ARTC2.2/P2RX7 signaling during cell isolation distorts function and quantification of tissue-resident CD8⁺ T cell and iNKT subsets

Abstract

Peripheral invariant natural killer T (iNKT) and CD8⁺ tissue-resident memory T cells (T_RM) express high levels of the extracellular ATP (eATP) receptor P2RX7 in mice. High eATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Since both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT and CD8⁺ T_RM. Indeed, ARTC2.2 blockade enhanced recovery of viable liver iNKT and T_RM. The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets and T_RM is unclear however, as is the impact of recovery from other non-lymphoid sites. Here, we performed a comprehensive analysis of ARTC2.2 and P2RX7 expression in iNKT and CD8⁺ T cells in diverse tissues, at steady-state and after viral infection. NKT1 cells and CD8⁺ T_RM express high levels of both ARTC2.2 and P2RX7 compared to NKT2, NKT17 and CD8⁺ circulating memory subsets. Using nanobody-mediated ARTC2.2 antagonism, we showed that ARTC2.2 blockade enhanced NKT1 and T_RM recovery from non-lymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability and proliferation of both populations. We also showed that short-term direct P2RX7 blockade enhanced recovery of T_RM, though, to a lesser degree. In summary, our data show that short-term in vivo blockade of the ARTC2.2/P2RX7 axis permits much improved flow cytometry-based phenotyping and enumeration of murine iNKT and T_RM from non-lymphoid tissues, and it represents a crucial step for functional studies of these populations.

Introduction

During preparation of ex vivo cell suspensions or in response to microbial infections, inflammation or tumor growth, high concentrations of the nucleotides ATP and NAD can be released from apoptotic, necroptotic or stressed cells (1). Extracellular ATP (eATP) stimulates P2RX7, which is a non-selective ligand-gated ion channel expressed by several immune cell types. Prior research focused on myeloid cells (2, 3), but P2RX7 is also expressed by lymphocyte populations (4–7). When activated by high concentrations of eATP, P2RX7 forms reversible non-selective pores that can mediate activation signals but can ultimately lead to cell death if eATP exposure persists (2, 8). ADP-ribosylation of P2RX7 by the ecto-enzyme ARTC2.2, on the other hand, induces irreversible pore formation and subsequent cell death. ARTC2.2 is activated by extracellular NAD (9). Importantly, ARTC2.2 activation-induced P2RX7 pore formation occurs at much lower concentrations of NAD compared to that of extracellular ATP (10). ARTC2.2 is catalytically active even when cells are at 4^oC (1). The subsequent formation of P2RX7 pores, however, only happens at temperature above 24^oC^, suggesting the effects of ARTC2.2-mediated ribosylation could be manifested during tissue processing that involves incubation at room temperature or 37^oC – such as the steps necessary for lymphocyte isolation from non-lymphoid tissues (11, 12). Indeed, previous studies have shown extensive cell death of T cell populations under these circumstances, especially cells expressing high levels of ARTC2.2 and P2RX7, like CD4⁺ T_reg cells (1). Moreover, even cells that survive isolation steps may be compromised for in vitro functional assays (13).

To tackle this issue, ARTC2.2-specific antagonist nanobodies to block the ARTC2.2/P2RX7 signaling axis were developed (9). Previous studies successfully used this strategy to recover lymphocytes with high expression of ARTC2.2, including T_reg and iNKT cells (13). Two recent reports showed that ARTC2.2 blockade also prevents the death of liver-resident memory (T_RM) CD8⁺ T cells during tissue preparation (14, 15). Overall, this indicates that T lymphocytes in non-lymphoid tissues are sensitive to death induced by activation of ARTC2.2 and P2RX7. Despite the pioneering nature of these reports, several questions remain. First, these studies focused on elevated frequency of live cells and short-term functional assays, rather than numeric comparisons of cells in the tissues. This made it hard to quantify to what extent ARTC2.2 blockade prevented loss of iNKT and T_RM cells. Specially in the case of T_RM cells, a severe underestimation of cell numbers detected by isolation and flow cytometric assays has been reported, in comparison to cell numbers calculated by immunofluorescence in situ (16) and it is unclear to what extent activation of the ARTC2.2/P2RX7 axis contributes to this.

Second, T_RM cells and iNKT cells are not homogeneous populations, with potential differences dictated both by differentiation state and tissue-specific microenvironmental signals. iNKT cells, for instance, are composed of functionally and transcriptionally distinct effector subsets, that include T-bet+ PLZF^low NKT1, PLZF^high NKT2 and RORγt⁺ PLZF^intermediate NKT17 cells (17–19). Notably, the previous ARTC2.2 blockade studies focused on liver and spleen iNKT cells, most of which are NKT1 cells. Whether other iNKT subsets co-express ARTC2.2 and P2RX7 and whether blockade of this pathway can rescue these cells is unexplored. As for T_RM cells, expression of CD69 and CD103 have been used as “signature” markers (20, 21). However, co-expression of CD69 and CD103 is not consistent among non-lymphoid tissues (21); moreover, parabiosis and in situ staining showed that some cells that do not express either of these molecules may nevertheless be bona fide T_RM cells (16). Whether these different sub-populations of T_RM cells vary in their susceptibility to ARTC2.2/P2RX7-mediated cell death is untested.

