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. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Hypertension. 2024 Jan 9;81(3):530–540. doi: 10.1161/HYPERTENSIONAHA.123.21819

P2X7-Mediated Antigen-Independent Activation of CD8+ T Cells Promotes Salt Sensitive Hypertension

Lance N Benson 1, Katherine S Deck 1, Christoph J Mora 1, Yunping Guo 1, Tonya M Rafferty 1, Lin-Xi Li 2, Lu Huang 2, J Tucker Andrews 2, Zhiqiang Qin 3, Daniel W Trott 4, Robert S Hoover 5, Yunmeng Liu 1,*, Shengyu Mu 1,*
PMCID: PMC10922507  NIHMSID: NIHMS1955456  PMID: 38193292

Abstract

Background:

CD8+ T cells (CD8Ts) have been implicated in hypertension. However, the specific mechanisms are not fully understood. In this study, we explore the contribution of the P2X7 receptor to CD8T activation and subsequent promotion of sodium retention in the kidney.

Methods:

We employed mouse models of hypertension. Wild-type (WT) were used as genetic controls, OT1 and Rag2/OT1 mice were utilized to determine antigen dependency, and P2X7-KO mice were studied to define the role of P2X7 in activating CD8Ts and promoting hypertension. Blood pressure was monitored continuously, and kidneys were obtained at different experimental endpoints. Freshly isolated CD8Ts from mice for activation assays and ATP stimulation. CD8T activation-induced promotion of sodium retention was explored in co-cultures of CD8Ts and mouse DCTs.

Results:

We found that OT1 and Rag2/OT1 mice, which are nonresponsive to common antigens, still developed hypertension and CD8T-activation in response to DOCA/salt treatment, similar to WT mice. Further studies identified the P2X7 receptor on CD8Ts as a possible mediator of this antigen-independent activation of CD8Ts in hypertension. Knockout of the P2X7 receptor prevented calcium influx and cytokine production in CD8Ts. This finding was associated with reduced CD8T-DCT stimulation, reversal of excessive salt retention in DCTs, and attenuated development of salt-sensitive hypertension.

Conclusion:

Our findings suggest a novel mechanism by which CD8Ts are activated in hypertension to exacerbate salt retention and infer that the P2X7 receptor on CD8Ts may represent a new therapeutic target to attenuate T cell-mediated immunopathology in hypertension.

Keywords: Hypertension, salt, T cells, Kidneys, P2X7, ATP, cytokines

Graphical Abstract

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Introduction:

T cells play an important role in the development of hypertension1–4. Recent studies from our laboratory also suggest a specific involvement of the CD8+ T cells (CD8Ts) in the pathogenesis of hypertension. We found that CD8Ts infiltrate the kidney and directly interact with distal convoluted tubular cells (DCTs). The subsequent upregulation of the sodium chloride cotransporter (NCC) results in enhanced sodium retention5,6.

Our studies also uncovered a potential mechanism that CD8Ts from hypertensive mice exhibit higher activity and production of interferon-gamma (IFNγ), which in turn primes the DCTs to express programmed death-ligand 1 (PDL1) and major histocompatibility complex6. These molecules co-signal to CD8Ts, resulting in an enhanced synapse-like direct interaction between the two cell types. This pathological interaction stimulates NCC, exacerbating hypertension7. These findings corroborate earlier reports that CD8Ts from hypertensive patients produce higher levels of IFNγ8. Moreover, they add insight into the events mediating activation of CD8Ts and subsequent IFNγ production, thereby contributing to the “defect” in the hypertensive kidney initially proposed by Guyton that impairs sodium excretion9,10. However, the precise stimuli responsible for CD8T activation during hypertension remain unclear. Therefore, the goal of the current study was to explore potential mechanisms leading to CD8T activation in hypertension.

Early study from our co-author has identified an oligoclonal population of CD8Ts invading the kidneys of hypertensive animals11. The presence of this oligoclonal population within the kidney indicates two non-exclusionary possibilities: 1) multiple unique antigens are presented driving CD8T activation and renal invasion, and/or 2) the activation of CD8Ts during hypertension is driven by a non-antigen dependent mechanism. Although several potential neo-antigens have been proposed that possibly activate T cells in hypertension12–14, the possibility of non-antigen-driven CD8T activation in hypertension has not been explored extensively. This limitation narrows our scope of viable therapeutic targets. Additionally, non-antigen-driven CD8T activation independent of T cell receptors (TCR) has been identified in multiple inflammatory pathways with relevance to diverse disease models15,16, inferring that such a mechanism may be physiologically feasible in hypertension.

