CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

Abstract

CD39 is an ectoenzyme, present on different immune cell subsets, which mediates immunosuppressive functions catalyzing ATP degradation. It is not known whether CD39 is expressed and implicated in the activity of CD8+ regulatory T lymphocytes (Treg). In this study, CD39 expression and function was analyzed in both CD8+ and CD4+CD25(hi) Treg from the peripheral blood of healthy donors as well as from tumor specimens. CD39 was found expressed by both CD8+ (from the majority of healthy donors and tumor patients) and CD4+CD25(hi) Treg, and CD39 expression correlated with suppression activity mediated by CD8+ Treg. Importantly, CD39 counteraction remarkably inhibited the suppression activity of CD8+ Treg (both from peripheral blood and tumor microenvironment) suggesting that CD39-mediated inhibition constitutes a prevalent hallmark of their function. Collectively, these findings, unveiling a new mechanism of action for CD8+ Treg, provide new knowledge on intratumoral molecular pathways related to tumor immune escape, which could be exploited in the future for designing new biological tools for anticancer immune intervention.

Fig. 1

Phenotypes of CD8+ Treg (a) and CD4+CD25^hi Treg (b) from a healthy subject (donor # 1). CD8+ Treg were generated in vitro from purified CD8+ T lymphocytes; CD4+CD25+ Treg were directly purified from the peripheral blood. Shown data are representative of the CD8+ and CD4+CD25^hi Treg phenotypic characterization performed with cells from all the other donors. In the dotplots Treg populations are in black. The percentages of CD39+ Treg are indicated in the histograms

Fig. 2

Relationship between suppression activity and CD39 expression in Treg from healthy subjects. CD8+ Treg were generated in vitro from purified CD8+ T lymphocytes; CD4+CD25+ Treg were directly purified from the peripheral blood. a Correlation between percent suppression activity and percent expression of CD39 on CD8+ Treg from 13 healthy subjects; b Correlation between percent suppressive activity and percent expression of CD39 on CD4+CD25^hi Treg from 10 healthy subjects; c Experiment of suppression activity by CD8+ Treg treated or not with the SASI_Hs02_00318598 CD39-specific siRNA performed with cells from donor # 3. The histograms refer to the proliferation activity of a responder PBMC stimulated with an anti-CD3 mAb cultured without CD8+ Treg (left panel), with CD8+ Treg (middle panel), or with CD8+ Treg treated with the inhibitory CD39-specific siRNA for 24 h before the test (right panel). The percentages of proliferating cells are shown in all the three panels while the percentages of suppression activity are indicated between parentheses in the middle and right panel, respectively. The experiment is representative of all the experiments performed with cells from the other donors. d Counteraction of CD8+ Treg suppression activity by a CD39-specific interfering RNA. In vitro generated CD8+ Treg were transfected or not with the SASI_Hs02_00318598 CD39-specific siRNA for 24 h before being tested for their suppression activity (big panel). The percentage of CD8+ CD28-CD127^loCD39+ Treg in control cells and in cells treated with the CD39-specific siRNA is shown in the insert. The results are expressed as mean ± SD of data from 4 independent experiments performed with cells from donors # 1, 2, 3, 8, respectively. *: P = 0.04. e Histogram showing CD39 expression on control CD8+ Treg untreated (dark gray) or treated with the liposomal transfection reagent in the absence of the CD39 specific siRNA (light gray)

Fig. 3

CD39 expression on intratumoral Treg subsets. a and c Phenotypic analyses on tumor-infiltrating CD8+ (a) and CD4+CD25^hi (c) Treg purified from tumor specimens of patient # 12. Shown data are representative of the CD8+ and CD4+CD25^hi Treg phenotypic characterization performed with cells from all the other tumor specimens. The percentages of CD39+ Treg are indicated. b Comparison of CD39 MFI between CD8+ Treg ex vivo generated from the peripheral blood of healthy donors and CD8+ Treg purified from tumor specimens; data are expressed as MFI on CD8+ Treg divided by MFI of CD39- cells in order to minimize the effects of interassay variations. d Comparison of CD39 MFI between CD4+CD25^hi Treg from the peripheral blood of healthy donors and CD4+CD25^hi Treg purified from tumor specimens; data are expressed as MFI on CD4+CD25^hi Treg divided by MFI of CD39- cells in order to minimize the effects of interassay variations. e Correlation between percent suppression activity and CD39 percent expression of CD8+ Treg from tumor specimens of all 19 patients enrolled in the study; f Correlation between percent suppression activity and CD39 percent expression of CD4+CD25^hi Treg from tumor specimens of patients # 3, 4, 5, 6, 8, 9, 10, 12, 13, 14

Fig. 4

CD39 expression on intratumoral Treg subsets and its relationship with their relative function. a Suppression activity of CD8+ Treg treated or not with the SASI_Hs02_00318598 CD39-specific siRNA: the experiment was performed with cells from patient # 12, and it is representative of all the experiments performed with cells from the other patients. The histograms refer to the proliferation activity of a responder PBMC (from one healthy donor) stimulated with an anti-CD3 mAb cultured without CD8+ Treg (left panel), with CD8+ Treg (middle panel), or with CD8+ Treg treated with the inhibitory CD39-specific siRNA for 24 h before the test (right panel). A control sample was also prepared culturing the anti-CD3 mAb-stimulated PBMC with CD8+ Treg treated with the liposomal transfection reagent used to transfect the cells with the siRNA but without the siRNA itself: in all the experiments, the suppression activity of these cells was comparable to that of untreated CD8+ Treg (not shown). The percentages of proliferating cells are shown in all the three panels while the percentages of suppression activity are indicated between parentheses in the middle and right panel, respectively. b Counteraction of CD8+ Treg suppression activity by a CD39-specific siRNA. CD8+ T lymphocytes purified from tumor specimens were transfected or not with the SASI_Hs02_00318598 CD39-specific siRNA 24 h before being tested for their suppression activity. The efficient downmodulation of the CD39 expression is shown in the insert. The results are expressed as mean ± SD of data from 4 independent experiments performed with cells from patients # 8, 9, 10, 12, respectively. c Histogram showing CD39 expression on control CD8+ Treg untreated (dark gray) or treated with the liposomal transfection reagent in the absence of the CD39 specific siRNA (light gray)