LPS ATP
4.2 EXPRESSION AND FUNCTION OF P2X7 ON HUMAN LYMPHOCYTES
4.2.3 P2X7 is preferentially expressed in effector and memory CD4 T cells
We next investigated at which stage of maturation/differentiation do T cells express higher levels of P2X7. To this end, we used CD27 and CD45RA to define naive (CD27+ CD45RA+), central memory (CM, CD27+ CD45RA-), effector memory CD45RA positive (TEMRA, CD27-CD45RA+) and effector memory (EM, CD27- CD45RA-) phenotypes in CD4 and CD8 T cells. In addition, we identified effector T cells based on the lack of expression of CD27 and CD28 (CD27-CD28- cells). The gating strategy for the identification of the distinct effector subsets is represented on Figure 15A. TEMRA CD4 T cells were not analysed due to insufficient cell number.
All cell subsets were significantly positive (although with low expression) for P2X7 (Figure 15B, C).
Interestingly, CM CD4 T cells showed higher expression of surface P2X7 than the whole population of CD4 T cells (Figure 15C). These results were confirmed by comparison of P2X7 staining after subtraction of IC (Figure 15D). We also observed that CM and EM cells expressed higher levels of P2X7 than naïve CD4 T cells (Figure 15D).
64 Figure 15. CD4 T cells with effector phenotype express higher levels of P2X7 than naïve cells. (A) Gating strategy for the identification of different T cell effector subtypes. (B) P2X7 expression (MFI) on the surface of naive (CD27+CD45RA+), central memory (CM, CD27+CD45RA-), effector memory (EM, CD27-CD45RA-) and effectors (CD27-CD28-) CD4 T cells. (C) Expression (MFI ± SD) of P2X7 and background signal detected on the IC on the surface of different CD4 effector subsets. (D) Expression of P2X7 (MFI ± SD) after subtraction of the MFI of the IC from the MFI of the P2X7 staining (MFI P2X7 - MFI IC) on the surface of different effector CD4 T cell effector subsets. Data are representative of one (B) or seven (C-D) healthy donors. P values were determined by paired Student’s t test, or by RM one-way ANOVA, followed by Bonferroni post-test: * = <0.05; ** = <0.01;
*** = <0.001; **** = <0.0001. Legend: blue = adaptive immune cells (C-D).
Similarly, we also determined the pattern of P2X7 expression within distinct Tregs subpopulations according to maturation status (Figure 14A); and observed that activated (HLA-DR+), memory (CD45RA) and CD39+ Tregs expressed higher levels of P2X7 than naïve Tregs (CD45RA+) (data not shown).
Since P2X7 is involved in the polarization of T cells towards the Th1 and Th17 phenotypes (Schenk et al., 2011; Salles et al., 2017), we assessed the expression of P2X7 on distinct Th subtypes defined
CD28
CD27
Effectors TCD4 A
P2X7
Counts
B
CD45RA
CD27
Naive
TEMRA TCD4 CM
EM
C
P2X7 expression [MFI]
EM
Naive CM
Effectors
Isotype control hP2X7 staining 0 T CD4 Naïve CM EM Effectors
300 600 900 1200
*** *** *** **** **
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Δ P2X7 expression [MFI]
Isotype control hP2X7 staining
T CD4 Naïve CM EM Effectors 0
300 600 900 1200
***
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**
*
65 according to expression of different chemokine receptors (Becattini et al., 2015). Th17 cells were identified as CCR6+ CCR4+ CXCR3-, Th1/Th17 cells as CCR6+ CCR4-CXCR3+, Th2 as CCR6- CCR4+ CXCR3 -and Th1 as CCR6- CCR4- CXCR3+ (Figure 16A). All T helper cell subtypes (Figure 16C) tend to express higher levels of P2X7 than whole CD4 cells, which contain the naïve subset, and P2X7 expression was significantly higher on Th1 and Th17 cells (Figure 16D).
Figure 16. Proinflammatory Th1 and Th17 cells express higher levels of P2X7. (A) Gating strategy for the identification of distinct T helper cells. (B) P2X7 expression (MFI) on the surface of Th17 cells (CCR6 -CCR4+CXCR3-), Th1/Th17 (CCR6-CCR4-CXCR3+), Th2 (CCR6+CCR4+CXCR3-) and Th1 cells (CCR6+CCR4-CXCR3+). (C) Expression of P2X7 (MFI ± SD) and background signal detected on the IC on the surface of distinct T helper cells.
