Key Points
The CD27 pathway on CD4+ T cells plays a role in driving skin autoimmunity.
CD27 signaling promotes survival by suppressing cell-extrinsic apoptosis.
Abstract
Signaling through CD27 plays a role in T cell activation and memory. However, it is currently unknown how this costimulatory receptor influences CD4+ effector T (Teff) cells in inflamed tissues. In the current study, we used a murine model of inducible self-antigen expression in the epidermis to elucidate the functional role of CD27 on autoreactive Teff cells. Expression of CD27 on Ag-specific Teff cells resulted in enhanced skin inflammation when compared with CD27-deficient Teff cells. CD27 signaling promoted the accumulation of IFN-γ and IL-2–producing T cells in skin draining lymph nodes in a cell-intrinsic fashion. Surprisingly, this costimulatory pathway had minimal effect on early T cell activation and proliferation. Instead, signaling through CD27 resulted in the progressive survival of Teff cells during the autoimmune response. Using BH3 profiling to assess mitochondrial cell priming, we found that CD27-deficient cells were equally as sensitive as CD27-sufficient cells to mitochondrial outer membrane polarization upon exposure to either BH3 activator or sensitizer peptides. In contrast, CD27-deficient Teff cells expressed higher levels of active caspase 8. Taken together, these results suggest that CD27 does not promote Teff cell survival by increasing expression of antiapoptotic BCL2 family members but instead acts by preferentially suppressing the cell-extrinsic apoptosis pathway, highlighting a previously unidentified role for CD27 in augmenting autoreactive Teff cell responses.
Introduction
Costimulatory receptors shape the initiation, magnitude, and quality of an immune response. The prototypical costimulatory receptor is CD28, and stimulation through this receptor is required for optimal T cell activation and productive immunity (1). Additional costimulatory receptors include the TNFR super family (TNFRSF), which includes OX40, CD30, 4-1BB, and CD27 (2). These TNFRSF family members lack proapoptotic death domains, and signaling upon engagement with their respective ligands induces the activation of the NF-κB and JNK pathways. Although the functions of these receptors can be complementary, the outcome of signaling through any given receptor is highly context dependent. It is therefore of interest to understand how individual TNFRSF receptors influence immune responses within different tissues and inflammatory contexts.
One particular TNFRSF member, CD27, is constitutively expressed by naive and memory T cells in secondary lymphoid organs as well as on activated B cells (3). The only known ligand for CD27 is CD70. Expression of CD70 is tightly regulated and only transiently expressed on dendritic cells, B cells, T cells, and NK cells after immune activation (4–6). Engagement of CD27 by CD70 induces the recruitment of the TRAF2 and TRAF5 adaptor proteins, which in turn activate either the JNK or NFKb signaling pathways (7). This costimulatory pathway influences T cell function in several disease models (8). In settings of viral infection, CD27 contributes to the effective generation of both primary and memory CD8+ T cell responses (9–12). CD27 can also promote T cell responses in the context of productive immunization (13–16). Finally, constitutive expression of CD70 on dendritic cells result in autoimmunity (17). The CD27 pathway is therefore a promising target for therapeutic intervention to either augment immune responses to infections and tumors or attenuate excessive inflammation in the setting of autoimmunity.
Signaling through CD27 can augment T cell responses, in part by promoting cell survival. In specific contexts, engagement of CD27 can prevent apoptosis in both human and mouse CD8+ T cells by increasing expression of antiapoptotic BCL2 family members (including BCL-XL) (18–20). CD27 can also promote survival by inducing the downregulation of FasL and suppressing extrinsic (or death receptor–mediated) apoptosis (21). In addition to regulating the expression of antiapoptotic proteins, CD27 may also promote T cell survival and maintenance by modulating the expression of both cytokines and cytokine receptors. During viral infection, CD27 can induce IL-2 expression and promote CD8+ T cell survival through autocrine IL-2 signaling (22). CD27 signaling has also been implicated in increasing the frequency of IL-7R expressing memory precursors (14). The mechanisms by which CD27 promotes T cell survival therefore appear to be highly complex and contextually dependent.
