Naive CD4 T cells inhibit CD28-costimulated R5 HIV replication in memory CD4 T cells -- Data Supplement - Supporting Text -- Proceedings of the National Academy of Sciences

Supporting information for Mengozzi et al. (September 18, 2001) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.21120509.

Introduction

In our article, we assert that the division of M1 memory CD4 cells was not altered when these cells were cocultured with other cells, including either naïve, M1, or M2 memory cells. These data support that assertion.

Methods
Quantification of Cell Division.
After stimulation with anti-CD3/CD28 beads, carboxy fluorescein succinimethyl (CFSE)-stained cells were analyzed by flow cytometry. Nonviable cells were excluded from the analysis based on light scatter characteristics, and the percentage of cells in CFSE fluorescence peaks was determined. A corrected percentage was calculated by dividing the percentage of cells in each division peak by the total number of CFSE⁺ cells (i.e., to determine the percentage of CFSE⁺ cells within each peak); the first peak (d₀) represents no divisions; the second peak (d₁) represents a single division, and so forth. Assuming that two cells of a given CFSE intensity arise from a single mitosis of one cell with a CFSE intensity immediately greater, the initial proportion of cells that gave rise to those in the different division peaks (d₁, d₂, d₃, d₄, d₅) was calculated by dividing the percentage of cells in divisions peaks d₁, d₂, d₃, d₄, and d₅ by 2, 4, 8, 16, and 32, respectively. The average number of cell divisions (division index) was estimated by the following formula: (1d₁ + 2d₂ + 3d₃ + 4d₄+ 5d₅)/d, where d represents the sum of the cells in all peaks (including d₀). Therefore, the division index represents the number of divisions, on average, that any cell in the starting population is expected to undergo during the culture period. Results and Discussion
M1 Proliferation Rate Is Not Inhibited in Cocultures.
To investigate whether inhibition of M1 HIV replication in cocultures with M2 or naïve cells might be caused by inhibition of M1 cell proliferation, we used the dye CFSE, which covalently binds to cellular proteins and allows measurement by flow cytometry of several rounds of cell divisions.

To confirm that CFSE can be used to monitor cell division induced by anti-CD3/CD28 beads, M1 cells were stained with CFSE and analyzed after 5 days in culture with or without anti-CD3/CD28 beads (Fig. 6 Upper). Six distinct peaks of CFSE fluorescence intensity, differing by factors of 2, were detected among stimulated M1 cells. The brightest peak corresponds to undivided cells, whereas the five remaining peaks identify cells that have undergone 1-5 divisions, respectively. Unstimulated cells show uniform retention of CFSE, indicating that no cell division occurred.

We determined whether M1 cell division was inhibited in cocultures. CFSE-stained M1 cells were cocultured with unstained M2, naïve, or M1 cells at 1:1 ratio and analyzed 5 days after stimulation with anti-CD3/CD28 beads (Fig. 6 Lower Right). Six peaks of CFSE fluorescence intensity could be detected in all the different coculture conditions. The seventh peak (from right to left) represents the autofluorescence of the unstained cells cocultured with the stained M1. Analysis of the data in terms of average number of cell divisions (division index, see Materials and Methods) indicated that M1 proliferation rate was unchanged by coculture with M2 or naïve T cells (Fig. 6 Lower Left). Thus, inhibition of M1 HIV replication in cocultures is not caused by inhibition of M1 cell division.