(D) Expression of P2X7 (MFI ± SD) after subtraction of the MFI of the IC to the MFI of the P2X7 staining (MFI P2X7 - MFI IC) on the surface of distinct T helper cells. Data are representative of one (B) or six (C-D) healthy donors. P values were determined by paired Student’s t test, or by RM one-way ANOVA, followed by Bonferroni post-test: * = <0.05; ** = <0.01; *** = <0.001. Legend: blue = adaptive immune cells.
CD3
CD4
TCD4
FSC-H
CXCR3
CCR4
Th17
Th1/Th17 TCD4 (CCR6+) A
Th1
P2X7
Counts
Th2 Th17
B
C D
CCR6
CCR6-TCD4
CCR6+
CXCR3
CCR4
TCD4 (CCR6-) Th2
Th1
Th1/Th17
Δ P2X7 expression [MFI]
P2X7 expression [MFI]
Isotype control hP2X7 staining
TCD4 Th1 Th2 Th17 Th1/Th17 0
300 600 900
1200 ** ** **
***
**
TCD4 Th1 Th2 Th17 Th1/Th17 0
200 400 600
*
*
*
Isotype control hP2X7 staining
66 In contrast to CD4 T cells, we did not find any differences on the expression of P2X7 on the different CD8 effector subsets, neither before nor after subtraction of the IC (Figure 17).
Figure 17. All CD8 T cell subsets express similar levels of P2X7 on the cell surface. (A) Gating strategy for the identification of different T cell effector subtypes. (B) P2X7 expression (MFI) on the surface of naive (CD27+CD45RA+), central memory (CM, CD27+CD45RA-), effector memory CD45RA positive (TEMRA, CD27 -CD45RA+), effector memory (EM, CD27-CD45RA-) and effectors (CD27-CD28-) CD8 T cells. (C) Expression (MFI ± SD) of P2X7 and background signal detected on the IC on the surface of different CD8 effector subsets. (D) Expression of P2X7 (MFI ± SD) after subtraction of the MFI of the IC from the MFI of the P2X7 staining (MFI P2X7 - MFI IC) on the surface of different effector CD8 T cell effector subsets. Data are representative of one (B) or seven (C-D) healthy donors. P values were determined by paired Student’s t test, or by RM one-way ANOVA, followed by Bonferroni post-test: **** = <0.0001. Legend: blue = adaptive immune cells.
In summary, CD4 T cells with an activated, effector or memory phenotype express higher levels of P2X7 within CD4 T cells. This pattern was also observed in Tregs, even though they show very low amounts of P2X7. In contrast, the effector phenotype does not correlate with the expression of P2X7 on CD8 T cells, which expressed similar amounts of P2X7 independently of their effector phenotype.
A
B
CD28
CD27
Effectors TCD8
CD45RA
CD27
Naive
TEMRA TCD8 CM
EM
TEMRA
Naive CM EM Effectors
P2X7
Counts Δ P2X7 expression [MFI]
P2X7 expression [MFI]
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Isotype control hP2X7 staining
T CD8 Naïve CM TEMRA EM Effectors 0
300 600 900 1200
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T CD8 Naïve CM TEMRA EM Effectors 0
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Isotype control hP2X7 staining
67
4.2.4 Innate-like lymphocytes exhibit higher levels of P2X7 than conventional T cells
Since NK cells express the highest levels of P2X7 within the lymphocyte compartment, we asked whether other innate lymphocytes or innate-like lymphocytes would also express higher levels of P2X7. In humans, 1-6% of circulating T cells belong to the γδ lineage, being T cells harbouring the Vδ2 chain the most abundant (50-90%) γδ T cell subtype. The gating strategy for the identification of γδ T cells is shown in Figure 18A.T cells from the γδ lineage expressed higher levels of P2X7 on their surface when compared to conventional CD4 and CD8 T cells (Figure 18). Within γδ T cells, Vδ2+ cells were the ones expressing the highest levels among all T cell subsets (Figure 18B). After subtraction of the unspecific signal, we confirmed the higher expression of P2X7 in both γδ T cells (all) and Vδ2+ cells compared to conventional T cells and Vδ2- cells (Figure 18D).
Another T cell population with a semi-invariant TCR and innate properties are MAIT cells, identified as CD3+ Vα7.2+ CD161+ in Figure 19A. Interestingly, MAIT cells exhibited similar expression of P2X7 than γδ T cells (Figure 19 B-D), and therefore higher than conventional T cells.
