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Keywords:

  • Allograft rejection;
  • T cell;
  • Treg

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Despite extensive studies on CD4+CD25+ regulatory T cells (Tregs), their application in adoptive transfer therapies is still not optimal in immune-competent wild-type (WT) animal models. Therefore, it is compelling to search for more potent Tregs for potential clinical application. Mounting evidence has shown that naturally occurring CD8+CD122+ T cells are also Tregs. However, their suppression in allograft rejection, efficiency in suppression and underlying mechanisms remain unclear. Using a murine allotransplantation model, we reported here that CD8+CD122+ Tregs were actually more potent in suppression of allograft rejection and underwent more rapid homeostatic proliferation than their CD4+CD25+ counterparts. Moreover, they produced more IL-10 and were more potent in suppressing T cell proliferation in vitro. Deficiency in IL-10 in CD4+CD25+ and CD8+CD122+ Tregs resulted in their reduced but equal suppression in vivo and in vitro, suggesting that IL-10 is responsible for more effective suppression by CD8+CD122+ than CD4+CD25+ Tregs. Importantly, transfer of CD8+CD122+ Tregs together with the administration of recombinant IL-15 significantly prolonged allograft survival in WT mice. Thus, for the first time, we demonstrate that naturally arising CD8+CD122+ Tregs not only inhibit allograft rejection but also exert this suppression more potently than their CD4+CD25+ counterparts. This novel finding may have important implications for tolerance induction.


Abbreviations
Abs

antibodies

CBD

common bile duct

LN

lymph nodes

MST

median survival time

Tregs

regulatory T cells

WT

wild-type

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Emerging evidence has shown that naturally occurring CD8+CD122+ T cells are also regulatory T cells (Tregs) that maintain T cell homeostasis, suppress aggressive T cell responses [1-6] and antitumor immunity [7], and control autoimmunity [8, 9]. We have also found that CD8+CD122+ T cells are memory-like Tregs [10]. Therefore, CD8+CD122+ Tregs well correspond to CD4+CD25+ Treg counterparts given that CD122 is the β subunit of IL-2 receptor on T cells while CD25 is the α subunit of the same receptor [11]. It now seems clear that both of the subunits are involved in the immune regulation mediated by Tregs. However, it remains unknown which subunit is more potent in mediating Treg suppression. In particular, CD8+CD122+ Tregs are relatively new and understudied in comparison with CD4+CD25+ counterparts. Since current Treg cell therapies using adoptive transfer of CD4+CD25+ cell population are not optimal in immune-competent wild-type (WT) animal models, it is very important to identify an efficient Treg population for more potent suppression of pathogenic immune responsiveness including alloimmune responses.

Here we sought to determine if naturally occurring CD8+CD122+ and CD4+CD25+ Tregs exhibit a differential pattern in their suppressive function. We found that CD8+CD122+ Tregs were more potent in suppression of allograft rejection and underwent faster homeostatic proliferation than their CD4+CD25+ counterparts. Moreover, they produced more IL-10 and were more potent in suppression of responder T cell proliferation in vitro than CD4+CD25+ Tregs. Most importantly, transfer of CD8+CD122+, but not CD4+CD25+, Tregs together with administration of recombinant IL-15 significantly prolonged islet allograft survival in the absence of a major immunosuppressive agent even in WT mice.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Mice and antibodies

WT BALB/c (H-2d), C3H/HeJ and C57BL/6 (H-2b) mice were purchased from National Cancer Institute (NIH, Bethesda, MD). Rag1−/−, IL-10−/− mice and Thy1.1+ congenic mice were all in B6 background and purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in a specific pathogen-free environment and all animal experiments were approved by the Animal Care and Use Committee of the University of Texas Health Science Center, Tyler, TX. Recombinant murine IL-15, IL-2 and anti-IL-10 mAb were bought from eBioscience (San Diego, CA) while activating anti-CD3 and anti-CD28 antibodies (Abs) were purchased from BD Biosciences (Bedford, MA).

Islet transplantation

Approximately 400 donor islets were transplanted into the subcapsular space of the right kidney of a recipient mouse as described in our previous studies [12-14]. Donor mice were anesthetized and a median laparotomy was performed. The common bile duct (CBD) was exposed and clamped. Three milliliters of collagenase V (1 mg/mL in phosphate buffered saline; Sigma, St. Louis, MO) was injected into the CBD to distend the pancreas. The pancreas was then removed, minced and subjected to stationary digestion by incubation in a 37°C water bath for 12 min [15], and then washed three times in ice-cold HBSS. The crude preparation was filtered through a hand-held 100 μm nylon cell strainer (BD Biosciences). The strainer was turned upside-down over a Petri dish and rinsed with HBSS to wash the islets into the dish. Islets were counted and picked up using a 23G needle. Recipient mice were rendered diabetic by a single injection of streptozotocin (180 mg/kg; Sigma) 10–12 days before transplantation. Primary graft function was defined as blood glucose under 200 mg/dL for 48 h after transplantation. Graft rejection was defined as a rise in blood glucose to >300 mg/dL for three consecutive days after primary function and routinely confirmed by histology showing cellular rejection.