Here, we provide an analysis of co-expression of ARTC2.2 and P2RX7 in different iNKT subsets and T_RM cells, in lymphoid and non-lymphoid tissues. In summary, we show that high expression of both molecules correlated with high susceptibility to ex vivo cell death during harvest procedures. Moreover, we found that blocking ARTC2.2 using nanobodies preserved the cell viability and in vitro function, favoring a more precise description of the numeric proportions of different subsets, as well as their exact functionality. Finally, we explored the possibility of using direct P2RX7 inhibition for the preservation of T cell numbers.

Materials and Methods

Mice and infections.

6- to 10-week old C57BL/6 (B6) and B6. SJL (expressing the CD45.1 allele) mice were purchased from Charles River (via the National Cancer Institute). P2rx7^−/− (MGI strain designation: 2386080) mice were obtained from Jackson Laboratories. LCMV-DbGP33-specific TCR transgenic P14 mice were fully backcrossed to B6 and P2rx7^−/− mice, with introduction of CD45.1 and CD45.2 congenic markers for identification. Mice were infected with LCMV Armstrong strain (2 × 10⁵ PFU, intraperitoneally (i.p.)). Animals were maintained under specific-pathogen-free conditions at the University of Minnesota. All experimental procedures were approved by the institutional animal care and use committee at the University of Minnesota.

Anti-ARTC2.2 nanobody treatments.

To prevent ADP-ribosylation of P2RX7 during harvest procedures, experimental mice were injected intravenously (i.v.) with 50 μg of the ARTC2.2 blocking nanobody (s+16a; T-reg Protector, BioLegend) diluted in 200 μl of PBS, 30 min prior to sacrifice (14). Control mice received vehicle (PBS) at the same time.

Administration of P2RX7 antagonists in mice.

For short-term blockade of P2RX7 during harvest procedures, experimental mice were injected i.v. with 200 μl of either 35 μM (22) of Brilliant Blue G (BBG; Sigma-Aldrich), or alternatively with 80 mg kg⁻¹ of A-438079 (Sigma-Aldrich, 0.5% DMSO in PBS) 30 min prior to mice sacrifice. Control mice received vehicle injections (PBS or 0.5% DMSO in PBS, respectively) at the same time.

Flow cytometry.

Lymphocytes were isolated from tissues including spleen, skin-draining lymph nodes, mesenteric lymph nodes, thymus, blood, lung, small intestine intestinal epithelium (IEL), small intestine lamina propria (LPL) and liver as previously described (16, 23), with the indicated mouse pre-treatments (vehicle, ARTC.2.2 nanobody or P2RX7 antagonists). Briefly, this involved digestion at 37^oC for 30 minutes in DTE (SI-IEL) or 30–45 minutes in Type I Collagenase (SI-LPL, Lung), with stirring, followed by Percoll gradient centrifugation at room temperature. For secondary lymphoid organs, processing was followed by red blood cell lysis with ACK lysis buffer, at room temperature. For discrimination of vascular-associated CD8⁺ T cells in non-lymphoid tissues, in vivo i.v. injection of PE-conjugated CD8α antibody was performed as described (24) 3 minutes prior to sacrifice. Direct ex vivo staining and intracellular cytokine staining were performed as described previously (23, 25). Briefly, cells were stimulated (as below) for 6 hours with GolgiPlug added for the final four hours. FoxP3Fix/Perm kit (eBioscience) was used for intracellular detection. Fluorochrome-conjugated antibodies were purchased from BD Biosciences, BioLegend, eBioscience, Cell Signaling Technology, Tonbo or Thermo Fisher Scientific. iNKT cells were detected using CD1d tetramers loaded with PBS-57 (provided by the NIH Tetramer Facility) and TCRβ staining, and the distinct iNKT subsets distinguished as described previously (17, 18); briefly, the NKT1 cells were defined as PLZF^low T-bet⁺, NKT2 cells were PLZF^high ROR-γt⁻ T-bet⁻ and NKT17 cells were PLZF^intermediate ROR-γt⁺. Polyclonal CD8⁺ T cells were identified as TCRβ⁺ CD8α⁺ CD4⁻. To detect LCMV-specific CD8⁺ T cell responses, tetramers were prepared as described previously (26). Among LCMV-specific CD8⁺ T cells, the following markers were used to distinguish these respective populations: T_CM (CD44⁺CD62L⁺), T_EM (CD44⁺CD62L⁻ CD127⁺), SLO T_RM (in spleen, LNs: CD44⁺CD62L⁻CD69⁺), T_RM (i.v. CD8α⁻CD69^+/−CD103^hi/int/lo). For survival assessment, cells were stained with Live/Dead (Tonbo Biosciences). For measurement of mitochondrial mass and membrane potential, cells were incubated with MTG (Thermo Fisher Scientific) and TMRE (Cell Signaling Technology) simultaneously for 15 min at 37^oC prior to ex vivo staining. For assessment of proliferation upon in vitro re-stimulation, cells were stained with Ki-67 (eBiosciences) after fixation using the Foxp3 Kit (Tonbo Biosciences). Flow cytometric analysis was performed on a LSR II or LSR Fortessa (BD Biosciences) and data was analyzed using FlowJo software (Treestar).