In this study, we employed OT1 mice, in which the transgenic TCR Vα2 and Vβ5 subunits precisely recognize only ovalbumin peptide SIINFEKL17,18. Given the evidence that in hypertensive animals and humans, CD8Ts exhibit higher activity6,8, we expect that challenging the OT1 mice with DOCA/salt in the absence of exposure to the ovalbumin antigen will allow us to determine whether the activation of CD8Ts during salt-sensitive hypertension relies on the classic antigen-TCR mechanism.

As further elaboration, elevation of cytosolic free calcium is essential and often sufficient for T cell activation19,20, inferring that calcium-permeable channels in the plasma membrane could potentially mediate non-antigen-driven activation of T cells. One such calcium channel, the P2X7 receptor, has been implicated in hypertension based on the demonstration that global knockout of this receptor blunts blood pressure (BP) elevation in three different mouse models of hypertension21,22. Nevertheless, the key cellular events by which P2X7 promotes BP elevation have not been identified. The specific physiological ligand for the P2X7 receptor is extracellular ATP (exATP). ExATP is a well-established pro-inflammatory signaling molecule that reportedly is increased in pre-clinical models and clinical cases of hypertension22. It has been demonstrated that P2X7 receptor contributes to CD8T auto-reactivity23; however, the relevance of the P2X7 receptor expressed by CD8Ts to the pathogenesis of hypertension has not been established. In this study, we propose a novel mechanism whereby exATP stimulation of the P2X7 receptor on CD8Ts results in their non-antigen-driven activation and production of IFNγ, which in turn, promotes the interaction of CD8Ts with renal tubular cells to induce NCC upregulation, increased sodium retention, and exacerbation of hypertension.

Materials and Methods:

All data in this study, analytical methods, and materials are available from the corresponding author upon reasonable request. Detailed methods & materials are available in the supplementary materials.

Results:

OT1 mice treated with DOCA/salt exhibit hypertension and enhanced CD8T activation.

In preliminary studies, we found that OT1 mice still exhibit similar BP elevations as WT mice in response to the administration of DOCA/salt despite the absence of the ovalbumin antigen (Figure S1). Consistent with our previous finding in WT mice6, CD8Ts isolated from DOCA/salt-treated OT1 mice demonstrated higher cytokine production compared to sham-treated OT1 mice (Figure S2a, gating strategy shown in Figure S3), indicating greater activation. Moreover, the mDCTs co-cultured with CD8Ts from hypertensive OT1 mice showed increased NCC-mediated sodium retention compared to those co-cultured with sham OT1 CD8Ts (Figure S2b). These results imply that even in the absence of ovalbumin-mediated classical antigen-TCR signaling, the OT1 CD8Ts exhibit activation in hypertension and drive increased NCC-mediated sodium retention in DCTs, events also observed by us earlier in wild-type CD8Ts5,6. This finding raises an exciting prospect that a non-antigen-dependent mechanism may be responsible for driving the BP-induced activation of CD8Ts that exacerbate hypertension.

To further consolidate this preliminary finding, we employed Rag2-KO+OT1-transgenic mice (Rag2/OT1), which exclusively possess OT1 CD8Ts without other T cell subtypes. As expected, DOCA/salt treatment (without ovalbumin peptide) induced blood pressure elevation in these mice (Figure 1A), similar to WT mice (Figure S4). Additionally, it led to increased renal infiltration of OT1 T cells into their kidneys compared to sham Rag2/OT1 mice (Figure 1B). Interestingly, staining the Rag2/OT1 T cells with post-activation marker CD44, we found that sham mice displayed approximately less than 10% of CD44 positive OT1 CD8Ts, a ratio increased to over 40% in DOCA/salt-induced hypertensive mice (Figure 1C). Furthermore, the enhanced activity of OT1 CD8Ts induced by DOCA/salt was confirmed by increased production of cytokines IFNγ and TNFα (Figure 1D).

Figure 1: Rag2/OT1 mice exhibit hypertension, T-cell activation and renal infiltration upon DOCA/salt treatment, despite the lack of antigen.