The molecular mechanisms responsible for CD27-mediated influence of T cell function have predominately been defined in CD8+ T cells. The role of CD27 signaling in CD4+ T cell responses remains poorly understood. In addition, how the CD27 pathway influences T cell responses outside of secondary lymphoid organs (i.e., in peripheral tissues such as the skin) remains to be defined. In the current study, we use a well-established model of cutaneous autoimmunity to study the role of CD27 signaling in CD4+ effector T (Teff) cell responses to tissue Ag (23).
Materials and Methods
Animals
All mice were bred and maintained in specific pathogen free facilities at University of California, San Francisco (UCSF) according to National Institutes of Health guidelines and experiments were approved by the Institutional Animal Care and Use Committee of UCSF. Cytokeratin 5 (K5)/ tetracycline-response element (TGO) BALB/C (23) mice were backcrossed to C57BL/6 mice for at least 10 generations and crossed to CD45.1 mice purchased from Jackson Laboratories to generate K5/TGO/CD45.1.2 recipients. CD27−/− (9) mice were generously donated by Jannie Borst and crossed to OTII mice. Control donors were generated by crossing OTII to the CD45.1 strain.
Adoptive transfer of T cells and disease development
Spleens and lymph nodes of donor wild-type (WT) OTII CD45.1 and CD27−/− OTII CD45.2 mice were harvested and processed. Single-cell suspensions underwent TAC lysis to remove RBCs, followed by a magnetic CD4+ isolation (STEMCELL Technologies). Between 2 × 105 and 3 × 106 OTII cells (as noted in figure legends) were adoptively transferred i.v. into gender-matched K5/TGO/CD45.1.2 recipients. Recipient mice were placed on a 1 mg/kg doxycycline chow diet to turn on Ag in the skin. For clinical diseases measurement, dorsal hair was first shortened with clippers before applying depilatory cream (Nair) to the shaved region for a period of exactly 30 s before wiping clean.
Skin processing for flow cytometry
Skin was processed and digested as previously described. Briefly, trunk skin was shaved, harvested, lightly defatted, and minced. Skin was then incubated in digestion media: RPMI 1640 with 2 mg/ml collagenase XI (catalog no. C9407; Sigma-Aldrich), 0.5 mg/ml hyaluronidase (catalog no. H3506; Sigma-Aldrich), and 0.1 mg/ml DNase (ICN Pharmaceuticals) for 45 min at 37°C in a shaking incubator. Digested skin was then washed with RPMI 1640, filtered, and then stained for flow cytometry.
Abs and flow cytometry
Cells were stained for surface Ags and a live/dead marker (Ghost Dye Violet 510; Tonbo Biosciences) in PBS with 2% calf serum and 1% penicillin/streptomycin for 30 min at 4°C. To stain for intracellular cytokines, 4 × 106 cells were stimulated with Tonbo restimulation mixture (catalog no. TNB-4975) in tissue culture media for 4 h. Cells were then washed, and surface Ags were stained in PBS with 2% FBS and 1% penicillin/streptomycin. Intracellular staining was performed using the eBioscience FOXP3 staining kit (catalog no. 00-5523-00). Abs used are listed in Supplemental Table I. Samples were run on a Fortessa (BD Biosciences) in the UCSF Flow Cytometry Core. FlowJo software (FlowJo) was used to analyze flow cytometry data.
BH3 profiling
BH3 profiling was performed as previously described (24). Cells were isolated from the skin draining lymph nodes (SDLNs) of mice that received WT and CD27−/− OTII cells followed by CD4 enrichment via magnetic bead isolation (STEMCELL Technologies). Cells were then stained with surface markers in 2% FBS and 1% penicillin/streptomycin. After the surface stain, cells were resuspended in magnesium, EGTA, and β-glycerophosphate buffer at 2 × 106 cells/ml. Cells were then treated with various peptide concentrations in the presence of 0.001% digitonin and incubated for 1 h at room temperature. After peptide exposure, cells were fixed in 4% PFA for 10 min, followed by neutralization with N2 buffer (1.7M Tris, 1.25M glycine, pH 9.1). Cells were then stained with anti–cytochrome c A647 (BD Pharmingen) in intracellular staining buffer (10% BSA, 2% Tween 20 in PBS) at 4°C overnight. Cells were analyzed by FACS the next day.