CD4+CD25+ and CD8+CD122+ Treg isolation

Spleen cells from 5- to 6-week-old naïve mice were pooled after lysing red blood cells. Cells were then stained with anti-CD4-PE, anti-CD25-FITC, anti-CD8-APC and anti-CD122-biotin Abs (BD Biosciences, Mountain View, CA), followed by streptavidin–PerCP, and CD4+CD25+ or CD8+CD122+ Tregs were sorted out using a FACSAria (BD Biosciences). The purity of the sorted cells was around 96%.

Analysis of T cell proliferation in vitro by [3H]-Thymidine uptakes

Naturally occurring CD4+CD25+ or CD8+CD122+ Tregs were sorted out from naïve WT B6 mice using FACSAria (BD Biosciences) and then were cultured alone or with nylonwood-enriched T cells from naïve B6 mice in 96-well plates (Corning Costar, Brooklyn, NY) in complete RPMI 1640 medium (10% FCS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin). Irradiated BALB/c spleen cells were also added to the culture to serve as both alloantigens and APC, as described in our previous study [16]. In antigen-specific studies, splenocytes derived from reconstituted Rag1−/− recipient mice that received Balb/C islets, Tregs and T cells 2 weeks ago were restimulated with irradiated Balb/C donor or third-party (C3H/HeJ) splenocytes in vitro. Four days later, cells were harvested and analyzed by a Scintillation counter (PerkinElmer, Meriden, CT). Cells were pulsed with [3H]-Thymidine for last 8 h before harvesting.

Treg proliferation in vivo via CFSE staining

For CFSE staining, CD4+CD25+ or CD8+CD122+ Tregs isolated from naïve WT B6 or Thy1.1+ mice were first labeled with CFSE dye (5 μM; Invitrogen, Carlsbad, CA) at 37°C for 8 min, washed, and then transferred to Rag1−/− mice (B6 background). Some recipients were also transplanted with Balb/C islets. Four days later, splenocytes of different recipient mice were isolated and stained separately with anti-CD4-PE, anti-CD8-PE or anti-CD90.1-PE (BD Biosciences), and analyzed by a FACSCalibur (BD Biosciences).

Measurement of cytokines in the supernatant

Purified CD4+CD25+ or CD8+CD122+ Tregs derived from WT mice were cultured in complete RPMI 1640 medium in 96-well plates in the presence of plate-bound anti-CD3 Ab alone, together with activating anti-CD28 Ab, or irradiated allogeneic splenocytes. Seventy-two hours later, IL-10 levels in the supernatant were detected by ELISA according to manufacturer's instructions (Invitrogen).

Statistical analysis

Comparisons of the mean were performed using the Student's t-test for two groups and ANOVA for multiple groups. The analysis of graft survival was conducted using Kaplan–Meier method (log-rank test). All analyses were performed using Prism5 software (GraphPad Software, La Jolla, CA). A value of p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

CD8+CD122+ Tregs are more efficient in suppression of allograft rejection than their CD4+CD25+ counterparts

Since both naturally occurring CD4+CD25+ and CD8+CD122+ cells are Tregs, we asked whether they differ in their efficacy in suppression. Rag1−/− mice that received unfractionated T cells with either CD4+CD25+ or CD8+CD122+ Tregs derived from naïve B6 mice were transplanted with islets from Balb/C donors. As shown in Figure 1A, we found that both CD4+CD25+ and CD8+CD122+ Tregs, at the ratio of 1:4 (Treg/T-responder), significantly delayed islet allograft rejection mediated by conventional T cells (median survival time [MST] = 39 vs. 14 days and 77 vs. 14 days, respectively, n = 7–9, both p < 0.001). However, transfer of CD8+CD122+ Tregs was more effective in the prolongation of islet allograft survival than that of CD4+CD25+ counterparts (ratio 1:4: MST = 77 vs. 39 days, n = 8–9, p = 0.008 and ratio 1:8: MST = 46 vs. 27 days, n = 7, p = 0.018), suggesting that CD8+CD122+ Tregs are more potent T suppressors than their CD4+CD25+ counterparts. As a control, transfer of CD8+CD122+ Tregs alone did not cause allograft rejection, indicating that they are true Tregs. To define antigen specificity in their suppression, splenocytes derived from Rag1−/− recipient mice that received islet allografts, T cells and Tregs 2 weeks ago were restimulated with donor or third-party splenocytes in vitro. As shown in Figure 1B, transfer of CD8+CD122+ Tregs suppressed in vitro T cell proliferative responses to both donor and third-party alloantigens (donor: 12.3 ± 1.6 vs. 22.4 ± 2.4, n = 4, p = 0.012; and third-party: 11.2 ± 1.1 vs. 18.8 ± 2.5, n = 5, p = 0.014), implying that their suppression is not antigen specific.

