Alpha tumor necrosis factor contributes to CD8+ T cell survival in the transition phase

Biochemical and Biophysical Research Communications 360 (2007) 702–707 www.elsevier.com/locate/ybbrc

Alpha tumor necrosis factor contributes to CD8+ T cell survival in the transition phase Meiqing Shi a

a,1

, Zhenmin Ye a,1, Keshav Sokke Umeshappa a, Terence Moyana b, Jim Xiang a,*

Research Unit, Saskatchewan Cancer Agency, Department of Oncology, University of Saskatchewan, 20 Campus Drive, Saskatoon, Sask., Canada S7N 4H4 b Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ont., Canada K1H 8L6 Received 20 June 2007 Available online 3 July 2007

Abstract Cytokine and costimulation signals determine CD8+ T cell responses in proliferation phase. In this study, we assessed the potential effect of cytokines and costimulations to CD8+ T cell survival in transition phase by transferring in vitro ovalbumin (OVA)-pulsed dendritic cell-activated CD8+ T cells derived from OVA-specific T cell receptor transgenic OT I mice into wild-type C57BL/6 mice or mice with designated gene knockout. We found that deficiency of IL-10, IL-12, IFN-c, CD28, CD40, CD80, CD40L, and 41BBL in recipients did not affect CD8+ T cell survival after adoptive transfer. In contrast, TNF-a deficiency in both recipients and donor CD8+ effector T cells significantly reduced CD8+ T cell survival. Therefore, our data demonstrate that the host- and T cell-derived TNF-a signaling contributes to CD8+ effector T cell survival and their transition to memory T cells in the transition phase, and may be useful information when designing vaccination.  2007 Elsevier Inc. All rights reserved. Keywords: Cytokine; TNF-a; Costimulation; CD8+ T cell survival; Memory T cell; Transition phase

CD8+ cytotoxic T lymphocytes (CTLs) contribute to the killing and clearance of virus-infected cells and tumors. After initial antigen (Ag) encounter, naı¨ve CD8+ T cells initiate a program for their autonomous clonal expansion and development into functional effectors and memory cells [1]. The in vivo CD8+ T cell response consists of three main phases [2]. These include (i) a proliferation phase of growth and differentiation of naı¨ve CD8+ T cells into effector T cells with lytic activity and cytokine production; (ii) a contraction (or transition) phase of transiting from a large population of effector T cells to a smaller population (5– 10%) of long-lived memory T (Tm) cells upon clearance of the infectious pathogen; and (iii) a maintenance phase of long-term Tm cells in the host. *

1

Corresponding author. Fax: +1 306 655 2635. E-mail address: [email protected] (J. Xiang). Shi and Ye made the same contribution to the study.

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.126

The magnitude and quality of the CD8+ T cell responses and subsequent memory pool is part influenced by the pathogen to which the immune system is responding. However, the host-derived antigenic, costimulatory and cytokine stimulations in the proliferation, transition and maintenance phases also play an important role in CD8+ Tm cell development, survival and recall expansion [2,3]. The critical effect of cytokines and costimulatory stimulations on priming CD8+ effector T cell response in the proliferation phase and maintaining CD8+ memory T cell survival in the maintenance phase has been extensively studied [2,3]. However, their effect on CD8+ effector T cell survival in the transition phase is less well defined. In this study, in vitro ovalbumin (OVA)-pulsed dendritic cells (DCOVA)-activated CD8+ T cells derived from OVA-specific T cell receptor transgenic OT I mice or OT I mice with deficiency of designated cytokines were