In vitro culture experiments.

To assess P2RX7 expression kinetics after initial activation, P14 splenocytes from naïve mice were stimulated for 72h with gp33 peptide (1 μM, KAVYNFATM, New England Peptide) and IL-2 (10 ng/ml). To measure the proliferation, survival and cytokine production of liver iNKT and spleen/small intestine memory (P14) CD8⁺ T cells, cells from experimental mice were isolated as described above and stimulated in vitro with vehicle (RPMI), PMA (20 ng/ml) + Ionomycin (1 μM), or gp33 peptide (1 μM). Cells were culture either for 4h, 24h or 72h. For all experiments, complete RPMI media (RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine) was used.

Statistical analysis.

Data were subjected to the Kolmogorov-Smirnov test to assess Gaussian distribution. Statistical differences were calculated by using unpaired two-tailed Student’s t-test or One-way ANOVA with Tukey post-test where indicated. All experiments were analyzed using Prism 5 (GraphPad Software). P values of <0.05 (*), <0.01 (**), <0.001 (***) or <0.0001 (****) indicated significant differences between groups, and non-significant differences were indicated with “ns”.

Results

ARTC2.2 and P2RX7 are preferentially co-expressed in peripheral NKT1 cells and CD8⁺ T_RM cells in lymphoid and non-lymphoid tissues

We first sought to evaluate the expression of P2RX7 and ARTC2.2 in subsets of invariant natural killer T cells (iNKT) from an array of lymphoid and non-lymphoid tissues. Previous studies indicate that ARTC2.2/P2RX7 activation strongly affects the viability of liver iNKT cells (27). Liver iNKT cells are predominantly NKT1, and we confirmed that liver NKT1 cells express high level of both ARTC2.2 and P2RX7 (Fig. 1a). Similar expression was seen on NKT1 from other non-lymphoid tissues and in the spleen and mesenteric lymph node (LN), while mature NKT1 in the thymus showed low expression of both ARTC2.2 and P2RX7 (Fig. 1b, 1c). Examination of NKT2 and NKT17 cell subsets (17), however, showed distinct expression patterns: NKT2 cells expressed high ARTC2.2 and P2RX7 in all sites, including the thymus, while NKT17 cells expressed low levels of both molecules whether recovered from thymus, LN, or lung (Fig. 1b and data not shown).

Fig.1. Expression of ARTC2.2 and P2RX7 in iNKT and CD8⁺ T cell subsets.

(a) Representative flow cytometry plot showing expression of ARTC2.2 and P2RX7 or isotype control in liver iNKT cells. (b) Representative plots showing expression of ARTC2.2 and P2RX7 in NKT1, NKT2, or NKT17 cell subsets from the indicated tissues. N/A means not analyzed (due to paucity of cells of that subset in the tissue) (c) Percentage of NKT1 cells that are ARTC2.2⁺P2RX7⁺ (as defined by the quadrants in (a)). (d) Expression of ARTC2.2 and P2RX7 in polyclonal CD8⁺ T cells from the indicated tissues (above) and in virus specific CD8⁺ T cells 4 weeks after infection (below). For the virus specific CD8 T cell analysis, P14 CD8 T cells were adoptively transferred into congenic recipient mice (2.5 × 10⁴ cells/mouse), followed by infection with LCMV-Armstrong (2 × 10⁵ PFU, i.p.). After 4 weeks, cells were isolated from the indicated tissues. (e) The percentages of ARTC2.2⁺P2RX7⁺ cells are shown for polyclonal CD8⁺ T cells in the thymus and spleen, as well as splenic memory P14 cell subsets (central memory – T_CM, effector memory – T_EM, spleen resident memory – SLO T_RM). (f) Percentages of memory P14 cells that were ARTC2.2⁺P2RX7⁺ in the different organs is shown, as well as polyclonal small intestine-resident CD8⁺ T cells. (a-f) Data are from 3 independent experiments, n=5–6 per experimental group.

We further assessed ARTC2.2 and P2RX7 expression in CD8⁺ T cells in unimmunized mice. While CD8 single-positive (CD8 SP) thymocytes expressed low levels of both molecules, peripheral naïve CD8⁺ T cells expressed ARTC2.2 but not P2RX7 (Fig. 1d, top). P2RX7 and ARTC2.2 were both highly expressed by most CD8⁺ T cells found in non-lymphoid tissues such as the small intestine at steady-state (Fig. 1d, top). As expected, these cells had a phenotype resembling antigen-experienced cells (Fig. S1a), thus suggesting CD8⁺ T cells activated by either antigen or homeostatic cytokines express higher levels of ARTC2.2 and P2RX7. Indeed, following cognate antigen-induced activation, P2RX7 expression rose in CD8⁺ T cells (Fig. S1b). Furthermore, we generated P14 immune chimeras, where congenic distinct host mice were adoptively transferred with P14 CD8⁺ T cells (bearing a TCR transgene specific for an LCMV epitope-ref [put in Ohashi ref]) and subsequently infected with LCMV. Virtually all memory P14 populations displayed ARTC2.2, but P2RX7 expression level varied widely in distinct memory sub-populations (Fig. 1d, bottom, and 1e): While effector memory cells (T_EM) showed low P2RX7 expression, a substantial fraction of central memory cells (T_CM), essentially all resident memory cells present in secondary lymphoid organs (SLO T_RM) and all T_RM in non-lymphoid tissues exhibited high P2RX7 expression (Fig. 1d, bottom, and 1f). Hence, the majority of P14 T_RM cells are P2RX7⁺ ARTC2.2⁺, suggesting they might also be susceptible to NAD-induced cell death.