BIM and PUMA peptides were dose titrated and used at final concentrations from 1 to 0.01 μM and 10 to 0.1 μM, respectively. DMSO was used as a vehicle control (maximal mitochondrial cytochrome c retention), whereas 15 μM alamethicin was used as a positive control by directly permeabilizing the mitochondria to induce full cytochrome c release. Percentage of cytochrome c release for each sample was calculated as % cytochrome c release = 1−(MFIsample–MFIALA)/(MFIDMSO–MFIALA), where MFI is the mean fluorescence intensity.
Caspase 8 staining
SDLNs were processed into a single-cell suspension and stained for 30 min with the CaspGLOW Fluorescein Active Caspase-8 (catalog no. 88-7005) staining kit and protocol. Following active caspase staining, cells were stained with surface markers and analyzed by FACS.
Statistics
Statistics were performed using GraphPad Prism software. The p values were calculated using two-tailed paired or unpaired Student t tests.
Results
CD27 expression by Ag-specific CD4+ Teff cells promotes skin inflammation
To study the role of CD27 in CD4+ T cell–mediated autoimmunity, we used a model of inducible Ag expression within the epidermis (23, 25, 26). This model involves two transgenes: one that has a K5 promoter driving the expression of a tetracycline trans-activator protein and one that drives the expression of the model Ag OVA under the control of a TGO. Mice with both K5 and TGO transgenes enable expression of OVA to be stringently induced in the basal layer of the epidermis upon treatment with doxycycline. The transfer of OVA-specific CD4+ T cells (OTII) into K5/TGO recipients treated with doxycycline induces a robust inflammatory dermatitis.
We used the K5/TGO model to elucidate the role of the CD27 pathway on autoreactive CD4+ Teff cells specific for a tissue Ag. CD27−/− mice were crossed to OTII-TCR transgenic mice, and resultant mice were used as a source of OVA-specific T cells that lacked CD27 expression. CD4+ T cells were isolated from either WT OTII or CD27−/− OTII mice and transferred into K5/TGO recipients. Mice were then treated with doxycycline to induce Ag expression in the epidermis. Recipient mice that received WT OTII cells developed a robust inflammatory dermatitis that progressively worsened over the course of 11 d (Fig. 1A, 1B). This dermatitis was accompanied by an increase in percentage and absolute numbers of neutrophils (CD45+CD11b+Ly6G+) and inflammatory macrophages (CD45+CD4−CD8−MHCII+CD11b+Ly6Chi) in skin (Fig. 1C, 1D). In contrast, mice receiving CD27−/− OTII cells had attenuated skin disease, reduced scaling, and decreased neutrophil and inflammatory macrophage influx in skin. These results demonstrate that CD27 expression on Ag-specific CD4+ Teff cells promotes autoimmunity to tissue Ag.
CD27 expression on CD4+ T cells promotes inflammation in response to tissue self-antigen. WT OTII or CD27−/− OTII cells were adoptively transferred i.v. into K5/TGO recipients. The day of transfer, mice were placed on doxycycline chow to induce Ag expression. (A) Representative photos of dorsal skin of mice given WT or CD27−/− cells 9 d after Ag induction. (B) Clinical disease scores after Ag induction. (C) Representative flow cytometric plots and quantification of neutrophil (gated on CD45+CD11b+Ly6G+) percentages and absolute numbers in skin. (D) Representative flow cytometric plots and quantification of percentages and absolute number of proinflammatory (Ly6C+) and noninflammatory (Ly6C−) macrophages (gated on Live CD45+ CD4–CD8–CD11b+MHCIIhi) in skin. Data are representative of two independent experiments with n = 5 mice per group. The p values are determined using an unpaired Student t test. *p < 0.05, ***p < 0.001, ns, p > 0.05.