Figure 1:

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A) Radio-biotelemetry was used to continuously monitor BP in Rag2/OT1 mice treated with DOCA/salt. Average systolic BP of baseline (days 0–3, Base, Blue) and endpoint (days 16–19, End, Red) were compared using t-test, n=5–6 per group. B) Flow cytometry study to examine T cells infiltrated into the kidney of Sham or DOCA/salt-treated Rag2/OT1 mice at the end point of a. n=5–6, gating strategy include singlets – cells – FVD – CD3, as shown in Figure S3. C) Flow cytometry was used to identify CD44-positive OT1 T cells in the spleens of Sham or DOCA-treated Rag2/OT1 mice. quantification compared using t-test, n=5–6, gating strategy shown in Figure S3. D) Cytokine production was determine in OT1 T cells isolated from hypertensive or normotensive Rag2/OT-1 mice using realtime-PCR. Data analyzed by t-test, n = 5–6 per group.

Collectively, these results suggest that an antigen-independent mechanism may play an important role in driving the activation of CD8Ts during the development of salt-sensitive hypertension, potentially contributing to the exacerbation of the condition.

Stimulation of exATP-P2X7 receptor increase IFNγ & TNFα production in CD8Ts.

To screen for the events responsible for the unconventional activation of CD8Ts in hypertension, we treated CD8Ts isolated from control wild-type mice with a pathophysiological concentration of different molecules commonly associated with hypertension, particularly salt-sensitive hypertension, to evaluate their ability to stimulate cytokine production in CD8Ts. As shown in Figure 2, treatment with additional NaCl, norepinephrine, angiotensin-II, or aldosterone failed to stimulate IFNγ transcription in our CD8Ts. In contrast, extracellular ATP (exATP), which has been reported to be increased in the circulation in clinical and experimental hypertension22, increased IFNγ transcription in our cultured CD8Ts (Figure 2A). This effect was also observed after the addition of the P2X7 receptor selective agonist BzATP (Figure 2A), implying that the P2X7 receptor may be the dominant P2X receptor subtype involved in CD8T activation. To confirm the involvement of the P2X7 receptor in CD8T activation, we treated CD8Ts isolated from WT or P2X7 KO mice with exATP or BzATP for 3 hours. Consistent with our hypothesis, both extracellular ATP and BzATP increased IFNγ and TNFα transcription in CD8Ts from WT, but not in P2X7 KO mice (Figure 2B–E). As expected, CD8Ts from OT1 mice also exhibited increased transcription of IFNγ and TNFα following P2X7 stimulation (Figure S5a–b). It is worth noting that, this ATP-P2X7 mediated activation and cytokine production is CD8T-specific, because the same ATP treatment to CD8-negative T cells failed to stimulate higher production of IFNγ and TNFα (Figure S6). These results suggest that stimulation of the P2X7 receptor is sufficient to increase IFNγ transcription in CD8Ts, but not other T cells, and P2X7 deficient CD8Ts do not exhibit increased cytokine transcription in response to extracellular ATP.

Figure 2: Extracellular ATP stimulates cytokine production in WT but not P2X7 KO CD8Ts.

Figure 2:

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A) Mouse CD8Ts were stimulated by 40 mM NaCl, 10 μM Norepinephrine, 100 nM angiotensin II, 100 nM aldosterone, 1 mM ATP, or 100 μM BzATP for 3 hours and IFNγ transcription was measured using rt-qPCR, data normalized to β-actin. Changes in IFNγ transcription were quantified by Brown-Forsythe ANOVA (P = 0.0018) followed by post hoc Dunnett’s T3 comparing each treatment group to the control. B-E) Primary isolated splenic CD8Ts from WT (B, D) or P2X7 KO (C, E) mice were treated for 3 hours with ATP or BzATP and analyzed via rt-qPCR for IFNγ and TNFα transcription, normalized to β-actin. *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001

ExATP-P2X7 stimulation drives calcium influx into CD8Ts.