image

Figure 1. Naturally arising CD8+CD122+ Tregs more effectively suppress islet allograft rejection than their CD4+CD25+ counterparts. (A) Rag1−/− (B6 background) mice that did not or did receive 2 × 106 unfractionated T cells without or with 0.5 × 106 (ratio 1:4) or 0.25 × 106 (ratio 1:8) CD4+CD25+ or CD8+CD122+ B6-derived Tregs 1 day earlier were transplanted with Balb/C islets (n = 7–9). Islet allograft rejection was observed. Transfer of CD8+CD122+ Tregs was more effective in the prolongation of islet allograft survival than that of CD4+CD25+ Tregs (Treg/T-responder ratio 1:4: MST = 77 vs. 39 days, n = 8–9, p = 0.008 and ratio 1:8: MST = 46 vs. 27 days, n = 7, p = 0.018). (B) Splenocytes derived from Rag1−/− recipient mice that received Balb/C islets, Tregs and T cells (ratio 1:4) 2 weeks ago were restimulated with irradiated donor or third-party (C3H/HeJ) splenocytes in vitro. Four days later, cell proliferation was measured by 3H-Thymidine uptakes. One representative of three separate experiments is shown. Transfer of CD8+CD122+ Tregs suppressed in vitro T cell proliferation to both donor and third-party alloantigens (donor: 12.3 ± 1.6 vs. 22.4 ± 2.4, n = 4, p = 0.012; and third-party: 11.2 ± 1.1 vs. 18.8 ± 2.5, n = 5, p = 0.014). CPM, counts per minute; MST, median survival time; Tregs, regulatory T cells.

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CD8+CD122+ Tregs underwent faster homeostatic proliferation in vivo than their CD4+CD25+ counterparts

Given that CD8+CD122+ Tregs were more potent in suppression of allograft rejection than their CD4+CD25+ counterparts, we speculated that the former underwent faster expansion or homeostatic proliferation than the latter. CFSE-stained CD8+CD122+ or CD4+CD25+ Tregs were adoptively transferred to Rag1−/− mice. Four days later, Tregs were analyzed for their proliferation based on their CFSE dye dilution. As shown in Figure 2A, CD8+CD122+ Tregs proliferated faster than their CD4+CD25+ counterparts (percentage of proliferated cells: 83 ± 10 vs. 54 ± 7, n = 4, p = 0.015). CD8+CD122+ Tregs also proliferated more quickly than CD4+CD25+ components (percentage of proliferated cells: 64 ± 12 vs. 33 ± 8, n = 4, p = 0.018) when recipient mice were co-transferred with conventional T cells and transplanted with islet allografts as well (Figure 2B). These findings suggest that CD8+CD122+ Tregs, in either absence or presence of alloantigenic stimulation, undergo quicker homeostatic proliferation and expansion than their CD4+CD25+ counterparts.

image

Figure 2. CD8+CD122+ Tregs undergo faster homeostatic proliferation in vivo than their CD4+CD25+ counterparts. (A) 0.5 × 106 CFSE-stained CD8+CD122+ or CD4+CD25+ Tregs were adoptively transferred to Rag1−/− mice. Four days later, splenocytes were isolated and Tregs were analyzed for their proliferation based on their CFSE dye dilution. Histograms were gated on CD4 or CD8 population. (B) Rag1−/− mice were transplanted with Balb/C islets and received 2 × 106 unfractionated T cells from wild-type B6 mice together with 0.5 × 106 CFSE-stained CD8+CD122+ or CD4+CD25+ Tregs derived from Thy1.1 congenic mice (B6). Four days later, splenocytes were stained for Thy1.1 surface marker and analyzed by FACS. Data are presented as mean ± SD. One representative of three independent experiments is shown. CD8+CD122+ Tregs underwent faster homeostatic proliferation than their CD4+CD25+ counterparts (percentage of proliferated cells: 83 ± 10 vs. 54 ± 7, n = 4, p = 0.015). The former also proliferated faster than the latter in transplanted recipients (percentage of proliferated cells: 64 ± 12 vs. 33 ± 8, n = 4, p = 0.018). Tregs, regulatory T cells.