ARTC2.2 blockade improves the yield of NKT1 cells in non-lymphoid tissues

Considering the high expression of both ARTC2.2 and P2XR7 in NKT1 cells, especially those residing in the non-lymphoid tissues (liver and small intestine), we asked whether tissue harvest and processing led to loss of NKT1 cells in an ARTC2.2-dependent pathway. To test this, we used a nanobody specific to ARTC2.2, previously shown to inhibit this enzyme’s function in T_regs (27). In keeping with previous reports (13, 27), we observed a significantly higher yield of NKT1 cells in liver of mice treated with anti-ARTC2.2 i.v 30 minutes prior to sacrifice (Fig. 2). The action of ARTC2.2 blockade was not limited to the liver, since we observed a notably higher yield (5 fold) of NKT1 cells in the IEL of treated mice (Fig. 2). Though not statistically significant, there was also a clear trend of higher yield of NKT1 cells in LPL by pre-treatment with anti-ARTC2.2 nanobody (Fig. 2). Importantly, ARTC2.2 blockade led to increased numbers of NKT1 cells recovered, which was not specifically reported in previous studies (13, 27). In contrast, we did not observe any difference in the recovery of NKT1 cells in thymus, spleen or mLN between mice injected with anti-ARTC2.2 nanobody or PBS (Fig. 2). It is important to note that tissue processing of liver and intestines (but not of thymus, spleen or LNs) require short-term (20–60min) incubations at room temperature (25^oC) or 37^oC, conditions that permit ARTC2.2-mediated P2RX7 pore formation. Together, our data suggest that non-lymphoid tissue processing might result in ARTC2.2-mediated loss of NKT1 cells, and blockade of this pathway is a crucial step for the recovery of optimal numbers of this cell population.

Fig. 2. Blockade of ARTC2.2/P2RX7 axis improves recovery of NKT1 cells in non-lymphoid tissues.

Number of NKT1 cells recovered in the indicated tissues from mice treated with anti-ARTC2.2 nanobody or PBS prior to tissue harvest. NKT1 cells are defined as TCRβ⁺ CD1d tetramer⁺ T-bet⁺. Blue bar represents cells from mice received PBS, red bar represents cells from mice received anti-ARTC2.2 nanobody. mLN=mesenteric lymph node; LPL=lamina propria; IEL=intestinal epithelial lymphocyte. Data are from 8 independent experiments with 2–6 mice in each experiment.

Anti-ARTC2.2 nanobody-mediated blockade improves yield of T_RM cells in non-lymphoid tissues and allow a more precise estimation of their population frequency ex vivo

We next evaluated whether blocking the ARTC2.2/P2RX7 signaling axis could likewise enhance recovery of CD8⁺ T_RM cells from different non-lymphoid tissues in LCMV immune mice. As reported before (14, 15), we found that nanobody pre-treatment led to an increased recovery of viable liver T_RM (Fig. 3a). This effect was not limited to the liver, since the numbers of T_RM in other non-lymphoid tissues was also elevated in mice injected with the anti-ARTC2.2 nanobody (Fig. 3a) and was associated with decreased cell death (Fig. S2a). We also observed an increase in numbers of CD44⁺ polyclonal CD8⁺ T cells from small intestine with nanobody treatment (Fig. S2b). Interestingly, Masopust and collaborators (16) reported that processing tissues for recovery of Trm significantly underestimates their numbers in non-lymphoid tissues, compared to in situ quantification using immunofluorescence. Our results suggest that ARTC2.2-mediated cell death during cell isolation can account for nearly all of this discrepancy in SI-IEL T_RM numbers and substantially corrects T_RM numbers from other tissues tested (Fig. 3b). Both CD103+ and CD103-SI-IEL T_RM express ARTC2.2, with slightly higher expression in CD103-T_RM (Fig. S2c). Consistently, recovery of both subsets was enhanced by ARTC2.2 nanobody treatment (Fig. 3c–d). Importantly, CD103-SI-IEL T_RM have a higher proportional representation in nanobody-treated samples (Fig. 3c), which corresponds with the finding that CD103-T_RM are underrepresented by conventional tissue extraction protocols (16). Our data indicates ARTC2.2-mediated cell death plays a role in this issue and suggests blockade of this enzyme not only induces increased recovery of T_RM numbers, but also offers a more faithful representation of T_RM subsets after tissue harvests.

Fig.3. Nanobody-mediated ARTC2.2 blockade enhances recovery of diverse T_RM cells in tissue processing.