P2X7 is an ATP-gated cation channel reported to mediate calcium influx in many cell types, but to our knowledge, its function in CD8T cells remains unexplored. Intriguingly, the processes of T cell activation, cytokine production, and cell expansion require the influx of extracellular calcium, which triggers downstream chromatin de-condensation and transcriptional changes19,20. Therefore, we investigated whether stimulation of the P2X7 receptor is sufficient to trigger calcium influx and activate CD8Ts. For these studies, we isolated CD8Ts from WT and P2X7 KO mice and pre-loaded them with the fluorescent intracellular calcium indicator Fluo-4. Using flow cytometry, we observed sustained rises in intracellular free calcium in CD8Ts from WT mice in response to either BzATP (Figure 3A–B) or ATP (Figure 3C–D), whereas CD8Ts from P2X7 KO mice failed to respond to either stimulus. As a negative control, we performed this assay in calcium-free conditions, which are known to prevent T cell activation. Accordingly, we found that exATP-mediated calcium influx was diminished in WT CD8Ts in calcium-free media (Figure 3A–D, PBS controls in Figure S7a–b). Collectively, these results suggest that the P2X7 receptor can drive sustained calcium influx in CD8Ts and mediates calcium influx in response to extracellular ATP.

Figure 3: Extracellular ATP treatment increases calcium influx in WT CD8Ts but not P2X7 KO CD8Ts.

Figure 3:

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Splenic CD8Ts from WT or P2X7 KO mice were pre-loaded with Fluo-4 dye and then stimulated with the P2X7 agonist BzATP (A, B) or ATP (C, D). Calcium influx in cells was analyzed by flow cytometry and quantitated by geometric mean fluorescent intensity (gMFI). Panel colors are indicative of recorded population density. Fluorescence from cells in HBSS (n= 5–7 per group) or calcium-free media (n = 3–4 per group) were quantified in 200-second segments in B) and D). Data were analyzed by Two-way ANOVA via mixed effect analysis followed by posthoc Sidak’s for comparison of gMFI at each time point for HBSS groups, and Dunnett’s for comparison of gMFI at each time point with baseline in calcium-free treatment groups. ns = not significant. *P < 0.05, **P < 0.01, ***P <0.001. Data are expressed ± SEM, with no visible error bars in d) due to small variability.

ATP-pretreated CD8Ts exhibit an increased ability to stimulate NCC function in DCTs.

We reported recently that pre-activated CD8Ts increase NCC-mediated sodium retention in co-cultured mDCTs, an event enabled by PDL1-mediated direct CD8T-DCT adhesion5,6. However, using PMA + ionomycin as a stimulus to activate CD8Ts is artificial and cannot explain the physiological stimuli that activate CD8Ts during the development of hypertension. The current study raised the possibility that increased ATP in hypertension may act as a P2X7 ligand to activate CD8Ts, we further predicted that P2X7 agonism of CD8Ts would increase NCC-mediated sodium retention in co-cultured renal tubular epithelial cells. To test this, we pre-stimulated CD8Ts with BzATP and washed it out prior to co-culturing them with mDCTs. The procedures of harvesting and assessing NCC-mediated sodium retention in mDCTs (Figure 4A) were the same as those reported by us previously6, and the gating strategy for flow cytometry is shown in Figure 4B. As expected, we observed that BzATP pre-stimulation of CD8Ts resulted in more mDCTs exhibiting greater NCC-mediated sodium retention compared to unstimulated CD8Ts, which was revealed as more mDCTs with elevated CoroNa Green-initiated mean fluorescent intensity (MFI) in Figures 4C–D. Furthermore, the mDCTs with high NCC-mediated sodium retention also demonstrated higher expression of PDL1, which is consistent with our previous report that activated CD8Ts stimulate mDCTs via PDL1-mediated CD8T-DCT interaction6. Notably, direct BzATP stimulation of mDCTs, without CD8T-co-culture, resulted in no change in NCC-mediated intracellular sodium or PDL1 expression (Figure S8a–b), indicating that ATP per se does not regulate NCC-mediated sodium resorption in renal tubule epithelial cells. Our findings suggest that pre-stimulation of the P2X7 receptor on CD8Ts is sufficient to drive increased CD8T-to-DCT stimulation, promoting NCC-mediated sodium retention in co-cultured DCT cells.

Figure 4: Pre-activation of CD8Ts with the P2X7 agonist results in increased NCC-mediated sodium retention in co-cultured DCTs.

Figure 4:

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A) Schematic of the co-culture experimental protocol. B) Gating strategy for mDCTs after selection and staining. C) Representative flow cytometry images showing an increase in CoroNa Green (measuring intracellular Na+) and PDL1 in mDCTs following co-culture with pre-stimulated CD8Ts compared to naïve CD8Ts. Quantified in D-F) with gating as shown for gMFI calculation (grey box, D, E) or percentage of both high intracellular sodium and high PDL1 (F). Analyzed via unpaired t-tests.