M. Shi et al. / Biochemical and Biophysical Research Communications 360 (2007) 702–707

adoptively transferred into wild-type C57BL/6 mice or mice with deficiency of genes coding for designated cytokines (IL-10, IL-12, IFN-c, and TNF-a) and costimulatory molecules (CD28, CD40, CD80, CD40L, and 41BBL). Subsequently, a kinetic survival of transferred CD8+ T cells in the recipients, which represents the transition from a large number of CD8+ effector T cells to a small population of CD8+ Tm cells in the transition phase, were examined. Materials and methods Reagents, antibodies, and animals. The following monoclonal antibodies (Ab) were purchased from BD-Biosciences (San Diego, CA): biotin-labeled rat anti-mouse CD8, CD11c (HL3), CD25 (7D4), CD40 (3/23), CD40L (MR1), CD54 (3E2), CD69 (H1.2F3), CD80 (16-10A), 41BB (1AH2), and Iab (AF6-120.1) antibodies (Abs). The biotin-labeled anti-41BBL (TSK-1) Ab was obtained from eBioscience (San Diego, CA). The FITC-conjugated avidin was obtained from Jackson ImmunoResearch Laboratory Inc., West Grove, Pennsylvania. Ovalbumin (OVA) and lipopolysaccharide (LPS) were obtained from Sigma (St. Louis, MO). The recombinant granulocyte macrophage colony-stimulating factor (GM-CSF), IL-2 and IL-4 were obtained from R&D Systems Inc., Minneapolis, MN. Female C57BL/6 mice were obtained from Charles River Laboratories (St. Laurent, Quebec, Canada). The OVA-specific T cell receptor (TCR) transgenic OT I mice and IL-10 / , IL-12 / , IFN-c / , TNF-a / , CD28 / , CD40 / , CD80 / , and CD40L / mice on C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, MA). The 41BBL / mice on C57BL/6 background were obtained from Amgen, Seattle, WA. The OT I/IFN-c / , OT I/ TNF-a / , OT I/CD40 / , and OT I/CD80 / mice were generated by backcrossing the designated gene deficient mice onto OT I mice for three generations; homozygosity was confirmed by polymerase chain reaction (PCR) according to Jackson laboratory’s protocols. All mice were housed in the animal facility at the Saskatoon Cancer Center and treated according to Animal Care Committee guidelines of University of Saskatchewan. Dendritic cell preparation. Bone marrow (BM)-derived dendritic cells (DCs) were generated in presence of GM-CSF (20 ng/ml) and IL-4 (20 ng/ ml) as described previously [4]. DCs derived from wild-type C57BL/6, CD40 / , and CD80 / , mice were pulsed with 0.5 mg/ml OVA overnight at 37 C in the presence of LPS (1 lg/ml) and referred to as DCOVA, (CD40 / )DCOVA and (CD80 / )DCOVA, respectively. OT I CD8+ T cell preparation. Spleens were removed from OT I mice or OT I mice with designated gene deficiency and mechanically disrupted to obtain a single-cell suspension. The erythrocytes were lysed using 0.84% ammonium chloride. Naı¨ve T cells were enriched by passage through nylon wool columns (C&A Scientific Inc., Mannose, VA). Naı¨ve OVAspecific CD8+ T cells were then purified by negative selection using antimouse CD4 (L3T4) paramagnetic beads (DYNAL Inc., Lake Success, NY). To generate OVA-specific active CD8+ T cells, naı¨ve CD8+ T cells (2 · 105 cells/ml) from OT I mice were stimulated for 3 days with irradiated (4000 rads) DCOVA (1 · 105 cells/ml) in presence of IL-2 (20 U/ml) [4]. These CD8+ T cells derived from OT I, OT I/IFN-c / , OT I/TNFa / , OT I/CD40 / , and OT I/CD80 / mice were termed CD8+ T, (IFN-c / )T, (TNF-a / )T, (CD40 / )T, and (CD80 / )T cells, respectively. Phenotypic characterization of DCOVA and OT I CD8+ T cells. The above DCOVA, naı¨ve and active CD8+ T cells derived from OT I mice were stained with a panel of Abs and analyzed by flow cytometry. Culture supernatants of the active CD8+ T cells were analyzed for cytokine expression using ELISA kits (R&D Systems Inc.), as previously described [4]. Adoptive transfer and tetramer staining. Wild-type C57BL/6 mice or mice with designated gene deficiency were i.v. injected in tail veins with 10 · 106 in vitro DCOVA-activated OVA-specific CD8+ T cells,

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(IFN-c / )T and (TNF-a / )T cells, respectively. Blood samples were taken from the tails of these mice or spleens were removed from these mice at different time points after the adoptive transfer. The blood samples or splenocytes were incubated with PE-conjugated H-2Kb/OVA257–264 tetramer and FITC-conjugated anti-CD8 Ab (Beckman Coulter, San Diego, CA) for 30 min at room temperature. The erythrocytes in blood samples were then lysed using lysis/fixed buffer (Beckman Coulter). The cells were washed and analyzed by flow cytometry [4].

