative diseases. In prion diseases, the main difficulty is that the prion protein is tolerated as a self protein, which prevents powerful immune responses. Passive antibody therapy is effective only during early, asymptomatic disease, well before diagnosis is made. If efficient immunotherapy of prion diseases is to be achieved, it is crucial to understand precisely how immune tolerance against the prion protein can be overcome and which effector pathways may delay disease progression. To this end, we generated a transgenic mouse that expresses the ß-chain of a T cell receptor recognizing a PrP epitope presented by the class II major histocompatibility complex. The fact that the constraint is applied to only one TCR chain allows adaptation of the other chain according to the presence or absence of tolerogenic PrP. We first show that transgene-bearing T cells, pairing with rearranged α-chains conferring anti-PrP specificity, are systematically eliminated during ontogeny in PrP+ mice, suggesting that precursors with good functional avidity are rare in a normal individual. Second, we show that transgene-bearing T cells with anti-PrP specificity are not suppressed when transferred into PrP+ recipients and proliferate more extensively in a prion-infected host. Finally, such T cells provide protection through a cell-mediated pathway involving IL-4 production. These findings support the idea that cell-mediated immunity in neurodegenerative conditions may not be necessarily detrimental and may even contribute, when properly controlled, to the resolution of pathological processes.

Figure 1. Expression of the TCR β-chain transgene in PrP+ and PrP– mice.

(A) Genomic PCR from a founder mouse. (B) FACS analysis of LN cells from Tg versus WT mice bred on a PrP+ or PrP– background showing histograms of the percentages of BV12⁺ T cells in gated CD4⁺ populations. (C) Over-expression of the BV12⁺ transgene starting at the single positive stage of thymic differentiation. Tg mouse (top row) and WT mouse (bottom row). (D) BV12⁺ cells within CD4⁺ or CD8⁺ LN T cells of Tg PrP+ and Tg PrP– mice. Each symbol represents an individual mouse. Horizontal bars show medians. Differences within subsets are not significant.

Figure 2. In vitro responses of CD4⁺ T cells from PrP+ or PrP– Tg mice.

(A) Proliferation against PrP_158–187 of PrP-primed CD4⁺ T cells from Tg (black circles) or WT (grey circles) PrP+ mice. T cells were collected 10 days after priming. Error bars show standard errors of triplicate cultures. Cultures were pulsed at day 5. (B) Proliferation of PrP-primed T cells from Tg or WT PrP+ mice treated with PC61 or an isotype control. T cells were challenged with 6 µM of PrP_158–187. Cultures were arrested as in (A). (C) In vitro proliferation of PrP-primed CD4⁺ T cells from Tg (black circles) or WT (grey circles) PrP– mice. Cultures were pulsed at day 3. Error bars show standard error of triplicate cultures. (D) PrP-primed CD4⁺ T cells from Tg mice respond massively and specifically to peptide PrP_158–187 but not to the irrelevant OVA_323-339 peptide. Conversely, priming with OVA_323–339 results in a moderate response to the peptide. (E) Primary responses to PrP_158–187 of naive T cells from Tg or WT PrP– mice. Numbers in legend correspond to peptide concentrations present in the micro-cultures. Cultures were pulsed at day 5. Error bars show standard deviations of triplicate wells. Each graph is representative of at least 3 independent experiments, with 3 to 4 mice per group.

Figure 3. Immunoscope analysis of TCR α-chains pairing with the BV12⁺ TCR β-chain.

(A) Focus on CDR3 diversity of TRAV13⁺ chains in naive or primed CD4⁺ cells from PrP+ and PrP– mice. (B) Identity between CDR3 sequences of TCR α-chains paired with the BV12⁺ TCR β-chain in PrP– mice and the CDR3 sequence of the original TCR α-chain identified in the T cell hybridoma. Each experiment is representative of 2 or 3 independent analyses, with a pool of at least 3 mice per test.

Figure 4. In vivo proliferation of CD4⁺ BV12⁺ cells.

(A) Dot-plot of CFSE-labeled CD4⁺ T cells after 3-day engraftment in mice boosted with PrP_158–187. Cells were gated in a Ly5.2⁺ window. (B) Same cells as in (A) but transferred into non-loaded PrP– mice. (C) Proliferation statistics on CD4⁺ BV12⁺ and BV12^- T cells displayed in (A). (D) Modelized representation of T cell divisions in various recipients. Digits in each histogram represent the “% Divide”. (E) Division indexes of CD4⁺ BV12⁺ cells transferred into various recipients: PrP– (n = 8), PrP+ (n = 11), peptide-loaded PrP– (n = 11), prion-infected (n = 12). Each symbol represents a single mouse. Horizontal bars show the means. Differences between the infected mice and the other groups were significant by one-way analysis of variance (p<0.005) and by Bonferroni's multiple comparison tests.

Figure 5. Adoptive transfer of minimal amounts of transgene-bearing CD4⁺ T cells delays scrapie onset and prolongs the clinical phase.

(A) Disease onset in untreated controls (white circles, dotted line), in recipients of BV12-negative peptide-boosted T cells (grey circles, dotted line), in non-boosted recipients of BV12⁺ cells (grey diamonds, solid line), and in boosted recipients of BV12⁺ cells (black diamonds, solid line). Differences were significant between the 4 groups by multivariate log rank test (p = 0.0073). They were also significant (p<0.005) between the two control groups and the boosted plus non-boosted recipients of BV12⁺ T cells. (B) The clinical phase of scrapie was significantly longer in mice receiving BV12⁺ CD4⁺ T cells, whether boosted or non-boosted, compared with mice receiving BV12-negative CD4⁺ T cells or left untreated (p<0.05 by Mann-Whitney test between the two groups receiving BV12⁺ T cells and the two groups receiving BV12-negative T cells or no T cells).

Figure 6. Anti-PrP T cells prevent the accumulation of PK-resistant PrP in secondary lymphoid organs.

(A) Western blots of spleen material at 90 dpi. (B) Normalization of western blots shown in (A). Numbers above bars correspond to lanes in (A).

Figure 7. Presence of infiltrating T cells in the brain of adoptively transferred mice.

(A) Frozen sections of anterior, medium, and posterior crosswise brain portions showing the presence lymphocytes staining positively with an anti-CD3 Ab. The EAE brain culled at the climax of MOG-induced disease served as a positive control (40X). (B) Summary of infiltrating T cell frequencies in brains of adoptively transferred mice. Each circle represents the frequencies of infiltrating T cell of an individual mouse, based on the analysis of at least 12 sections from anterior, median and posterior brain portions. Arrows indicate infiltration values at terminal stage while the other dots show infiltration values at 90 dpi.

Figure 8. Humoral and cell-mediated responses of adoptively transferred T cells.

(A) Anti-PrP Abs against cell-surface PrPc. Sera were collected at 90 and 120 dpi. They are presented collectively since the time of collection had no impact on MFI values. Cell-bound Abs was revealed by immunofluorescence. Black circles represent sera from non-transferred mice, black diamonds sera from mice transferred with BV12-negative T cells, gray circles sera from mice transferred with BV12⁺ T cells boosted and non-boosted, black triangles are positive controls from PrP– Tg mice primed with peptide PrP_158–187. MFIs (at the 1∶100 dilution) between positive controls and the 3 other groups were significantly different by the Kruskal-Wallis test. (B) In vitro proliferation of spleen T cells collected at 120 dpi from recipient mice. Error bars show the standard deviations of triplicates. Each column represents an individual mouse. (C) ELISPOT assay of IL-4-secreting T cells collected at 120 dpi. (D) Same as in (C) for IFN-γ secretors.