(a-e) P14 CD8 T cells were isolated from the indicated tissues, with injection of ARTC2.2 blocking nanobodies (Nanobody) or PBS. (a) P14 cell numbers per organ comparing the PBS and Nanobody-treated groups. (b) Side-by-side comparison between the fold differences observed in the results from (a), and the fold differences between flow cytometry vs quantitative immunofluorescence (QIM) observed by Steinert et al. (2015). (c) Representative flow cytometry plots showing expression of CD69 and CD103 in SI LPL P14 cells from PBS and Nanobody-treated mice. (d) Numbers of CD103⁺ and CD103⁻ P14 cells from small intestines from PBS and Nanobody-treated mice. (e) Representative flow cytometry plots showing the percentages of CD8α i.v.⁺ (non-resident) vs i.v.⁻ (resident) P14 cells in the lungs of PBS and Nanobody-treated mice. (f-g) Mice were transferred with equal numbers of WT and P2rx7^−/− P14 cells (identified by distinct congenic markers), followed by LCMV-Arm infection. (f) Representative flow cytometry plots of SI IEL P14 cells from Nanobody- or PBS-treated, co-transferred mice. (g) Numbers of WT (filled bars) and P2rx7^−/− (open bars) P14 cells in the SI IEL of co-transferred mice, with PBS or Nanobody treatment. Data are from 3–4 independent experiments, n=6–15 per experimental group.

Furthermore, we observed that nanobody injection significantly enhanced recovery of lung T_RM (Fig. 3a). Indeed, lung showed the highest discrepancy between flow cytometry and quantitative immunofluorescence in determination of T_RM cell numbers (16) (Fig. 3b). Therefore, it may not be surprising that a majority of lung T_RM cells underwent ARTC2.2-mediated cell death during tissue preparation. Following conventional tissue processing procedures, only 5–10% of lung virus-specific CD8⁺ T cells are identified as i.v.⁻ (i.e. likely to be in the lung parenchyma), while quantitative immunofluorescence estimates the frequency of the i.v.⁻ population is 25–30% (16). In lungs processed after ARTC2.2 nanobody treatment, nearly 30% of specific CD8⁺ T cells were i.v.⁻ (Fig. 1e), in close agreement with the quantitative immunofluorescence approach. This cements the notion that anti-ARTC2.2 blockade prior to organ harvest renders a faithful representation of T_RM subsets in several non-lymphoid tissues.

We recently reported that expression of P2RX7 is necessary for T_RM development in non-lymphoid tissues (7). Superficially, this appears to be at odds with the increased susceptibility to NAD-induced cell death for T_RM cells described here. In our previous study we used ARTC2.2 nanobody blockade prior to tissue harvest (7), based on the expectation that P2RX7 deficiency would avert cell death mediated by ARTC2.2 (13). To directly test this idea, we co-transferred equal numbers of WT and P2rx7^−/− P14 cells into recipient mice, which were then infected with LCMV and, 8 weeks later, cells were recovered from the SI-IEL with or withoutanti-ARTC2.2 nanobody treatment just prior to harvest. Without treatment, we saw no advantage in WT compared to P2rx7^−/− T_RM numbers (Fig. 3e–f). In contrast, ARTC2.2 blockade resulted in an approximately 5-fold increased recovery of WT relative P2rx7^−/− P14 T_RM cells (Fig. 3f). Importantly, there was no difference in the numbers of P2rx7^−/− P14 T_RM in PBS-versus nanobody-treated recipient mice (Fig. 3f), confirming that P2RX7 expression dictates the susceptibility of T_RM to ARTC2.2-induced cell death.

Anti-ARTC2.2 nanobody blockade preserves CD62L expression in T_CM cells upon 37^oC incubation

In previous studies, ARTC2.2 blockade prior to harvest prevented shedding of CD62L in iNKT cells and Tregs (9). T_CM cells also express CD62L and are found primarily in secondary lymphoid organs and blood (28). Of note, some staining protocols and tissue processing experiments with these organs involve incubations at room temperature or 37^oC, such as peptide/MHC tetramer enrichment or staining of chemokine receptors (29–31). We observed that a considerable portion of T_CM cells express high levels of both ARTC2.2 and P2RX7 (7) (Fig.1). Hence, we tested whether incubation of spleen cells from P14 immune chimeras at 37^oC would influence the staining and numbers of CD62L⁺ T_CM cells detected. We observed a significant decrease in percentages of CD62L⁺ P14 cell in samples incubated at 37^oC, while this was prevented by anti-ARTC2.2 blockade (Fig. S3a, S3c). This is likely due to CD62L shedding rather than T_CM cell death, since the total P14 cell numbers were not altered by nanobody treatment (Fig. S3b).

In non-lymphoid tissues, most memory CD8⁺ T cells are resident (16) and virtually all have been characterized as CD62L⁻, based chiefly on flow cytometric analysis (20). Nevertheless, recent studies indicate CD62L⁺ memory CD8⁺ T cells can migrate into peripheral tissues (16, 32). Given that most non-lymphoid tissue processing protocols involve 37^oC incubation steps, we asked whether anti-ARTC2.2 blockade could improve detection of migrating CD62L⁺ memory CD8⁺ T cells in non-lymphoid tissues. Indeed, we detected significantly higher numbers of CD62L⁺ P14 cells in the small intestines of nanobody-treated mice compared to their PBS-treated counterparts (Fig. S3d). Together, our data show that, akin to iNKT cells and Tregs, anti-ARTC2.2 blockade prior to tissue harvest permits a more precise assessment of CD62L⁺ CD8⁺ T_CM cells in both lymphoid and non-lymphoid tissues.