CD27 promotes the accumulation of IFN-γ– and IL-2–producing autoreactive CD4+ Teff cells
We next evaluated how CD27 expression influenced the accumulation of Ag-specific CD4+ T cells in skin and SDLN upon Ag induction. WT OTII or CD27−/− OTII cells were adoptively transferred into K5/TGO recipients, and mice were treated with doxycycline. After 9 d of Ag induction, skin and SDLNs were harvested and the accumulation of Ag-specific T cells was quantified. In skin, CD27−/− OTII cells accumulated at lower percentages and absolute numbers compared with WT OTII cells (Fig. 2A), and a similar trend was seen in SDLNs, although this was not statistically significant (Fig. 2B). We have previously shown that in this model, OVA-specific T cells almost exclusively express IFN-γ (25). We therefore quantified expression of this cytokine via intracellular cytokine staining by flow cytometry. When compared with WT OTII cells, CD27−/− OTII cells produced significantly less IFN-γ and IL-2 (Fig. 2C, 2D). Taken together, these results suggest that CD27 expression promotes the accumulation of cytokine-producing autoreactive CD4+ Teff cells in response to autoantigen expression in skin.
CD27 promotes accumulation of cytokine-producing Ag-specific CD4+ T cells in response to tissue self-antigen. WT OTII CD45.1 or CD27−/− OTII CD45.2 cells were adoptively transferred i.v. into K5/TGO CD45.1.2 recipients. The day of transfer, mice were placed on doxycycline chow to induce Ag expression. Skin and SDLNs were harvested at day 9 to quantify the accumulation and cytokine production from adoptively transferred T cells. (A and B) Representative flow cytometric plots (gated on live CD4+ Vb5+ cells) and quantification of percentages and absolute numbers of transferred Vb5+ cells in the (A) SDLN and (B) skin. (C and D) Representative flow cytometric plots and quantification of (C) IFN-γ– and (D) IL-2–producing OTII cells in the SDLN. Data are representative of three independent experiments with n = 5 mice per group. The p values are determined using an unpaired Student t test. *p < 0.05, **p < 0.01, ns, p > 0.05.
CD27 promotes autoreactive Teff cell accumulation in a cell-intrinsic manner
To determine if CD27 signaling promotes autoreactive CD4+ T cell accumulation in a cell-intrinsic or cell-extrinsic fashion, WT OTII and CD27−/− OTII cells were combined at a 1:1 ratio and adoptively transferred into K5/TGO recipients. Accumulation of WT and CD27−/− OTII cells was quantified in SDLNs at days 3, 5, 7, and 10 after Ag induction (Fig. 3). On days 3 and 5 after Ag induction, both WT and CD27−/− OTII cells were present at equivalent percentages, suggesting that CD27 does not markedly influence the early CD4+ T cell response to tissue Ag. However, by day 7, WT OTII cells were present at ∼20-fold higher levels compared with CD27−/− OTII cells, and this difference was maintained at day 10. These results suggest that CD27 signaling promotes autoreactive Teff cell accumulation in a cell-intrinsic fashion and that this effect is most pronounced ∼7–10 d after Ag presentation.
CD27 promotes CD4+ T cell accumulation in a cell-intrinsic manner. (A) Experimental model. WT OTII CD45.1 and CD27−/− OTII CD45.2 cells were combined at a 1:1 ratio, and cells were adoptively transferred into K5/TGO CD45.1.2 recipients and placed on doxycycline chow. (B) Representative flow cytometric plots of the percentages of WT and CD27−/− OTII cells at specific timepoints after Ag induction (gated on live CD4+). (C) Quantification and ratios of WT/CD27−/− cells in the SDLN at days 3, 5, 7, and 10 after Ag induction. Statistics calculated using an unpaired Student t test to compare percentage of WT versus CD27−/− cells at each timepoint. Data are representative of two independent experiments with three mice per timepoint. Graphs depict mean ± SD, and p values are determined using an unpaired Student t test. **p < 0.01, ***p < 0.001, ns, p > 0.05.
CD27 promotes autoreactive Teff cell survival but not proliferation
To determine the mechanism by which CD27 promotes the accumulation of Ag-specific CD4+ T cells in our adoptive transfer model, we first quantified proliferation. WT OTII CD45.1 or CD27−/− OTII CD45.2 cells were CFSE labeled, combined at a 1:1 ratio, and cotransferred into K5/TGO recipients. Recipients were then placed on doxycycline to induce Ag expression. SDLNs were harvested 4 d after transfer and CFSE dilution was quantified by flow cytometry. WT and CD27−/− OTII cells displayed similar levels of proliferation with no differences was observed in either the proliferative or division index of each population (Fig. 4A). We also quantified Ki67 expression as a metric of cell cycling. Both WT and CD27−/− cells expressed similar levels of Ki67 (Fig. 4B). Consistent with these results, both WT and CD27−/− cells expressed similar levels of CD69, a validated marker of early T cell activation (Fig. 4C). These results suggest that signaling through CD27 does not promote Teff cell accumulation by augmenting early T cell activation, entry into the cell cycle, or cumulative cell proliferation.