Results and discussion Phenotypic characterization of OT I CD8+ T cells The DCOVA generated in this manner were mature DCs displaying (i) typical morphologic features of DCs (data not shown), and (ii) expression of I-Ab, costimulatory molecules (CD40, CD80 and 41BBL) and adhesion molecules (CD54 and CD11c) (Fig. 1A). As shown in Fig. 1B, active CD8+ T cells generated by in vitro coculture with DCOVA up-regulated CD25, CD69 and TCR, indicating that they are active CD8+ OVA-specific T cells. They also expressed costimulatory molecule receptors CD28, CD40L and 41BB. In addition, they showed some expression of costimulatory molecules CD40 and CD80, whereas naı¨ve CD8+ T cells did not express any CD40 and CD80, indicating that these CD40 and CD80 molecules on active CD8+ T cells may be derived from DCOVA by in vitro DCOVA activation via internalization and recycling of synapsecomprised molecules [5]. To confirm it, naı¨ve CD8+ T cells derived from OT I/CD40 / and OT I/CD80 / mice were activated with irradiated DCOVA, (CD40 / ) DCOVA and (CD80 / ) DCOVA for 3 days in vitro, respectively, and then analyzed by flow cytometry. As shown in Fig. 1C, DCOVA-activated, but not (CD40 / ) DCOVA and (CD80 / ) DCOVA-activated CD8+ T cells derived from OT I/CD40 / and OT I/CD80 / mice expressed CD40 and CD80, respectively, indicating that active CD8+ T cells with endogenous deficiency of CD40 and CD80 acquire CD40 and CD80 molecules from DCOVA, which is consistent with our recent report [6]. As shown in Fig. 1D, these active CD8+ T cells secreted IFN-c (1.5 ng/ml/106 cells/24 h) and TNF-a (2.2 ng/ml/106 cells/24 h), but no IL-4, indicating that they are type 1 CD8+ T cells. Except for the respective gene deficiency, the phenotypic profiles of CD8+ (IFN-c / )T and (TNF-a / )T cells were similar to that of CD8+ T cells (data not shown). Host-derived cytokine signaling and costimulation may contribute to CD8+ effector T cell survival in the transition phase Host-derived cytokines such as IFN-c, IL-10, IL-12, and TNF-a may provide signals essential for both priming of CD8+ T cell responses in the proliferation phase and maintaining Tm cells in the maintenance phase. For example, IFN-c contributes to CD8+ T cell priming [7], homeostasis and contraction [8]. Stimulation of Ag in vivo in absence of IL-12 affected CD8+ T cell clonal expansion and cytolytic

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Fig. 1. Phenotypes of DCs and OT I CD8+ T cells. (A) DCOVA and (B) naive and active CD8+ T cells derived from OT I mice were stained with a panel of biotin-labeled antibodies followed by FITC-avidin (solid lines), and analyze by flow cytometry. The isotype-matched irrelevant antibodies were used as controls (dotted lines). (C) DCOVA-activated CD8+ T cells derived from OT I/CD40 / and OT I/CD80 / mice were stained with biotin-labeled anti-CD40 and CD80 Abs followed by FITC-avidin (solid lines). (CD40 / )DCOVA- and (CD80 / )DCOVA-activated CD8+ T cell derived from OT I/CD40 / and OT I/CD80 / mice, respectively, were also stained as described above (thick dotted lines), and analyzed by flow cytometry. The isotype-matched irrelevant antibodies were used as controls (thin dotted lines). (D) The supernatants of active CD8+ T cells were measured for cytokine secretion by ELISA. Values represent the mean of triplicates from two experiments.

effector function in the proliferation phase [9] and generation of effector and memory T cells [10]. TNF-a can stimulate T cell proliferation [11], provide costimulatory survival signal for CD8+ T cells in priming CD8+ T cell responses [12], and prevent T cell apoptosis derived from activation-induced cell death [13]. IL-10 can rescue T cells from apoptotic cell death [14] and promote the maintenance of CD8+ T cells in vivo in the maintenance phase [15]. Host-derived adhesion/costimulatory molecules may also provide signals essential for both priming of CD8+ T cell responses and maintaining Tm cells. For example, costimulation via CD28/CD80 interactions can promote the generation of effector and Tm cell responses in the proliferation phase [16]. Recently, it has been shown that CD28 is not required for the generation and maintenance of CD8+ memory T cells [17]. CD40 is a TNFR family member expressed on DCs, which binds to CD40L on active T cells [18]. CD4+ T cells are known to provide help in CD8+ T cell priming and CD8+ Tm cell mainte-

nance via CD40/CD40L interactions [16]. In absence of CD4+ T cell help, CD40/CD40L interactions are required for to stimulate maximal T cell responses [19]. 41BBL is the ligand of 41BB, a costimulatory member of the TNFR family and is expressed on DCs [20]. 41BBL signaling induced CD8+ T cell expansion, cytokine production and development of cytotoxic function [21], and facilitated robust CD8+ Tm cell recall responses [22]. However, the potential effect of the above cytokines and costimulations on CD8+ T cell survival in the transition phase is still unclear. Host-derived TNF-a signaling, but neither IL-10, IL-12, and IFN-c signalings nor CD28/CD80, CD40/CD40L, and 41BB/41BBL interactions contributes to CD8+ effector T cell survival in the transition phase The major aim of the present study is to assess the potential contribution of host-derived cytokines and