Anti-ARTC2.2 nanobody blockade preserves phenotype, functionality and viability of iNKT cells

Activation of ARTC2.2/P2RX7 signaling axis also induces the loss of other cell surface molecules (9). Consistent with a previous study (27), we observed that in vivo treatment with anti-ARTC2.2 nanobody prevented loss of CD27 in both splenic and liver iNKT cells (Fig. 4a). Furthermore, we evaluated other surface markers that have been used for phenotyping of iNKT and CD8⁺ T cells. We observed that cell surface levels of CD69 and P2RX7 in iNKT cells were substantially preserved by in vivo treatment of anti-ARTC2.2 nanobody, while expression of ARTC2.2 itself, CD122 and CD4 were not affected (Fig. 4a). The nanobody-mediated preservation of CD69 and P2RX7 expression in iNKT cells are novel findings and suggest that some previous studies of iNKT cells may not faithfully describe their phenotype ex vivo. Whether ARTC2.2 activation alters other surface markers in iNKT cells is still unclear and will be a focus of future research.

Fig. 4. Blockade of ARTC2.2/P2RX7 axis preserves surface molecules, cytokine production and viability of iNKT cells.

(a) Representative flow cytometry plots for surface expression of CD27, CD69, P2RX7, ARTC2.2, CD122 and CD4 in spleen and liver NKT1 cells from mice treated with anti-ARTC2.2 nanobody or PBS. (b-d) Liver mononuclear cells isolated from mice treated with anti-ARTC2.2 nanobody or PBS, were stimulated in vitro with presence of PMA/Ionomycin for 4 hours. (b) Numbers (left column) and frequency of NKT1 cells among total T cells (TCRβ⁺) (right column) after in vitro stimulation. (c) Representative flow cytometry plots for IFN-γ production in NKT1 cells after in vitro stimulation. (d) Frequency (left column) and number (right column) of IFN-γ+ NKT1 cells after in vitro stimulation. (e-f) Liver mononuclear cells isolated from mice treated with anti-ARTC2.2 nanobody or PBS, were cultured in vitro for 24 or 72 hours. (e) Frequency of live iNKT cells (viability dye negative) after in vitro culture for 24 hours (upper row) or 72 hours (bottom row). (f) Frequency (left column) and number (right column) of live iNKT cells after in vitro culture for 24 hours (upper row) or 72 hours (bottom row). Data are from 4 independent experiments, n=2–10 mice per experiment.

Next, we asked whether blockade of ARTC2.2/P2RX7 signaling axis would improve functional properties of iNKT cells after in vitro stimulation. Liver mononuclear cells from mice treated with PBS or anti-ARTC2.2 nanobody were cultured in vitro with the presence of PMA/Ionomycin for 4 hours. The treatment of anti-ARTC2.2 nanobody prior to tissue harvest not only led to much better recovery of iNKT cells after short term in vitro stimulation (Fig. 4b), but also a substantially higher frequency of IFN-γ production by those NKT1 cells (Fig. 4c–d).

Recently, there has been an increasing interest in harnessing iNKT cells for immunotherapy, which usually requires prolonged in vitro culture for transduction and/or expansion. Therefore, we tested the possibility that blockade with anti-ARTC2.2 nanobody might enhance the feasibility of culturing iNKT cells in vitro. Indeed, we observed that in vivo treatment of mice with anti-ARTC2.2 nanobody enhanced the number and viability of iNKT cells after short term in vitro culture by about 5-fold (Fig. 4e–f). Taken together, these data show that in vivo treatment of anti-ARTC2.2 nanobody to block ARTC2.2/P2RX7 signaling pathway prior to isolation significantly improves viability and functionality of iNKT cells for in vitro stimulation and cell culture.

Anti-ARTC2.2 nanobody blockade preserves the viability and function of T_RM cells in vitro

We also tracked the effect of anti-ARTC2.2 nanobody on the expression of cell surface molecules in CD8⁺ T cell T_RM. As for iNKT cells, expression levels of CD69, CD44 and especially P2RX7 were increased on T_RM following transient ARTC2.2 blockade, and expression of ARTC2.2 itself was also elevated (Fig. 5a, Fig. S4a). Of note, nanobody pre-treatment caused no changes in the expression of these molecules in memory CD8⁺ T cells isolated from spleen, harvested at 4^oC (data not shown). We next sought to define if ARTC2.2 blockade preserves the function of T_RM cells during in vitro assays (Fig. 5b). In line with our ex vivo assays (Fig. 2) and a recent report with liver T_RM cells (14, 15), injection of nanobody prior to non-lymphoid tissue preparation led to a substantial increase in the recovery of viable SI-IEL T_RM, and a small increase in the viability of spleen memory cells (Fig. 5c, S4c), following 4h of in vitro re-stimulation. Moreover, we observed an increased frequency of IFN-γ-producing P14 T_RM, especially following pharmacological stimulation using PMA/ionomycin (as is often used to assess T cell function independent of antigen specificity) (Fig. 5d–e). Memory CD8⁺ T cells rapidly increased surface P2RX7 level after stimulation in vitro (Fig. S4b), which is consistent with this receptor playing a role in memory CD8⁺ T cell re-activation (7), but at the same time rendering these cells susceptible to death by NAD/ARTC2.2.