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CD8+CD122+ Tregs are also more potent in suppression of T cell proliferation in vitro than their CD4+CD25+ counterparts

To determine if CD8+CD122+ Tregs can also suppress T cell proliferation in vitro more effectively than their CD4+CD25+ counterparts, one-way mixed lymphocyte reactions were set up using nylonwood-enriched T cells from B6 mice as responders and irradiated Balb/C splenocytes as stimulators with or without B6-derived Tregs. As shown in Figure 3, CD8+CD122+ Tregs, at various ratio of Treg/T-responder, inhibited T cell proliferation in vitro more potently than their CD4+CD25+ counterparts (1:4, counts per minute [CPM]: 7.2 ± 1.1 vs. 11.6 ± 1.2, n = 4, p = 0.021; 1:8, CPM: 11.0 ± 0.8 vs. 15.8 ± 1.3, n = 4, p = 0.022; and 1:16, CPM: 16.5 ± 1.5 vs. 21.9 ± 1.9, n = 4, p = 0.025).

image

Figure 3. CD8+CD122+ Tregs vigorously inhibit T cell proliferation in vitro. One-way mixed lymphocyte reactions were set up using unfractionated T cells from B6 mice as responders and irradiated Balb/C splenocytes as stimulators with or without B6-derived CD8+CD122+ or CD4+CD25+ Tregs at various ratios of Treg/T-responder. Data are presented as mean ± SD. One representative of three independent experiments is shown. CD8+CD122+ Tregs, at all ratios of Treg/T-responder, inhibited T cell proliferation more potently than their CD4+CD25+ counterparts (1:4, CPM: 7.2 ± 1.1 vs. 11.6 ± 1.2, n = 4, p = 0.021; 1:8, CPM: 11.0 ± 0.8 vs. 15.8 ± 1.3, n = 4, p = 0.022; and 1:16, CPM: 16.5 ± 1.5 vs. 21.9 ± 1.9, n = 4, p = 0.025). CPM, counts per minute; Tregs, regulatory T cells.

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CD8+CD122+ Tregs produced more IL-10 in vitro than their CD4+CD25+ counterparts

CD8+CD122+ Tregs exert their suppression largely by producing cytokine IL-10 [1-3, 10]. We therefore asked whether CD8+CD122+ and CD4+CD25+ Tregs would secrete similar levels of IL-10 upon activation by activating anti-CD3 and anti-CD28 Abs or allogeneic splenocytes. As shown in Figure 4, activated CD8+CD122+ Tregs produced more IL-10 in the supernatant than their CD4+CD25+ counterparts (anti-CD3 groups: 2.70 ± 0.28 vs. 1.26 ± 0.22, n = 4, p = 0.002; anti-CD3+ anti-CD28 groups: 7.80 ± 1.65 vs. 3.72 ± 0.73, n = 4, p = 0.002; and Allo-APC: 5.10 ± 0.63 vs. 2.81 ± 0.42, n = 4, p = 0.004).

image

Figure 4. CD8+CD122+ Tregs produced more IL-10 in the supernatant than CD4+CD25+ component upon activation. FACS-sorted CD8+CD122+ or CD4+CD25 Tregs from B6 mice were cultured in the presence of plate-bound anti-CD3 Ab and anti-CD28 Ab or allogeneic Balb/C splenocytes (Allo-APC). Seventy-two hours later, IL-10 levels in the supernatant were determined by ELISA. Data are presented as mean ± SD. One of three experiments is shown. CD8+CD122+ Tregs produced more IL-10 than CD4+CD25+ Tregs (anti-CD3 group: 2.70 ± 0.28 vs. 1.26 ± 0.22, p = 0.002; anti-CD3+ anti-CD28 group: 7.80 ± 1.65 vs. 3.72 ± 0.73, p = 0.002; and Allo-APC: 5.10 ± 0.63 vs. 2.81 ± 0.42, n = 4, p = 0.004). Ab, antibody; Tregs, regulatory T cells.

