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. 2009 Jan;85(1):88-97.
doi: 10.1189/jlb.0208107. Epub 2008 Oct 10.

The cytolytic enzymes granyzme A, granzyme B, and perforin: expression patterns, cell distribution, and their relationship to cell maturity and bright CD57 expression

Pratip K Chattopadhyay  1 , Michael R Betts, David A Price, Emma Gostick, Helen Horton, Mario Roederer, Stephen C De Rosa
Affiliations

The cytolytic enzymes granyzme A, granzyme B, and perforin: expression patterns, cell distribution, and their relationship to cell maturity and bright CD57 expression

Pratip K Chattopadhyay et al. J Leukoc Biol. 2009 Jan.

Abstract

Cytolytic enzymes (CEs) are critical mediators of anti-viral and -tumor immunity; however, as a number of molecules belong to this enzyme family, our understanding of CEs remains limited. Specifically, it remains unclear what combinations of granzymes and perforin (Perf) are expressed by various immune cells and how CE content relates to cellular differentiation. Using polychromatic flow cytometry, we simultaneously measured expression of the most common human CEs [granzyme A (gA), granzyme B (gB), and Perf] alongside markers of alphabeta and gammadelta T cell maturation (CD45RO, CCR7, CD27, CD57). Additionally, we measured CE content in NK cell subsets (defined by their expression of CD16 and CD56). We found that among a wide variety of immune cells, CE content was linked to cellular maturity. Moreover, common expression patterns were shared across cell types, such that gB+ cells always contained gA, and Perf+ cells were primarily gA+ gB+. Most importantly, CD57 expression correlated strongly with simultaneous expression of gA, gB, and Perf. Thus, the use of CD57 provides a means to easily isolate viable cells with high cytolytic potential, without the need for lethal fixation/permeabilization techniques.