Fig. 5. Pre-harvest ARTC2.2 blockade preserves IEL T_RM functions and viability during in vitro procedures.

(a) Median fluorescence intensity (MFI) values for ARTC2.2, P2RX7, CD69 and CD44 in liver P14 cells from mice treated with Nanobody or PBS. (b) Experimental design for the in vitro experiments done with spleen and SI IEL P14 cells. (c) Percentages (left) and numbers/well (right) of PBS vs Nanobody-treated SI IEL P14 cells after 4h of in vitro culture without further stimulation (RPMI), PMA/Ionomycin, or gp33 peptide. (d) Representative flow cytometry plots showing IFN-γ production in SI IEL P14 cells from PBS and Nanobody-treated mice, stimulated in vitro with PMA/Ionomycin (4h). (e) Percentages (left) and numbers/well (right) of IFN-γ⁺ SI IEL P14 cells after 4h of in vitro culture without stimulation (RPMI), PMA/Ionomycin or gp33 peptide. (f) Percentages (left) and numbers/well (center), as well as the numbers of IFN-γ⁺/well (right) P14 cells after 24h of in vitro culture without any stimulation (RPMI) or with αCD3/αCD28. Data are from 2–4 independent experiments, n=4–13 per experimental group.

Prolonged in vitro stimulation and/or manipulation of T_RM is difficult because of the high cell death rate (20, 33, 34), which has made in vitro assessment of prolonged T_RM cell re-activation and assays on immunometabolism impractical. Strikingly, anti-ARTC2.2 nanobody prior to cell collection led to a significant increase in T_RM cell viability after 24h of in vitro culture, in either the presence or absence of re-stimulation (Fig. 5f, left and center). ARTC2.2 inhibition prior to in vitro stimulation led to a striking rescue of IFN-γ-producing T_RM, which were increased by more than 100-fold (Fig. 5f, right), and a more moderate increase in viability of re-stimulated splenic memory P14 cells (Fig. S4d). This trend continued at 72h of culture, where we observed increased viability of T_RM cells following in vivo nanobody treatment (Fig. S4e–S4f) and this population showed enhanced cytokine-production and proliferation compared to controls (Fig. S4g, S4h). The major hallmarks of T_RM cell secondary immune responses after local antigen challenge are increased in situ cytokine production (35, 36) and proliferation (37). Thus, transient ARTC2.2 blockade prior to harvest makes reliable in vitro assessment of T_RM cell function practical.

Transient blockade of P2RX7 also improves recovery of T_RM cells in non-lymphoid tissues

NAD/ARTC2.2-induced cell death in T_RM cells occurs through the activation of P2RX7 (Fig. 3f). A recent report showed P2RX7 blockade during in vitro assays prevents CD4⁺ follicular helper T cell death (38). We assessed whether in vivo P2RX7 blockade just prior to harvest could also prevent T_RM cell death and improve recovery. Blockade of P2RX7 with the non-specific inhibitor Brilliant Blue G (BBG) and the specific synthetic inhibitor A-438079 (39) by intravenous injection 30 minutes prior to sacrifice led to recovery of increased numbers of small intestine P14 T_RM cells (Fig. 6a), albeit to a lesser extent than that of using ARTC2.2 nanobody treatment (Fig. 3). Like ARTC2.2 blockade, P2RX7 blockade also led to an increased representation of CD103⁻ T_RM cells (Fig. 6b), suggesting that direct P2RX7 blockade can be a viable option for ex vivo analysis of T_RM subsets.

Fig. 6. Short-term P2RX7 pharmacological blockade is an alternative for rescuing T_RM cells during ex vivo procedures.

(a-b) P14 memory cells were isolated from the small intestine of mice after treatment with PBS, Brilliant Blue G (BBG) or A-438079. (a) Numbers of i.v.⁻ (resident) small intestine P14 cells in mice treated with PBS, BBG or A-438079. (b) Representative flow cytometry plots showing CD69 and CD103 expression in SI LPL P14 cells from mice treated with PBS, BBG or A-438079. (c-d) Mice were injected prior to sacrifice with PBS, A-438079 or Nanobody, and organs harvested and processed as described before. (c) Mitotracker green (MTG) median fluorescence averages of spleen (left) and SI IEL (right) P14 cells from mice treated with PBS or A-438079. (d) MTG (left) and Tetramethylrhodamine (TMRE) (right, normalized to MTG) median fluorescence averages of SI IEL P14 cells from mice treated with PBS or ARTC2.2 Nanobody. Data are from 2–3 independent experiments, n=3–10 per experimental group.