CD27 promotes CD4+ T cell survival in response to tissue self-antigen. (A) WT OTII CD45.1 and CD27−/− OTII CD45.2 cells were CFSE labeled, combined at a 1:1 ratio, and adoptively transferred into K5/TGO CD45.1.2 recipients placed on doxycycline chow to induce Ag expression. Four days after transfer, SDLNs were harvested and CFSE dilution of the transferred WT and CD27−/− populations (gated on Live CD4+ and then CD45.1 or CD45.2, respectively) was quantified. (B) Representative flow cytometric plots and quantification of percentages of Ki67+ WT or CD27−/− OTII cells 4 d after transfer. (C) Representative flow cytometric plots and quantification of percentages and MFI of CD69 in WT or CD27−/− OTII cells 4 d after transfer. (D) Representative flow cytometric plots and quantification of cell death in WT or CD27−/− OTII cells as assessed by Annexin V and viability dye incorporation. Data are representative of at least two independent experiments with n = 5 or more mice. The p values are determined using a paired Student t test. *p < 0.05, **p < 0.01, ns, p > 0.05.
We next assayed whether signaling through CD27 affected Teff cell survival in our model. Similar to above, both WT OTII CD45.1 and CD27−/− OTII CD45.2 cells were adoptively transferred at a 1:1 ratio into K5/TGO recipients, followed by Ag induction. Survival was assessed at days 5 and 7 after transfer by quantifying the percentage of dead and dying cells, using Ghost dye (an amine reactive viability dye) and Annexin V staining (Fig. 4D). A significant increase in dead or dying Teff cells (GHOST+Annexin V+) was observed in the CD27−/− OTII population compared with WT, and this trend increased with time after Ag induction (Fig. 4D). Taken together, these results suggest that CD27 augments the accumulation of autoreactive CD4+ T cell responding to tissue Ag by promoting cell survival rather than activation or proliferation.
Signaling through CD27 suppresses the extrinsic cell death pathway to promote autoreactive Teff cell survival
One mechanism by which multiple TNFRSF receptors promote survival is through the induction of antiapoptotic BCL2 family members (20, 27–30). These antiapoptotic proteins suppress cell death by antagonizing both multidomain proapoptotic proteins (BAX and BAK) as well as activator BH3-only proteins (BID and BIM). In this manner, antiapoptotic BCL2 family members can prevent mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c (31). Opposing antiapoptotic proteins are sensitizer BH3-only proteins. This group of BH3 family members bind to antiapoptotic proteins, inhibiting their function and allowing for the release of activators and eventual activation of BAX and BAK. This process is referred to as the mitochondrial or intrinsic apoptotic pathway, and whether a cell undergoes apoptosis is ultimately regulated by the balance of proapoptotic, antiapoptotic, and sensitizer BH3-only family members (reviewed in Ref. 31). CD27 has been reported to induce the expression of antiapoptotic proteins including BCL-XL and MCL-1 (18–20). Thus, we hypothesized that signaling through CD27 promotes Teff cell survival by suppressing MOMP through the induction of antiapoptotic proteins. To test this hypothesis, we used BH3 profiling. BH3 profiling is a powerful technique that can be used to assess mitochondrial priming (24). This technique involves exposing cells to peptides that mimic the BH3 domains of endogenous activator or sensitizer BH3-only proteins and then measuring the degree of cytochrome c release from mitochondria. Peptide mimics of global sensitizers BIM and PUMA will inhibit all members of the antiapoptotic family. BIM peptide mimics will also directly interact with BAX and BAK, whereas PUMA cannot. This technique can therefore be used to compare differential sensitivity of individual populations to mitochondrial apoptosis by measuring cytochrome c release in response to various BH3-only peptides (32). If a certain population of cells is more primed for mitochondrial apoptosis, such a population should release more cytochrome c in response to exposure to an activator BH3 peptide such as BIM. Differences in response to treatment with PUMA further illustrate that a population exhibits a dependence on antiapoptotic family members for survival.