Expression of P2X7 in CD8Ts is critical for the progressive BP elevation in salt-sensitive hypertension.

ExATP is the only known natural endogenous ligand that activates P2X724,25. Previous studies have documented that the circulating exATP level in hypertensive humans and animals is higher compared to their normotensive counterparts22. However, whether hypertension augments P2X7 expression in CD8Ts, another mechanistic consideration, has yet to be studied. We explored this possibility by comparing CD8T P2X7 protein expression between CD8Ts isolated from untreated WT mice or WT mice treated with DOCA/salt for two weeks. Expression was quantified using modern western technology Protein Simple (Figures 5A & Figure S9a). The level of P2X7 expression was increased significantly in CD8Ts from hypertensive mice compared to CD8Ts from normotensive mice (Figure 5A & Figure S9a). Using flow cytometry, we further confirmed a similar pattern in the CD8T cell surface expression of the P2X7 receptor (Figure S9b).

Figure 5: Expression of P2X7 in CD8Ts and the impact of P2X7 knockout in DOCA/salt-induced salt-sensitive hypertension.

Figure 5:

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A) Representative Simple Western for P2X7 expression in CD8Ts from WT mice treated with DOCA/salt or sham, P = 0.0324. B) Radiotelemetry recording of systolic BP in WT (grey trace) and P2X7 KO (blue trace) mice receiving DOCA/salt treatment. Seven days after DOCA/salt initiation, P2X7 KO mice were randomly selected to receive saline (remain blue trace) or naïve WT CD8Ts (red trace). C) Quantification of BP in b) at days 0–1 (baseline), 9–10 (7 days post-DOCA/salt, before restoring WT CD8Ts), and 17–18 (7 days after adoptive transfer of WT CD8Ts). D) Quantifying T cells in kidneys taken from experimental P2X7 KO mice at the study endpoint. Each dot represents the mean of 4 images taken per kidney. See Figure S10 for representative images. For BP and T cell counting data, data analyzed via t-test or one-way ANOVA followed by posthoc Tukey’s. ns = not significant, * P < 0.05, ** P < 0.01, *** P <0.001. E) Kidneys obtained from P2X7 KO DOCA or P2X7 KO DOCA + WT naïve CD8T mice at the experimental endpoint (see Figure 5b) were stained and quantified for NCC expression. Each dot represents the mean intensity values of 4 images taken per kidney section, P = 0.00002502.

To further validate the critical role of P2X7-mediated CD8T activation in the pathogenesis of hypertension, we utilized P2X7 knockout (P2X7KO) mice. Earlier reports indicated that global P2X7 KO mice exhibit an attenuated BP elevation in response to DOCA/salt treatment21,22, but the specific cell type responsible for this depressor effect was not identified. Based on our new findings that the P2X7 receptor activates CD8Ts to promote NCC-mediated sodium retention by mDCTs, we hypothesized that the P2X7 receptor on CD8Ts contributes to the progressive BP elevation in DOCA/salt hypertension. To test this hypothesis, we administered DOCA/salt to WT and P2X7KO mice and observed that the development of hypertension was blunted in P2X7 KO compared to WT mice (Figure 5B–C). Remarkably, adoptive transfer of sham WT CD8Ts restored DOCA/salt-induced hypertension in P2X7KO mice to a same level as WT mice, suggesting an essential pressor role of CD8T-P2X7 in the pathogenesis of salt-sensitive hypertension (Figure 5B–C). Moreover, restoring WT naïve CD8Ts to DOCA/salt-treated P2X7KO mice also significantly increased the number of renal-invading T cells (Figure 5D and Figures S10 & S11) and elevated renal NCC expression (Figure 5E). Taken together, these findings indicate that the P2X7 on CD8Ts critically contributes to the progression of salt-sensitive hypertension.

CD8Ts from DOCA/salt-treated P2X7 KO mice do not display increased activation and fail to generate salt-sensitive hypertension in WT recipients.

We reported earlier that DOCA/salt treatment results in the activation of CD8Ts in WT mice, leading to enhanced cytokine production and an exaggerated NCC expression by co-cultured mDCTs, resulting in increased sodium retention6. Here, we present evidence that these effects are abolished by P2X7-deficiency. Accordingly, DOCA/salt treatment of P2X7KO mice did not increase cytokine production (IFNγ and TNFα) in CD8Ts from P2X7KO mice (Figures 6A–B), unlike its effect on WT mice6. As hypothesized, CD8Ts isolated from DOCA/salt-treated P2X7KO mice were unable to promote NCC-mediated intracellular sodium in co-cultured mDCTs (Figure 6C).