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Figures

Fig. 1.
Fig. 1.
Patterns of CE and CD57 expression within various cell populations from healthy donors. (A) gA, gB, Perf, and CD57 expression was compared across CD4+ and CD8+ αβ T cells and γδ T cells expressing vδ1 or vδ2 TCR genes. The left plots show expression of gA and gB, and the three columns of plots on the right show the expression of Perf and CD57 for the granzyme-defined subset. The dotted lines separate no expression, low expression, and high expression of Perf or CD57. CE expression patterns were remarkably consistent across these diverse cell populations. (B) The frequency of cells expressing various combinations of CEs is shown for seven healthy donors. The left plots show the percent of the indicated T cell subset expressing one or both granzymes. The plots on the right show the percent of the granzyme-defined subset expressing CD57 or Perf (low or high expression).
Fig. 2.
Fig. 2.
CE content relates to cellular differentiation. Patterns of CE expression are shown for various CD8+ T cell subsets from a representative donor. The top left graph shows the expression of CCR7 and CD45RO on CD8+ T cells, defining naïve cells, effector cells, and two memory subsets. For each cell type except central memory, three graphs show expression of gB versus gA (top graphs in column groups of three), Perf versus CD57 (middle graphs in column groups of three), and Perf versus CD27 (bottom graphs in column groups of three). CEs were rarely expressed within cells from the “naïve” CD45RO– CCR7+ population. Non-naïve subsets were heterogeneous for CE expression. Central memory cells had comparatively smaller gA+ gB+ populations than effector cells but had more gA+ gB– cells. Effector cells were mostly gA+ gB+ Perf+. For central memory cells, the bottom two graphs show gB versus CD27 on the left and Perf versus CD57 on the right, where two gB/CD27-defined subsets are shown as overlay on total CD8+ T cells. The few cells that coexpress Perf and CD57 are those cells that do not express CD27 (shown in red). These are likely not “true” central memory cells.
Fig. 3.
Fig. 3.
Frequency of CE-expressing cells within various CD8+ T cell subsets, across four healthy donors. (A) Using 10-color flow cytometry, cell subsets were identified according to their expression of CD45RO, CCR7, CD27, and CD57 and roughly split into naïve, central memory, memory, and effector cell populations, as shown along the x-axis. The frequency of cells within each naïve or memory/effector subset expressing a variety of CE combinations is shown for each cell subset. These data are shown from left to right and are color-coded according to the top left key. Within naïve cells, most cells lacked expression of gA, gB, and Perf. Within effector cell populations, most cells expressed gA, gB, and Perf. (B) Data are displayed by CE subset with one pie chart for each subset. The pie slices show the median frequency of cells within the CE subset expressing each combination of naïve/memory-defining markers, as shown in the bottom keys. Cool colors are used for naïve and central memory, and warm colors are used for memory and effector. The cells not expressing CEs clearly consist of mostly naïve and central memory cells, and the gA+ gB+ Perf+ populations are largely memory and effector cells.
Fig. 4.
Fig. 4.
Frequency of CE-expressing cells within CD4+ αβ, CD8+ αβ, and γδ T cell subsets in 10 healthy donors. Cell subsets were defined by expression of CD45RO and CD27 as shown in the bottom right graph; however, results were similar to those shown for the more finely defined CD8+ T cell subsets (i.e., including CCR7 to define subsets) in Figure 3. The left plots show the percent of the indicated T cell subset expressing or not expressing CD45RO and/or CD27. The middle plots show the proportion of each naïve/memory subset that is expressing the CE markers as shown in the right legend. Naïve (N) cells typically lacked CE expression, and effector cells (E) commonly expressed gA, gB, and CD57. CM, Central memory; M, other memory.
Fig. 5.
Fig. 5.
CE expression within NK cell subsets. (A) The three NK cell subsets defined by their expression of CD16 and CD56 are shown in three rows, and the expression of gB versus gA and Perf versus CD57 is shown for each. The NK subsets expressed varying levels and combinations of CEs. (B) The top plot shows the frequency of each NK subset for seven healthy donors. The middle and bottom plots show the proportion of each NK subset expressing the CEs indicated in the keys.
Fig. 6.
Fig. 6.
CE expression by antigen specificity. (A) Cells were stained with pMHCI multimers to detect T cells specific for CMV and EBV. The top and middle rows of graphs show data for one individual, and the bottom row shows data for another individual. The right graphs show the expression of Perf versus CD57 for the granzyme-defined subsets shown in the middle column of graphs. (B) CMV- and EBV-specific T cells followed the characteristic patterns of CE expression described for overall CD8+ T cell populations; however, these populations differed in CE content. The upper left graphs shows the proportion of CMV- and EBV-specific cells expressing the CEs shown on the x-axis. The upper right graphs show the proportion of the gA-expressing (Grz-defined) subsets that express CD57 or Perf. The gA– gB– CD57– subsets are not shown as a result of low frequency. The lower left graphs show the proportion of CMV- and EBV-specific cells expressing the memory markers shown on the x-axis. The lower right graphs show the proportion of the naïve/memory subsets that express the CEs as shown in the key. These differences could be accounted for by differences in maturation of CMV- and EBV-specific cells. CMV-specific cell populations, which contained more CD45RO– CD27– effector memory cells, also had higher levels of gA+ gB+ CD57+ cells (red bars) than EBV-specific T cells.
Fig. 7.
Fig. 7.
HIV-specific T cells have variable expression of granzymes and Perf. (A) Representative data from a HIV-infected subject with responses to the A03-restricted RK9 peptide. Upper graphs show overall CD8+ T cells, and lower graphs show RK9 pMHCI multimer+ cells. The left graphs show the expression of the granzymes, and the right graphs show the expression of Perf versus CD57 for the granzyme-defined subsets. The gate identifies bright Perf-staining cells. RK9-specific CD8+ T cells were typically gA+ or gB+ but rarely expressed Perf. (B) CE expression within overall CD8+ T cells (blue) or RK9-specific T cells (red) is shown for five HIV+ individuals with varying CD4+ T cell counts and viral load. The right graph shows the proportion of the gA/gB-coexpressing cells that expresses bright levels of Perf. Although trends were not statistically significant, it appeared that the RK9-specific CD8+ T cell population was enriched for gA+ gB+ cells but had lower levels of Perfbright cells than the overall CD8+ T cell population. Note that there is one individual (*) who has a relatively high proportion of RK9-specific cells expressing gB without gA. In all other individuals examined in this study, this subset was absent or present at very low frequency.
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