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Suppression of alloimmune responses by CD4+CD25+ and CD8+CD122+ Tregs is comparable when both lack IL-10

We then determined if IL-10 is responsible for more potent suppression by CD8+CD122+ Tregs than their CD4+CD25+ counterparts. Rag1−/− mice, which received unfractionated IL-10-replete T cells with either CD4+CD25+ or CD8+CD122+ Tregs derived from IL-10-deficient B6 mice, were transplanted with islets from Balb/C donors. Some recipients that received IL-10-replete CD4+CD25+ or CD8+CD122+ Tregs were also treated with neutralizing anti-IL-10 Abs. As shown in Figure 5A, IL-10-deficient CD8+CD122+ and CD4+CD25+ Tregs similarly extended islet allograft survival (MST = 28 vs. 24 days, n = 7, p = 0.247) and there was also no difference in allograft survival between anti-IL-10-treated CD8+CD122+ and CD4+CD25+ Treg groups (MST = 29 vs. 24 days, n = 6, p = 0.367), indicating that production of IL-10 is responsible for the superiority of CD8+CD122+ Tregs in suppression to their CD4+CD25+ counterparts. However, both Tregs still significantly delayed islet allograft rejection even if they lacked IL-10 (MST = 28 vs. 13, p = 0.002; and 24 vs. 13 days, p = 0.015, both n = 6–7), implying that other mechanisms, in addition to IL-10, may be also involved in their suppression. To further define if Treg suppression in vitro is dependent on their production of IL-10, T cells from WT B6 mice were stimulated by irradiated Balb/C splenocytes in the presence of WT or IL-10-deficient CD8+CD122+ or CD4+CD25+ Tregs. Four days later, T cell proliferation was measured by 3H-Thymidine uptakes. As shown in Figure 5B, suppression by CD8+CD122+ or CD4+CD25+ Tregs was significantly reduced (CD8+CD122+: 16.0 ± 1.5 vs. 7.5 ± 1.2, p = 0.006; and CD4+CD25+: 17.6 ± 1.8 vs. 12.5 ± 1.7, p = 0.012) when both lacked IL-10. But even without IL-10, their suppression was not totally abolished compared to T cells alone (CD8+CD122+: 16.0 ± 1.5 vs. 24.4 ± 3.1, p = 0.007; and CD4+CD25+: 17.6 ± 1.8 vs. 24.4 ± 3.1, p = 0.021).

image

Figure 5. Capability of CD8+CD122+ Tregs to suppress allograft rejection is similar to that of their CD4+CD25+ counterparts when both lack IL-10. (A) Rag1−/− mice, which received unfractionated IL-10-replete T cells (2 × 106) alone or together with 0.5 × 106 CD4+CD25+ or CD8+CD122+ Tregs derived from IL-10-deficient or IL-10-replete B6 mice, were transplanted with islets from Balb/C donors 1 day later (n = 6–8). Some recipients of IL-10-replete CD4+CD25+ or CD8+CD122+ Tregs were also treated intraperitoneally with anti-IL-10 mAb at 0.1 mg on days 0, 2, 4 and 6 after transplantation. IL-10-deficient CD8+CD122+ and CD4+CD25+ Tregs similarly extended islet allograft survival (MST = 28 vs. 24 days, n = 7, p = 0.247) and there was also no difference in allograft survival between anti-IL-10-treated CD8+CD122+ and CD4+CD25+ Treg groups (MST = 29 vs. 24 days, n = 6, p = 0.367). (B) T cells from WT B6 mice were stimulated by irradiated Balb/C splenocytes in the presence of WT or IL-10-deficient CD8+CD122+ or CD4+CD25+ Tregs with a Treg/T-responder ratio of 1:4. Four days later, T cell proliferation was measured by 3H-Thymidine uptakes. Suppression by CD8+CD122+ or CD4+CD25+ Tregs was significantly reduced (CD8+CD122+: 16.0 ± 1.5 vs. 7.5 ± 1.2, p = 0.006; and CD4+CD25+: 17.6 ± 1.8 vs. 12.5 ± 1.7, p = 0.012) when both lacked IL-10. CPM, counts per minute; MST, median survival time; Tregs, regulatory T cells; WT, wild-type.

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IL-15 promotes CD8+CD122+ Treg expansion and enhances their suppression of allograft rejection even in WT mice

To investigate whether transfer of CD8+CD122+ Tregs also suppresses allograft rejection in WT recipient mice, they were adoptively transferred to WT B6 mice that received Balb/C islets. As shown in Table 1, transfer of either CD8+CD122+ or CD4+CD25+ Tregs failed to prolong islet allograft survival in immune-competent WT recipients (MST = 15 vs. 13 days, n = 7, p = 0.236; and 14 vs. 13 days, n = 7, p = 0.839). However, administration of recombinant IL-15 significantly delayed islet allograft rejection in WT recipients that received CD8+CD122+ (MST = 26 vs. 13 days, n = 7–8, p = 0.006) but not CD4+CD25+ Tregs (MST = 16 vs. 13 days, n = 7–8, p = 0.101), indicating that IL-15 enhances CD8+CD122+ Treg function. As a control, administering recombinant IL-2 together with transfer of either CD8+CD122+ or CD4+CD25+ Tregs did not significantly inhibit islet allograft rejection in WT recipients (Table 1).