Figure 6: P2X7 deficient CD8Ts are not activated in DOCA/salt model and failed to cause salt-sensitive hypertension.

Figure 6:

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A) CD8Ts were isolated from the spleens of P2X7 KO mice and treated for 3 hours with PMA & Ionomycin in the presence of Brefeldin A, stained for intracellular IFNγ and TNFα production and analyzed via flow cytometry. Quantified in B). C) Representative flow cytometry images of mDCTs co-cultured with P2X7 KO CD8Ts isolated from DOCA/salt- or sham-treated mice and analyzed for intracellular sodium using CoroNa Green. D) Systolic BP in P2X7 KO mice that received adoptive transfer of CD8Ts isolated from spleens of DOCA/salt-treated WT mice. E) Systolic BP in WT mice that received adoptive transfer of CD8Ts isolated from spleens of DOCA/salt-treated P2X7 KO mice. Mean three-day baseline systolic BP was compared with the last three-day mean systolic BP via an unpaired t-test. ns = not significant, **P < 0.01, ***P < 0.001, r = recipient, d = donor

To validate the physiological impact of our findings, particularly the direct effect of CD8T cell-specific P2X7 on BP regulation, we performed adoptive transfer of CD8Ts from DOCA/salt-treated WT donor mice to sham P2X7KO recipient mice, as well as vice versa, from DOCA/salt-treated P2X7KO donor mice to sham WT recipient mice (Figure 6D–E). Our results revealed that CD8Ts from DOCA/salt-treated WT mice successfully caused salt-sensitive hypertension in P2X7KO recipient mice when 1% NaCl was added to drinking water three days after adoptive transfer (Figure 6D). Conversely, CD8Ts from DOCA/salt-treated P2X7KO mice failed to generate salt-sensitive hypertension in WT recipient mice (Figure 6E).

Discussion:

The new findings of our study have identified an important mechanism for the antigen-independent activation of CD8Ts during the development of hypertension. Many studies have indicated that the immune system and particularly T cells play a critical role in the development of hypertension1,3, and the activation of T cells in hypertension has been observed by us and others4,6,8. However, the upstream key player(s) responsible for activation of CD8Ts during hypertension remained unsolved6. In this present study, we provide strong evidence that stimulation of the P2X7 receptor on CD8Ts results in calcium influx and CD8T activation, exacerbating salt-sensitive hypertension. Our study thereby fills an important gap in knowledge about the immune signaling pathways that promote BP elevation.

Previously, skewing of the CD8T TCR repertoire has been reported in kidneys of mice with angiotensin II-induced hypertension11. However, this skewing was not observed in the spleen nor were there consensus dominant TCR clones observed in the kidneys of all hypertensive animals11. This presence of an oligoclonal population of CD8Ts with many TCR clones in the kidneys of hypertensive mice11 suggests the possibility of either multiple unique neo-antigens or non-antigen-driven CD8T activation. Yet our laboratory and others have observed increased activation of circulating CD8Ts in multiple pre-clinical models of hypertension, a finding confirmed in hypertensive human6,8,26. For this reason, we hypothesized that activation of CD8Ts in hypertension may not require antigen recognition. This hypothesis is supported by our findings in DOCA/salt-treated Rag2/OT1 mice. In the absence of the SIINFEKL peptide, which is required for OT1 CD8T activation, Rag2/OT1 mice still developed the same level of BP elevation in response to DOCA/salt treatment as WT mice. They also exhibited similar CD8T activation, T cell infiltration of the kidney, and cytokine production relative to their WT counterparts. These data strongly suggest that transgenic OT1 CD8Ts still contribute to hypertension despite the lack of antigen recognition.

Extracellular ATP, which can present locally at high levels27, has been found to be elevated in hypertension22. Moreover, the P2X7 receptor, which is activated by ATP and contributes to immune inflammatory responses28,29, has been implicated in the development of hypertension21. We, therefore, tested whether P2X7 stimulation on CD8Ts can result in their activation. Our findings revealed that exATP stimulation of P2X7 on CD8Ts significantly elevated cytosolic free calcium, which is required for T cell activation19,20 and IFNγ production. These key events have been reported by us and others to play a pivotal role in the pathogenesis of hypertension6. Our findings also demonstrate that P2X7-activated CD8Ts exhibit an enhanced ability to increase NCC-mediated sodium retention in co-cultured mDCTs compared to naïve CD8Ts, extending our earlier finding that PMA/ionomycin-stimulated CD8Ts can increase NCC-mediated sodium retention in tubular cells6. Collectively, our findings indicate that P2X7 agonism is sufficient to stimulate CD8T activation, leading to downstream interactions that exacerbate salt retention.