M. Shi et al. / Biochemical and Biophysical Research Communications 360 (2007) 702–707

costimulations to CD8+ T cell survival in the transition phase. In this study, CD8+ effector T cells were adoptively transferred to wild-type C57BL/6 as well as designated cytokine gene deficient mice. Double staining for Kb/ OVA tetramer and CD8 was then performed to determine the numbers of OVA-specific CD8+ T cells in peripheral blood and spleens of mice at different time points after the adoptive transfer. As shown in Fig. 2A and Table 1, OVA-specific CD8+ T cells detected in mouse peripheral blood at day 6 subsequent to the adoptive transfer into the wild-type C57BL/6 mice accounted for 30% of the total CD8+ T cell population. The absolute number of tetramerpositive CD8+ T cells in the spleen, which were derived from transferred CD8+ T cells, was 3.0 · 106 OVA-specific CD8+ T cells per spleen. The numbers of OVA-specific CD8+ T cells detected in mouse peripheral blood then gradually dropped to around 8% and 3% of the total CD8+ T cell population in day 12 and the first month subsequent to (Exp. I of Table 1), but stably maintained for at least 3 months (data not shown) after the adoptive transfer. The numbers of detected OVA-specific CD8+ T cells in peripheral blood or spleens of mice with IL-10, IL-12, and IFN-c gene KO at different time points after the adoptive transfer were similar to that in wild-type C57BL/6 mice (Exp. I of Table 1 and Fig. 2A), indicating that the deficiency of mouse IL-10, IL-12, and IFN-c does not affect CD8+ T cell survival. Interestingly, OVA-specific CD8+ T

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cells detected in peripheral blood of mice with TNF-a gene KO at day 6 subsequent to the adoptive transfer accounted for 19% of the total CD8+ T cell population, which is around 60% of OVA-specific CD8+ T cells detected in wild-type C57BL/6 mice. However, the numbers quickly dropped to around 0.6% of the total CD8+ T cell population and completely lost at day 12 and day 30 subsequent to the transfer, respectively (Exp. I of Table 1), indicating that the survival of CD8+ T cells is significantly decreased in mice with TNF-a gene KO compared to that in wild-type C57BL/6 mice (p < 0.05). Thus, our data demonstrate that the host-derived TNF-a plays an important role in CD8+ T cell survival in the transition phase. Since the above active CD8+ T cells expressed the costimulatory molecule receptors CD28, CD40L, and 41BB as well as the acquired costimulatory molecules CD40 and CD80, the above CD8+ effector T cells were then adoptively transferred into wild-type C57BL/6 as well as mice with designated gene deficiency (CD80 / , CD40 / , 41BBL / , CD40L / , and CD28 / ) to assess the potential importance of CD28/CD80, CD40/CD40L, and 41BB/ 41BBL interactions in adoptively transferred CD8+ T cell survival. As shown in Fig. 2A and Exp. I of Table 1, the numbers of detected OVA-specific CD8+ T cells in peripheral blood or spleens of mice with CD28, CD40, CD80, CD40L, and 41BBL gene KO at different time points after the adoptive transfer were all comparable to that in

A

B

Fig. 2. CD8+ effector T cell survival after the adoptive transfer. (A) C57BL/6 mice and mice deficient in genes indicated were i.v. injected with CD8+ T cells. (B) C57BL/6 mice were i.v. injected with CD8+ T cells derived from OT I mice or OT I mice deficient in genes indicated, respectively. Six days later, the blood samples or the spleen cells were doubled-stained with PE-Kb/OVA tetramer and FITC-CD8 Ab and analyzed by flow cytometry. The frequency of OVA-specific CD8+ T cells in peripheral blood and whole spleen is presented as the percentage in total CD8+ population (top numbers) and the absolute number of OVA-specific CD8+ T cells per spleen, respectively. The values in parentheses represent the standard errors. The results presented are representative of two separate experiments with 4–6 mice per group. *P < 0.05 versus cohorts in C57BL/6 mice or transferred with CD8+ T cells (Student’s t test).