We next assessed P2X7 on the whole population of innate lymphoid cells (ILCs), defined as CD3 -CD127+CD161+, and the two major NK cell subsets: NK dim (CD3- CD56dim) and NK bright cells (CD3- CD56high). The gating strategy for the identification of all immune cell populations is represented in Figure 19A. We observed higher expression of P2X7 on ILCs and both NK subsets than αβ T cells (Figure 19E, F).
68 Figure 18. γδ T cells express higher levels of P2X7 than conventional T cells. (A) Gating strategy for the identification of different immune cell populations. (B) P2X7 expression (MFI) on the surface of CD4 (CD3+CD4+), CD8 (CD3+CD8+), γδ (CD3+TCRγδ+), Vδ2+ (CD3+TCRγVδ2+), Vδ2- (CD3+TCRγVδ2-) T cells. Number of cells was normalised. (C) Expression of P2X7 (MFI ± SD) and background signal detected on the IC on the surface of distinct T cell subsets. (D) Expression of P2X7 (MFI ± SD) after subtraction of the MFI of the IC from the MFI of the P2X7 staining (MFI P2X7 - MFI IC) on the surface of T cell subsets. Data are representative of one (B) or seven (C-D) healthy donors. P values were determined by paired Student’s t test, or by RM one-way ANOVA, followed by Bonferroni post-test: ** = <0.01; *** = <0.001; **** = <0.0001. Legend: orange = innate-like lymphocytes // blue = adaptive immune cells.
TCRγδ
Vδ2
Vδ2+
Tγδ
Vδ2-TCD8 Single cells
CD3
TCRγδ
Tαβ Tγδ
TCD8
CD4
CD8 TCD4
Tαβ A
B TCD4
P2X7
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Tγδ Vδ2- Vδ2+
Isotype control hP2X7 staining
P2X7 expression [MFI] Δ P2X7 expression [MFI]
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TCD4 TCD8 Tγδ Vδ2- Vδ2⁺ 0
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TCD4 TCD8 Tγδ Vδ2- Vδ2⁺ 0
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hP2X7 staining hP2X7 staining Isotype control
69
CD3 TCRγδ
´CD3-Tαβ CD45+ cells
CD127
CD45
Contains ILCs
CD3-CD161
CD56
ILCs Contains ILCs
CD161
Vα7.2
MAIT Tαβ A
CD56 FSC-H NK dim
CD3-NK bright
B
ILCs
Tαβ Tγδ
NK bright MAIT
NK dim
P2X7
Counts Δ P2X7 expression [MFI]
P2X7 expression [MFI]P2X7 expression [MFI] Δ P2X7 expression [MFI]
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E F
Isotype control hP2X7 staining
hP2X7 staining hP2X7 staining hP2X7 staining
Isotype control Tαβ Tγδ MAIT
0 400 800 1200 1600
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Tαβ Tγδ MAIT 0
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Tαβ Tγδ ILCs NK dim
NK bright 0
400 800 1200 1600
****
**** ****
****
****
Tαβ Tγδ ILCs NK dim
NK bright 0
400 800 1200 1600
**
**
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***
70 Figure 19. Innate-like lymphocytes and innate like cells express higher levels of P2X7 than conventional T cells. (A) Gating strategy for the identification of different immune cell populations. (B) P2X7 expression (MFI) on the surface of αβ (CD3+TCRγδ-), γδ (CD3+TCRγδ+), MAIT (CD3+CD161+Vα7.2+), ILCs (CD3- CD127+ CD161+), NK dim (CD3-CD56dim) and NK bright cells (CD3-CD56high). Number of cells was normalised. (C and E) Expression of P2X7 (MFI ± SD) and background signal detected on the IC on the surface of distinct immune cells. (D and F) Expression of P2X7 (MFI ± SD) after subtraction of the MFI of the IC from the MFI of the P2X7 staining (MFI P2X7 - MFI IC) on the surface of distinct immune cells. Data are representative of one (B) or seven (C-F) healthy donors. P values were determined by paired Student’s t test, or by RM one-way ANOVA, followed by Bonferroni post-test: ** = <0.01; *** = <0.001; **** = <0.0001. Legend: red= innate immune cells // orange = innate-like lymphocytes // blue = adaptive immune cells.
Our data demonstrates that innate cells (NK cells, ILCs) and innate-like lymphocytes (MAIT cells, γδ T cells) express higher levels of P2X7 on the cell surface than conventional T cells. Therefore, P2X7 expression reveals a distinct pattern that correlates to the “innateness” of the lymphocytes.