Table 1. IL-15 enhances CD8+CD122+ Treg suppression of allograft rejection in WT mice
Treatment groupsSurvival time (days)MST
  1. 1 × 106 FACS-sorted CD4+CD25+ or CD8+CD122+ Tregs derived from naïve B6 mice were transferred to WT B6 mice that were then transplanted with islets from Balb/C donors 1 day later (n = 7–8). Some recipients were administered intraperitoneally with 0.25 μg of recombinant murine IL-2 or IL-15 on days 0, 2, 4 and 6 after islet transplantation. Administration of recombinant IL-15 significantly delayed islet allograft rejection in WT recipients that received CD8+CD122+ (MST = 26 vs. 13 days, n = 7–8, p = 0.006) but not CD4+CD25+ Tregs (MST = 16 vs. 13 days, n = 7–8, p = 0.101). MST, median survival time; Tregs, regulatory T cells; WT, wild-type.

Control7, 8, 13, 13, 14, 17, 2113
CD4+CD25+ Treg9, 12, 12, 14, 18, 20, 2114
CD8+CD122+ Treg8, 10, 15, 15, 16, 24, 2515
CD4+CD25+ Treg+ IL-210, 11, 14, 16, 21, 24, 2916
CD8+CD122+ Treg+ IL-29, 12, 14, 17, 19, 25, 2717
CD4+CD25+ Treg+ IL-159, 13, 14, 16, 16, 22, 23, 2816
CD8+CD122+ Treg+ IL-1511, 17, 18, 25, 26, 37, 43, 6026

To understand why IL-15 promotes naturally occurring CD8+CD122+, but not CD4+CD25+, Treg suppression, we determined their phenotypes. As shown in Figure 6A, both CD4+CD25+ and CD8+CD122+ subsets express CD132 (γ chain). But CD4+CD25+ components do not express CD122 and are IL-15Rαlow while CD8+CD122+ subsets do not express CD25 but are IL-15Rαhigh. This may help explain why we found that allostimulated CD4+CD25+ Tregs proliferated in vitro in response to IL-2 but not IL-15 (IL-2: 6.5 ± 0.6 vs. 1.2 ± 0.2, p = 0.006; IL-15: 1.4 ± 0.2 vs. 1.2 ± 0.2, p = 0.715) while CD8+CD122+ Tregs did the opposite (IL-2: 2.2 ± 0.4 vs. 1.7 ± 0.2, p > 0.05; IL-15: 7.9 ± 1.4 vs. 1.7 ± 0.2, p = 0.004; Figure 6B).

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Figure 6. Natural CD8+CD122+ Treg phenotypes and their responses to IL-2 and IL-15. (A) Spleen cells from wild-type B6 mice were stained and analyzed by FACS. Histograms were gated on CD4+CD25+ or CD8+CD122+ populations. Dotted line represents isotype controls. Other groups include CD122 (solid line on the top panel), CD25 (solid line on the lower panel), CD132 (bold line) and IL-15Rα (dashed line). (B) CD4+CD25+ or CD8+CD122+ Tregs derived from naïve B6 mice were stimulated in vitro by irradiated Balb/C splenocytes in the presence of IL-2 or IL-15 (0.1 μg/mL). Control situation was stimulation by the same splenocytes without IL-2 or IL-15. Four days later, cell proliferation was determined by 3H-Thymidine uptake. One of three separate experiments is shown. CD4+CD25+ Tregs proliferated in response to IL-2 but not IL-15 (IL-2: 6.5 ± 0.6 vs. 1.2 ± 0.2, p = 0.006; IL-15: 1.4 ± 0.2 vs. 1.2 ± 0.2, p = 0.715) while CD8+CD122+ Tregs did the opposite (IL-2: 2.2 ± 0.4 vs. 1.7 ± 0.2, p > 0.05; IL-15: 7.9 ± 1.4 vs. 1.7 ± 0.2, p = 0.004). CPM, counts per minute; Tregs, regulatory T cells.