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Table 1 Kinetic study of CD8+ effector T cell survival in mice after the adoptive T cell transfer Recipients

Detection of PE-tetramer and FITC-CD8 positive T cells (% ± SD) Day 6

Day 12

Day 30

Exp. Ia C57BL/6 IL-10 KO IL-12 KO IFN-c KO TNF-a KO CD28 KO CD40 KO CD80 KO CD40L KO 41BBL KO

29.72 ± 2.61 28.38 ± 2.17 27.81 ± 4.72 25.74 ± 2.35 18.57 ± 0.13* 28.92 ± 0.36 24.68 ± 0.21 26.92 ± 0.36 25.51 ± 0.67 28.12 ± 0.25

8.24 ± 0.91 7.13 ± 0.89 5.45 ± 0.67 6.77 ± 0.44 0.57 ± 0.06* 7.11 ± 0.92 7.34 ± 0.85 5.11 ± 0.92 7.37 ± 0.95 6.78 ± 0.72

2.88 ± 0.24 2.33 ± 0.25 2.72 ± 0.32 2.66 ± 0.31 0.06 ± 0.01* 2.90 ± 0.36 3.01 ± 0.24 2.66 ± 0.39 2.99 ± 0.27 2.71 ± 0.29

Exp. II CD8+ T cells CD8+ (IFN-c / )T cells CD8+ (TNF-a / )T cells

29.72 ± 2.61 27.36 ± 1.91 16.62 ± 2.10*

8.24 ± 0.91 7.29 ± 0.26 1.14 ± 0.27*

2.88 ± 0.24 2.69 ± 0.15 0.08 ± 0.01*

a

In Exp. I, the blood samples were taken from mice at different time points (day 6, 12, and 30) after adoptive transfer of CD8+ T cells. In Exp. II, C57BL/6 mice were i.v. injected with CD8+ effector T cells, CD8+ (IFN-c / )T cells and (TNF-a / )T cells, respectively. The blood samples were stained with PE-conjugated H-2Kb/OVA257–264 tetramer and FITCconjugated anti-CD8 Ab and analyzed by flow cytometry. OVA-specific CD8+ T cells detected in mouse peripheral blood at different time points after the adoptive transfer were accounted for percentage of the total CD8+ T cell population. *P < 0.05 versus cohorts in C57BL/6 mice or transferred with CD8+ T cells (Student’s t test). One representative experiment of two was shown.

total CD8+ T cell population, which is around 56% of OVA-specific CD8+ T cells detected in C57BL/6 mice. The numbers also quickly dropped to around 1% of the total CD8+ T cell population and completely lost at day 12 and day 30 subsequent to the transfer, respectively (Exp II of Table 1), indicating that the donor T cell-derived TNF-a also play an important role in CD8+ T cell survival in the transition phase. Collectively, our results demonstrate that the hostderived signals provided by IL-10, IL12, IFN-c, CD40, CD80, and 41BBL are not essential for survival of CD8+ T cells in the transition phase. That the host and T cellular IFN-c deficiency does not alter CD8+ T cell survival is consistent with a recent report by Hollenbaugh and Dutton [23]. However, the host and donor CD8+ T cell-derived TNF-a signalings after priming contribute to CD8+ T cell survival and their transition to memory T cells. Recently, it has been reported that membrane TNF-a can delay T cell activation-induced cell death by activation of AKT and NF-jB [13], which leads to elongated T cell survival. Memory, the ability of the immune system to respond with greater efficiency to a second insult by the same pathogen, is a cardinal feature of the adoptive immune system. The generation of immunological memory is the ultimate goal of vaccination. An understanding of how T cell memory is developed and maintained is crucial for the rational design of vaccine. Therefore, these findings may be useful information when designing vaccination. Acknowledgments

wild-type C57BL/6 mice, indicating that the deficiency of mouse CD28, CD40, CD80, CD40L, and 41BBL, did not significantly affect CD8+ T cell survival and the frequency of CD8+ memory T cell development. T cell-derived TNF-a, but not IFN-c signaling contributes to CD8+ effector T cell survival in the transition phase Since CD8+ effector T cells secreted IFN-c and TNF-a, we wanted to assess whether the donor T cell-derived TNF-a has any effect on CD8+ T cell survival in the transition phase. CD8+ T cells derived from OT I/IFNc / and OT I/TNF-a / mice were adoptively transferred to wild-type C57BL/6. As shown in Fig. 2B and Exp. II of Table 1, OVA-specific CD8+ T cells at day 6 subsequent to the adoptive transfer accounted for 30% of the total CD8+ T cell population in the peripheral blood and 3.0 · 106 OVA-specific CD8+ T cells per spleen. The numbers of detected OVA-specific CD8+ T cells with IFN-c gene deficiency in peripheral blood or spleens of mice at different time points after the adoptive transfer were comparable to that in C57BL/6 mice, indicating that the deficiency of donor CD8+ T cell’s IFN-c does not affect CD8+ T cell survival. Interestingly, OVA-specific CD8+ T cells with TNF-a gene deficiency detected in peripheral blood at day 6 subsequent to the adoptive transfer accounted for 17% of the

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