We performed BH3 profiling on WT and CD27−/− OTII cells isolated from the SDLNs of recipient K5/TGO mice 4 d after Ag induction. Isolated cells were exposed to various concentrations of peptides or vehicle controls, then fixed and stained for mitochondrial cytochrome c. Assay validation confirmed that both BIM and PUMA peptide exposure induced a dose-dependent release of mitochondrial cytochrome c (Fig. 5). Strikingly, in contrast to our hypothesis, both BIM and PUMA induced equal cytochrome c release in both WT and CD27−/− OTII cells at all concentrations tested, suggesting that these populations are equally primed for MOMP (Fig. 5). Thus, differences in survival between autoreactive WT and CD27−/− OTII cells in our model is most likely not attributed to activity of antiapoptotic BCL2 family members.
CD27 signaling on CD4+ T cells preferentially promotes survival via the extrinsic apoptosis pathway. WT CD45.1 and CD27−/− CD45.2 OTII cells were combined at a 1:1 ratio and adoptively transferred into K5/TGO CD45.1.2 recipients, followed by doxycycline administration. Four days after Ag induction, SDLNs were harvested. After CD4 enrichment, BH3 profiling was performed using specific concentrations (as indicated in figure) of BIM (activator) peptide, PUMA(sensitizer) peptide, DMSO (vehicle), or ALA (positive control). Cytochrome c release was quantified by flow cytometry. (A) Representative flow cytometric plots of cytochrome c staining in either WT (top) or CD27−/− (bottom) cells, gated on live CD4+, then either CD45.1 or CD45.2, respectively. (B) Quantification of percentage cytochrome c release after various peptide or vehicle treatments. (C) Flow cytometric staining and quantification of active caspase 8 expression in either WT (CD45.1+) or CD27−/− (CD45.2+) cells, after gating of Live CD4+Vb5+. Data are representative of three independent experiments with n = 4 mice. Graphs depict mean ± SEM. ****p < 0.0001.
The other major mechanism that influences survival is the extrinsic, or death receptor–mediated, apoptosis pathway. The ligation of death receptors, such as TNFR, CD95, or TRAIL1, can induce the activation of caspase 8 via proteolytic cleavage (reviewed in Ref. 33). Caspase 8 activation leads to the downstream caspase cascade that ultimately results in apoptosis. To determine if CD27 expression suppressed the extrinsic apoptosis pathway, we quantified active caspase 8 by flow cytometry. At day 4 after Ag induction, CD27−/− OTII cells expressed significantly higher levels of active caspase 8 compared with WT controls (Fig. 5C). These results suggest that CD27 promotes Teff cell survival by suppressing the extrinsic apoptosis pathway.
Discussion
Developing therapeutics that target the immune system are of great interest to treat both autoimmunity and cancer. The latest generation of cancer immunotherapies targeting immunoregulatory receptors on T cells (i.e., checkpoint inhibitor therapy) have had enhanced response rates in many tumors compared with conventional therapies. Notably, when checkpoint inhibitors are used in combination, response rates are higher than either therapy alone (34). In addition to “removing the brakes” from the immune response, it is also of great interest to understand how to “add fuel to the fire” and augment T cell responses. One way to achieve this is through targeting costimulatory receptors, including those of the TNFRSF (35). TNFRSF receptors play crucial roles in augmenting T cell responses by promoting activation, expansion, survival, differentiation, and memory. It is therefore of fundamental importance to understand how these receptors function in different disease settings and within different tissues. In the current study, we demonstrate that one TNFRSF member, CD27, enhances cutaneous immune responses by promoting autoreactive CD4+ Teff cell survival by suppressing the extrinsic (death receptor–mediated) apoptosis pathway.
