Measuring T cell immunity to influenza vaccination in children after haemopoietic stem cell transplantation

Abstract

Quantitative assessment of immunogen-specific T cell responses may provide a meaningful surrogate marker of functional immunity in patients following haemopoietic stem cell transplantation (HSCT). We developed a flow-cytometric assay to quantify antigen-specific T cell immunity to influenza-A and studied the T cell response to influenza vaccination in five children, 3-21 months post-HSCT. All patients showed an increase in influenza-A-specific CD4(+) immunity following vaccination while none had a detectable IgG response to the vaccine. This assay proved sufficiently sensitive to evaluate changes in T cell memory in immunocompromised individuals and could be used to better characterize post-HSCT immune reconstitution.

Fig 1

Evaluating T cell immunity to influenza-A in healthy donors and HSCT patients. (A) CFSE-labelled PBMC from a representative healthy donor cultured in the absence (left) or presence (right) of influenza-A antigen (7 μg/ml) for 7 d. Plots are gated on CD8⁺ cells, and percentages refer to frequency of proliferating/activated T cells detected by loss of CFSE fluorescence and gain of HLA-DR expression. (B) CFSE-labelled PBMC from an HLA-A*0201 donor cultured as in A and stained with HLA-A*0201-MP₅₈ tetramer. Plots are gated on CD8 and numbers refer to the percentage of CD8⁺ cells in the quadrants shown. (C) CFSE-labelled PBMC from an HLA-DR*0401 donor cultured in the absence (left) or presence (right) of influenza-A antigen (7 μg/ml) for 7 d. Interleukin 2 (IL2) (5 U/ml) was added on day 3 of culture. Cells were stained with HLA-DR*0401-HA₃₀₆ tetramer. Plots are gated on CD4 and numbers refer to the percentage of CD4⁺ cells in the quadrants shown. (D) CD4⁺ cells from an influenza-A responding healthy donor were sorted based on expression of the markers shown and cultured with autologous monocytes pulsed with influenza-A antigen. Proliferation is expressed as a percentage of the maximum proliferation seen in the total CD4⁺ population. Proliferation of sorted populations with unpulsed monocytes was <0.1%. (E) CFSE-labelled PBMC from UPN 01 cultured with influenza-A before (left) or after (right) influenza vaccination. Plots are gated on CD4⁺ lymphocytes. (F) PBMC from patients obtained pre- or post-influenza vaccination were CFSE-labelled and cultured with influenza-A (left), CMV (middle) or tetanus (right) antigen for 7 d. Graphs depict the percentage of CD4⁺ cells that were CFSE dim and HLA-DR⁺ following stimulation with antigen (values for UPN 01 are shown as red circles; UPN 02, green; UPN 03, blue; UPN 04, purple; UPN 05, black). Proliferation in control conditions was <0.3% and subtracted from the corresponding value for antigen-specific proliferation.