Although P2X7 is also expressed in renal tubules, we demonstrated that direct P2X7 stimulation on DCTs had no effect on NCC-mediated sodium retention. We also found that the P2X7 receptor of CD8Ts, both total protein and cell surface expression, is significantly increased in hypertension, which presumably serves to amplify the exATP–stimulated signaling pathway in CD8Ts and its downstream effects. Further research is needed to elucidate the particular mechanisms that drive P2X7 expression by CD8Ts during salt-sensitive hypertension.

To further demonstrate the crucial contribution of the P2X7 receptor to CD8T activation during hypertension, we adoptively transferred P2X7-expressing, naïve CD8Ts from WT mice to P2X7KO mice receiving DOCA/salt treatment. Earlier reports have indicated that global P2X7 deficiency attenuates BP in several experimental hypertension models21,22; however, the particular cell type(s) and mechanism(s) by which P2X7KO suppresses BP were not clarified. Here, by restoring WT naïve CD8Ts by adoptive transfer to P2X7KO mice, we were able to provoke DOCA/salt-induced renal T cell infiltration and NCC upregulation, ultimately restoring systolic BP to the same high level as WT mice. Collectively, these data strongly suggest that the P2X7 receptor expressed by CD8Ts is required for CD8T cell activation and the elevation of BP during the pathogenesis of salt-sensitive hypertension.

Alternative sources of T cell activation have been proposed in hypertension, including but not limited to gut microbiota differences 30, renal tubular-derived IL-1831, and isoketal neo-antigens expressed on dendritic cell MHC-I12. It is worth noting that our experiments only suggest the existence of a non-antigen-dependent pathway but do not rule out mechanistically these other potential alternative mechanisms of T cell activation. Indeed, CD8Ts harboring several specific TCRβ subunits did undergo some degree of clonal expansion in the kidney while others did not11, and mice deficient in CD11C+ cells of myeloid-derived lineage exhibit blunted BP to pressor stimuli13. Importantly, our in-vitro experiments demonstrate that the P2X7 pathway augments CD8T inflammation and induces sodium retention, as our results indicating that adoptive transfer of WT cells induces hypertension in P2X7 mice indicate that this novel pathway contributes to CD8T-driven hypertension regardless of antigen dependence or other activating stimuli.

Crosstalk among various cell types of the immune system is required for a robust response and maintenance of inflammation, and blocking multiple cytokines or chemokines can blunt the development of hypertension7,32. As such, more research is needed to clarify the interrelationship between antigen-independent CD8T activation and the recruitment of other immune cells that may contribute to hypertension. Additionally, the P2X7 receptor is expressed on dendritic cells and plays a role in dendritic cell activation and inflammasome formation33. Therefore, it is likely that global gene deletion of P2X7 also blunts dendritic cell activation and involvement in hypertension. Nevertheless, our findings that the adoptive transfer of naïve P2X7 expressing CD8Ts from WT mice to P2X7KO mice receiving DOCA/salt restores the development of salt-sensitive hypertension implies that the P2X7 receptor on CD8Ts is sufficient to promote their activation and elevate BP under our conditions.

Identifying the source of exATP stimulating CD8T P2X7 receptors during hypertension is a critical study that waits to be performed. In this regard, artificially maintaining a low perfusion pressure in one kidney of an anesthetized rat by servo-control reduces T cell invasion of the perfused kidney3. Conversely, acute increases in renal perfusion pressure increase exATP signaling in the kidney in-vivo34. As such, it is possible that the kidneys per se may provide some of the exATP leading to P2X7 stimulation and T cell activation. One pitfall to such a hypothesis is the finding that circulating T cells are activated in hypertensive humans and animals6,8,26, implying that renal-derived exATP is not the only source of activating exATP and/or that other systemic stimuli underlie CD8T activation during hypertension. Indeed, the gut also exhibits relatively high exATP levels, which are known to alter immune function and microbiome homeostasis, effects at least partly mediated by P2X735,36. Dysfunction of the gut microbiome has been linked to hypertension37. Sheer stress also can drive ATP release from endothelial cells38, and elevated BP increases tangential pressure on endothelial cells39,40, implicating the vascular endothelium as another potential source of exATP during hypertension. Finally, vesicles stored in pre-synaptic neurons contain high concentrations of ATP for release as a neurotransmitter41, and the nervous system is recognized to regulate T cell function in hypertension42,43. Thus, substantial studies seeking to identify the source of exATP that activates CD8Ts during hypertension will be confounded by the multiple tissue sources of this P2X7 agonist.