Our findings are consistent with prior studies demonstrating that CD27 plays a major role in promoting T cell survival (14, 18–20, 22). Several studies have demonstrated that this pathway increases the expression of antiapoptotic BCL-2 family members in CD4 and CD8 T cells, including BCL-2 and BCL-XL (18–20). We used BH3 profiling to test whether CD27 signaling suppressed sensitivity to mitochondrial-induced cell death by increasing the expression of antiapoptotic BCL2 proteins in autoreactive CD4+ Teff cells responding to tissue self-antigen (24). If increasing the expression of antiapoptotic proteins was the major mechanism by which CD27 promoted Teff survival, CD27-deficient cells would be more sensitive to cytochrome c release upon treatment with either BIM (activator) or PUMA (sensitizer) peptides. Notably, we did not observe any difference in priming between CD27-sufficient or CD27-deficient cells. Although our study does not necessarily rule out a role for CD27 in promoting survival through increasing the expression of antiapoptotic proteins, our results strongly suggest that this is not the major mechanism in CD4+ Teff cells. This is consistent with previous work demonstrating that forced overexpression of BCL2 family members in CD8+ T cells alone or in combination was not enough to compensate for CD27 deficiency (19), suggesting that CD27 can promote survival through multiple mechanisms.
We found that CD27 expression on CD4+ Teff cells promotes survival via suppressing the extrinsic apoptosis pathway. Death receptor ligation results activation of caspase 8, which in turn initiates the downstream caspase cascade that ultimately leads to apoptosis. CD27-sufficent cells expressed lower levels of active caspase 8 compared with CD27-deficient cells. This finding, combined with our results demonstrating that Ag-specific Teff cells are equally sensitive to BH3 peptide-induced MOMP with and without CD27, strongly suggests that CD27 promotes survival by suppressing activation of the extrinsic apoptosis pathway.
Our results are consistent with other models of tissue autoimmunity, demonstrating that blocking CD27 suppresses experimental colitis and lung allergy (36, 37). In contrast to these studies, it has been shown that CD27 deficiency augments autoimmune disease in experimental autoimmune encephalomyelitis (38). Although these studies appear to be contradictory, CD27 is now known to play different roles in CD4+ T cell function depending on the inflammatory context. Multiple studies have demonstrated that although CD27 augment Th1 responses (15, 18, 39–41), signaling through this pathway attenuates Th17 responses (38, 41). The K5/TGO model used in this study induces a strong Th1 response, as defined by IFN-γ and IL-2 production from the transferred Ag-specific Teff cells, with minimal IL-17 production (25). We observed a decrease in IFN-γ and IL-2 production from CD27-deficient cells after Ag induction, with no difference in IL-17 (data not shown). Thus, our results support the current literature, demonstrating that CD27 enhances Th1 responses. The functional consequence of CD27 signaling in Th17-driven cutaneous inflammatory responses (such as psoriasiform inflammation or Candida albicans infection) remain to be determined. Interestingly, alterations in the CD70–CD27 pathway have been associated with disease severity in patients with systemic lupus erythematosus, a predominantly Th1-mediated disease with numerous skin manifestations (42). Thus, the mouse model used in our study and our major findings may have relevance to human systemic lupus erythematosus.
In conclusion, in this study we demonstrate that CD27 plays a critical role in augmenting cutaneous autoimmunity by promoting autoreactive CD4+ T cell survival. These results suggest that manipulating the CD27 pathway may be a promising therapeutic approach in autoimmune or malignant diseases of the skin.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We acknowledge and thank C. Benetiz and Y. Hu for assistance with animal husbandry. Flow cytometry data were generated with the assistance of a Diabetes Research Center grant to the UCSF Parnassus Flow Cytometry Core.
Footnotes
This work was supported primarily by grants to M.D.R.: National Institutes of Health (NIH) K08-AR062064, Burroughs Wellcome Fund CAMS-1010934, NIH DP2-AR068130, NIH R21-AR066821, a National Psoriasis Foundation Translational Grant, and a Dermatology Foundation Stiefel Scholar Award in Autoimmune and Connective Tissue Diseases.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- K5
- cytokeratin 5
- MFI
- mean fluorescence intensity
- MOMP
- mitochondrial outer membrane permeabilization
- SDLN
- skin draining lymph node
- Teff
- effector T
- TGO
- tetracycline-response element
- TNFRSF
- TNFR super family
- UCSF
- University of California, San Francisco
- WT
- wild-type.
- Received March 13, 2019.
- Accepted May 29, 2019.
- Copyright © 2019 by The American Association of Immunologists, Inc.