Granzyme B mediated function of Parvovirus B19-specific CD4(+) T cells

Abstract

A novel conception of CD4(+) T cells with cytolytic potential (CD4(+) CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19-specific CD4(+) CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19-specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19-VP2 virus-like particles. The cytolytic potential was determined by enzyme immunoassay-based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19-seropositive individuals. T-cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4(+) T cells. CD4(+) T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19-specific CD4(+) T cells showed expected co-expression of GrB, perforin and interferon gamma (IFN-γ). Unexpectedly, also a strong co-expression of GrB and interleukin 17 (IL-17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus-specific CD4(+) CTLs co-expressing CD56 antigen. Our results suggest a role for CD4(+) CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis.

Figure 1

Correlation analysis. (a, b) Correlation between B19-specific IFN-γ and GrB responses among B19-seropositive (a) and -seronegative (b) individuals. (c, d) HBoV1 versus B19-specific GrB responses among the B19-seropositive (c) and -seronegative (d) subjects. Antigen concentrations were 1.5 μg ml⁻¹. Spearman's correlation test was used.

Figure 2

B19-specific perforin responses. Perforin responses in seven B19-seropositive (open circles) and three seronegative (closed circles) subjects with B19 VP2 VLPs, Candida albicans and PHA.

Figure 3

Correlation between B19-specific GrB and perforin responses. Correlation between B19-specific GrB and perforin responses among B19-seropositive (open quadrangles) and seronegative (closed quadrangles) individuals tested at antigen concentration 1.5 μg ml⁻¹. Spearman's correlation test was used.

Figure 4

B19 VP2-specific cytolytic CD4⁺ T-cell responses after T-cell subset depletion. PBMC of seven B19-seropositive (I to VII) individuals were depleted of either CD4⁺ or CD8⁺ T cells and subjected to stimulation with B19 VP2-VLPs. T-cell responses were assessed by GrB (a) and perforin (b) ELISA.

Figure 5

Effect of HLA-specific MAbs on B19 VP2-specific cytolytic CD4⁺ T-cell responses. (a, b) PBMC of three B19-seropositive individuals were blocked with HLA class II-specific MAbs and stimulated with B19-VP2 for GrB (a) and perforin (b) assessment. (c, d) PBMC of two B19-seropositive individuals with moderate (c) or strong (d) GrB responses were blocked with HLA class I-specific MAb W6/32 or with HLA DR-specific antibody L243 (MAbs) and stimulated with B19-VP2 for GrB assessment.

Figure 6

Direct cytotoxicity versus GrB secretion among seven B19-seropositive subjects. Cells were incubated for 5 days. Cytotoxicity and GrB responses among seropositive subjects (open circles) and seronegative subjects (closed circles) were determined simultaneously. B19 VLP was used at a concentration of 1.5 μg ml⁻¹.

Figure 7

Expression of surface markers and intracellular proteins in B19-specific PBMC. (a) PBMC from a subject ‘K' were incubated for 5 days with B19-VLPs (left and right panels) or with media alone (middle panel). Then B19-specific cells with highest intracellular GrB signal and forward scatter were gated (gate K5, right panel) for further analysis. Such cells were absent in unstimulated cells stained with GrB-antibody (middle panel) or B19 stimulated cells stained with GrB-isotype control antibody (left panel). (b) IL-17, IFN-γ, perforin, CD4 and CD56 expression signals in K5-gated cells are shown. (c) Left panel: total ungated PBMC stimulated with B19 VLPs and stained with isotype controls for CD4 and CD56 antigens. Right panel: co-expression of CD4 and CD56 antigens in B19-specific PBMC in gate K5.