Collectively, our data suggest a novel mechanism wherein the P2X7 receptor mediates CD8T activation leading to increased IFNγ production, renal invasion of CD8Ts, and interaction with DCTs, thereby stimulating NCC upregulation and function. The resulting excess sodium retention contributes to the pathogenesis of salt-sensitive hypertension. The identification of this mechanism not only provides new insight into the pathogenesis of hypertension but also identifies the P2X7 signaling pathway as a novel therapeutic target that could be leveraged to develop new antihypertensive medications that disrupt the deleterious interaction between CD8Ts and the kidney.

Perspectives:

In this study, we uncovered an antigen-independent mechanism that, through the ATP-P2X7 signaling pathway, CD8+ T cells become activated in hypertensive mice. This activation leads to excessive salt retention in the kidney, which in turn exacerbates salt-sensitive hypertension. Our findings shed light on a promising research direction that dysregulation of energy metabolism may contribute to the pathogenesis of hypertension, via enhancing the activation of immune cells.

Supplementary Material

Supplemental Figures
Supplement - Methods & Materials

Novelty and Relevance:

What Is New?

This study marks the first report of the discovery of an antigen-independent mechanism responsible for the activation of CD8+ T cells during the development of hypertension.

What Is Relevant?

P2X7 has been associated with salt-sensitive hypertension; however, the specific cell types targeted and the precise mechanisms involved have remained unclear. This study has successfully identified a critical role for ATP-P2X7 signaling in mediating the activation of CD8+ T cells, thereby contributing to the development of salt-sensitive hypertension.

Pathophysiological Implications?

This study suggests that dysregulation of energy metabolism may trigger the activation of immune cells, ultimately contributing to the development of salt-sensitive hypertension. To prevent or reverse this mechanism, it is crucial to identify the source of excessive ATP. Given the heterogeneous nature of hypertension’s pathogenesis, it is likely that various tissues or types of cells are involved in the release of ATP during the development of hypertension. Therefore, further research in this direction is of utmost importance to gain a comprehensive understanding of this complex process.

Acknowledgments:

We thank Drs. Nancy Rusch and Philip Palade in the Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences (UAMS), for their contributions to data discussion and editing of the article.

Sources of Funding:

This study was supported by National Institutes of Health (NIH) R01 HL146713 (S.M.), American Heart Association (AHA) 23TPA1076467 (S.M.), 15BGIA25730047 (S.M.), a UAMS Medical Research Endowment Award (S.M.) and a UAMS Bronson Foundation Award (S.M.). L.B. is supported by an American Heart Association Predoctoral Fellowship 23PRE1023017. Both L.B. and K.D. received support from the Systems Pharmacology and Toxicology Predoctoral Training Program T32 GM106999. Other support included National Institutes of Health R01 AI139124 (L.X.L), R21 AI175738 (L.H.), P20 GM103625 (L.X.L., L.H.), R01 AG060395 (D.W.T.), K01 AG061271 (D.W.T.), American Heart Association 940023 (D.W.T.), American Lung Association CA-828143 (L.H.) and a UAMS Sturgis Foundation Award (L.H.).

Abbreviations

P2X7

Purinergic Receptor P2X 7

RAG2

Recombination Activating Gene 2

mDCT

Mouse Distal Convoluted Tubule

DOCA

Deoxycorticosterone acetate

NCC

Sodium Chloride Co-Transporter

PDL1

Programmed Death Ligand 1

MHC-I

Major Histocompatibility Complex Class 1

BzATP

Benzoylbenzoyl-ATP triethylammonium salt

gMFI

Geometric Mean Fluorescent Intensity

PMA

Phorbol Myristate Acetate

Footnotes

Disclosures:

All authors report no conflicts to disclose.

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Supplementary Materials

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