We previously discovered a fundamental role of P2RX7 in controlling the establishment and function of circulating and resident memory CD8⁺ T cells (7). Importantly, we demonstrated that in vitro P2RX7 blockade for as short as 6h induced defects on CD8⁺ T cell survival, metabolism and function like those observed in P2rx7^−/− CD8⁺ T cells (7). Therefore, we evaluated whether pre-harvest P2RX7 blockade would also lead to metabolic alterations in memory CD8⁺ T cells. Our data show that short-term in vivo P2RX7 blockade induced a decrease in mitochondrial mass (measured by Mitotracker Green staining) in both spleen and small intestine P14 cells (Fig. 6c). In contrast, ARTC2.2 blockade prior to harvest did not induce alterations in mitochondrial mass or membrane potential, as measured by TMRE staining (Fig. 6d). Recently, P2RX7-specific antagonist nanobodies were developed (40), which might offer a better opportunity for direct blocking this receptor to yield a more accurate estimate of tissue-resident lymphocyte numbers. Additionally, it was reported that P2RX7 antagonists improve the functionality of P2RX7-expressing CD4⁺ T cells in vitro (38). Based on our findings, transient blockade of P2RX7 could be used to quantify T_RM subsets, but its use should be interpreted with caution for prolonged in vitro culture or functional assays of T_RM, for which transient ARTC2.2 blockade would be the preferred approach.

Discussion

Early studies indicated that the activation of the ARTC2.2/P2RX7 pathway during ex vivo preparation of a single cell suspension could lead to profound apoptosis and dysfunction in T_regs and liver iNKT cells (13). However, the expression of ARTC2.2 and P2RX7 in iNKT cells or CD8⁺ T_RM cells in tissues other than liver has not been extensively evaluated. Moreover, whether ARTC2.2/P2RX7 signaling might lead to disproportionate changes in cell number, phenotype or function of specific subsets of iNKT and CD8⁺ T_RM during tissue processing had not been evaluated. In the current study, we established a detailed analysis of P2RX7 and ARTC2.2 expression in iNKT and CD8⁺ T cells and short-term blockade of this ARTC2.2/P2RX7 axis shortly before tissue harvest could significantly improve the recovery and ex vivo function of iNKT cells and CD8⁺ T_RM. Our results suggest that both ARTC2.2 and P2RX7 are highly expressed in peripheral but not thymic NKT1 cells. This might be due to the environmental cues present only in periphery. Retinoic acid reportedly induces P2RX7 expression in intestinal lymphocytes (41), being a potential factor that influence the expression of ARTC2.2 and P2RX7 in iNKT and CD8⁺ T cells residing in non-lymphoid tissues. Whether retinoic acid itself or similar molecules drive expression in other tissues is still unknown and will certainly be a subject of future studies. Moreover, it is worth mentioning that cell-intrinsic factors might also contribute to this phenomenon, as NKT2 cells seem to express high levels of ARTC2,2 and P2RX7 regardless in the thymus or periphery, while NKT17 cells do not express these receptors in either the thymus or periphery. Altogether, these results demonstrate considerable heterogeneity in the expression of P2RX7 and ARTC2.2 across various sub-populations of T cells (even among iNKT subsets), suggesting differential susceptibility to NAD-induced and ATP—induced cell death and signaling. In the future, it will be important to determine whether iNKT differentiation signals override local environmental cues for expression of P2RX7 and ARTC2.2, and whether activation of these receptors is involved in shaping the representation of distinct iNKT cell subsets.

The physiological role of P2RX7 in T cell function and homeostasis is still the subject of intense investigation. It has been reported that P2RX7 activation can favor mouse CD4⁺ T cell IL-2 production following re-stimulation, and the differentiation of Th17 cells, but restrains generation of T_reg and T_FH (5, 42, 43). Moreover, we recently reported P2RX7 is crucial for establishment of long-lived memory CD8⁺ T cell populations, including T_RM cells (7). Interestingly, our data presented here suggest a “Jekyll and Hyde” function of this receptor for T_RM biology, in which high expression of P2RX7 can be beneficial in most cases (e.g. for memory establishment), but potentially detrimental if these cells encounter situations (such as highly inflammatory environments), in which ARTC2.2 activation by NAD may be anticipated. Indeed, a recent report showed P2RX7 to be detrimental for liver and SI IEL T_RM in the presence of sterile inflammation (44). It will be important to expand these studies and determine how long-term survival of various tissue-resident T cell populations, as well as their in vivo function, is affected by inflammatory challenge in a P2RX7/ARTC2.2 dependent way. Likewise, ARTC2.2 and P2RX7 are highly expressed by some subsets of iNKT cells, yet the impact of this expression on differentiation and function of these populations is unclear. Furthermore, it will be interesting to explore the role of P2RX7 and ARTC2.2 in mucosal associated invariant T (MAIT) cells (19, 29, 45–48), another innate like T cell population, in future studies.

Taken together, our results help cement the notion that high expression of ARTC2.2 and P2RX7 act as biomarkers for susceptibility to cell death during tissue isolation in murine models. This was confirmed by our comprehensive ARTC2.2 blockade study. Beyond the scope of previous reports, we showed here inhibition of ARTC2.2-mediated cell death not only preserves viability of non-lymphoid tissue T cell populations (iNKT cells and T_RM cells) but allows a more accurate representation of different subsets amongst those recovered lymphocytes. Moreover, we show this blockade permits the accurate assessment of functionality of tissue-resident T cells in vitro, which opens several future possibilities, for example metabolism assays and transduction of T cell populations isolated from non-lymphoid sites – both of which have been technically challenging. In summary, we report that short-term blockade of ARTC2.2/P2RX7 activation during cell isolation is a crucial technical step for the optimal study of the biological characteristics of iNKT and T_RM cells in non-lymphoid tissues and should be utilized when practical.