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To measure CD8+CD122+ and CD4+CD25+ Treg expansion after their transfer, they were sorted out from Thy1.1+ mice and transferred to WT recipients that received Balb/C islets. One week later, they were enumerated by FACS analysis. As shown in Figure 7A, administration of IL-15 significantly increased the number of Thy1.1+CD8+ Tregs in various organs (spleen: 6.88 ± 0.71 vs. 3.67 ± 0.63; draining lymph nodes (LN): 3.50 ± 0.45 vs. 1.67 ± 0.33; and kidney: 4.85 ± 0.66 vs. 2.48 ± 0.39, n = 4, two-tailed, p = 0.025). However, IL-15 did not increase the number of Thy1.1+CD4+ Tregs (spleen: 2.08 ± 0.35 vs. 1.90 ± 0.32; draining LN: 1.07 ± 0.31 vs. 0.90 ± 0.22; and kidney: 1.55 ± 0.24 vs. 1.28 ± 0.27, n = 4, two-tailed, p > 0.05). At the time of rejection, both Treg subsets were still detectable. Only the number of Thy1.1+CD8+ Tregs in the kidney, but not spleens and LNs, was significantly increased by IL-15 treatment (kidney: 1.9 ± 0.35 vs. 1.02 ± 0.22, n = 4, p = 0.027; Figure 7B). On the other hand, IL-15 treatment expanded both PD-1+ and PD-1 components of endogenous CD8+CD122+ subsets 1 week after transplantation (PD-1+ group, spleen: 15.03 ± 1.42 vs. 8.17 ± 0.99; draining LN: 5.83 ± 0.64 vs. 3.30 ± 0.49; and kidney: 9.60 ± 0.76 vs. 15.00 ± 0.47, n = 4, two-tailed, p = 0.006; and PD-1- group, spleen: 18.93 ± 0.98 vs. 10.75 ± 1.73; draining LN: 7.22 ± 0.74 vs. 4.32 ± 0.71; and kidney: 12.40 ± 0.74 vs. 6.85 ± 0.66, n = 4, two-tailed, p = 0.007; Figure 7C). Transferred Thy1.1+CD8+CD122+ Tregs remained CD122high, CD25low and largely PD-1 1 week after transplantation (Figure S1).

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Figure 7. IL-15 promotes CD8+CD122+ Treg expansion after transplantation. 1 × 106 CD4+CD25+ or CD8+CD122+ Tregs derived from naïve Thy1.1+ mice (B6 background) were transferred to wild-type B6 recipients that were transplanted with Balb/C islets. One week later (A) or at the time of rejection (B), Thy1.1+CD4+ or Thy1.1+CD8+ cells from spleens, draining lymph nodes (dLN) and kidneys of recipient mice were enumerated by FACS. One week after transplantation, both PD-1+ and PD-1− subsets of endogenous CD8+CD122+ Tregs were also enumerated by FACS (C). One representative of three separate experiments is shown. IL-15 significantly increased the number of transferred Thy1.1+CD8+, but not Thy1.1+CD4+, Tregs 1 week after transplantation or at the time of rejection. Tregs, regulatory T cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Previous studies have shown that both CD4+CD25+ and CD8+CD122+ T cells are Tregs.

In search for more potent T suppressors for potential clinical application, we evaluated efficacy of naturally occurring CD8+CD122+ versus CD4+CD25+ Tregs in their suppression. We found that CD8+CD122+ Treg cells were much more effective in suppression of allograft rejection and underwent faster homeostatic proliferation than their CD4+CD25+ counterparts. Moreover, they produced more IL-10 and were more potent in suppression of T cell proliferation in vitro than CD4+CD25+ Tregs. Most importantly, transfer of CD8+CD122+, but not CD4+CD25+, Tregs together with administration of recombinant IL-15 significantly prolonged allograft survival in the absence of a major immunosuppressive agent in WT mice.

CD8+CD122+ Tregs correspond to CD4+CD25+ counterparts because CD122 is the β subunit of IL-2 receptor on T cells while CD25 is the α subunit of the same receptor [11]. Interestingly, CD8+CD122+ Tregs accounts for nearly 50% of CD8+ T cells in mice at the age of first 2 weeks. They decline in numbers and reach the bottom with approximately 10% 7–9 weeks after birth [1]. Therefore, like naturally occurring CD4+CD25+ Tregs, they probably originate from thymic emigrates during young age of up to 9 weeks. Thereafter they gradually increase over time, perhaps because mice encounter environmental antigens that generate antigen-specific memory CD8+CD122+ T cells [17-19]. In this study, we isolated CD8+CD122+ cells from 5- to 6-week-old mice due to their abundance in young mice, and found that they were actually Tregs. It remains to be defined if CD8+CD122+ T cells from aged mice are also Tregs. We speculate that they comprise more T-memory than Tregs in older mice. However, it is unclear if these memory T cells from aged mice are allospecific or just bystanders to a specific donor antigen.

We have previously demonstrated that naturally occurring CD8+CD122+ Tregs are FoxP3-negative [10], which differs from natural CD4+CD25+FoxP3+ Tregs. On the other hand, recent studies have also shown that inducible CD8+ Tregs are FoxP3-positive and can suppress allograft rejection or graft-versus-host disease [20-23]. The relationship between natural CD8+CD122+ Tregs and induced CD8+FoxP3+ Tregs is unclear and warrants further investigation in the future. A new study has shown that CD8+CD122+ Tregs contain clonally expanded cells with the same TCR sequences [24]. It is unknown if those CD8+ Tregs that have reportedly suppressed allograft rejection and graft-versus-host disease are also CD122+ [25-27].

IL-10 plays an important role in the immunosuppression mediated by both CD4+CD25+ [28-30] and CD8+CD122+ [2, 10] Tregs. We found that CD8+CD122+ Tregs produced more IL-10 than their CD4+CD25+ counterparts and that lack of IL-10 in both Tregs resulted in equal suppression, suggesting that higher level of IL-10 produced by CD8+CD122+ Tregs is responsible for their superiority in suppression to their CD4+CD25+ counterparts. On the other hand, the suppression by IL-10-deficient CD4+CD25+ or CD8+CD122+ Tregs is still significant, though largely reduced, indicating that other mechanisms, in addition to IL-10, may be involved in the suppression mediated by both Tregs.

T cell homeostatic proliferation in a steady state is essential for both maintaining basic immunity against infections and preventing autoimmune diseases. It is generally accepted that conventional CD8+ T cells undergo faster homeostatic proliferation than their CD4+ counterparts. Here for the first time we found that CD8+CD122+ Tregs also underwent quicker homeostatic proliferation than their CD4+CD25+ counterparts. Therefore, rapider expansion of CD8+CD122+ Tregs than their CD4+CD25+ counterparts is likely another mechanism by which CD8+CD122+ Tregs exert a more potent suppression than their CD4+CD25+ counterparts, because expanded CD8+CD122+ Tregs would compete for spaces/niches with conventional lymphocytes. Perhaps that is why CD8+CD122+ Treg suppression in our experimental models is not antigen specific.

Despite extensive studies on CD4+CD25+ regulatory T cells (Tregs), their adoptive transfer therapies in an immune-competent wild-type (WT) animal remains unsatisfactory. Our results demonstrated that transfer of CD8+CD122+ Tregs together with administering recombinant IL-15 at low doses significantly prolonged allograft survival even in WT mice. This finding is encouraging and has important clinical implications for Treg therapies of autoimmune diseases and transplant rejection. IL-15 is a unique cytokine that is essential for the generation and survival of memory CD8+ T cells [31, 32] while administration of recombinant IL-15 boosts their number [17, 33]. A recent interesting study by Yu et al [34] has shown that administration of IL-15 enhances anti-tumor immunity when combined with agents that block both CD4+CD25+ and CD8+CD122+ Tregs. In their studies, IL-15 likely enhanced conventional effector/memory CD8+ T cell function but not CD8+CD122+ Treg suppressive activity because Tregs were suppressed by blocking CTLA4 and PD-1 signaling pathways. In our transplantation studies, we wanted to boost Tregs instead of blocking their functionality. We successfully utilized low doses of IL-15 to promote CD8+CD122+ Treg suppressive function to inhibit allograft rejection, suggesting that the functionality of memory CD8+ T cells targeting allografts in our model is not significantly increased by treatments with low doses of IL-15, or is enhanced to a less extent than that of CD8+CD122+ Tregs. On the other hand, transfer of either CD4+CD25+ or CD8+CD122+ Tregs together with IL-2 infusion did not delay allograft rejection in WT mice. IL-2 likely enhances both Treg and conventional T cell function, ending up with an unaltered alloimmune balance.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

This study was supported by a grant from National Basic Research Program of China (2013CB966900) to YX. and a grant from Juvenile Diabetes Research Foundation International (JDRF) to Z.D.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ajt12515-sm-0001-SuppData-S1.pdf133KFigure S1: CD8+CD122+ Treg phenotypes after transplantation. CD8+CD122+ Tregs were isolated from Thy1.1 congenic mice and transferred to WT B6 mice that were transplanted with Balb/C islets. One week later, splenocytes of recipients were isolated, stained and analyzed for Thy1.1+ cell phenotypes by FACS. Histograms are gated on CD8+Thy1.1+ cell population. They maintained CD8+CD122+ Treg phenotypes and were still CD122high and CD25low.

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