CD4+ and CD8+ T Cell Survival Is Regulated Differentially by Protein Kinase C θ, c-Rel, and Protein Kinase B
This information is current as Samuel D. Saibil, Russell G. Jones, Elissa K. Deenick, of September 27, 2021. Nicole Liadis, Alisha R. Elford, Mitchell G. Vainberg, Heather Baerg, James R. Woodgett, Steve Gerondakis and Pamela S. Ohashi J Immunol 2007; 178:2932-2939; ;
doi: 10.4049/jimmunol.178.5.2932 Downloaded from http://www.jimmunol.org/content/178/5/2932
References This article cites 69 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/178/5/2932.full#ref-list-1 http://www.jimmunol.org/
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology
CD4؉ and CD8؉ T Cell Survival Is Regulated Differentially by Protein Kinase C, c-Rel, and Protein Kinase B1
Samuel D. Saibil,†‡ Russell G. Jones,§ Elissa K. Deenick,*†‡ Nicole Liadis,*‡ Alisha R. Elford,‡ Mitchell G. Vainberg,*‡ Heather Baerg,‡ James R. Woodgett,* Steve Gerondakis,¶ and Pamela S. Ohashi2*†‡
An effective immune response requires the expansion and survival of a large number of activated T cells. This study compared the role of protein kinase C (PKC) and associated signaling molecules in the survival of activated primary CD4؉ vs CD8؉ murine T cells. We demonstrate that the absence of PKC resulted in a moderate survival defect in CD4؉ T cells and a striking survival defect of CD8؉ T lymphocytes. CD8؉ T cells lacking the c-Rel, but not the NF-B1/p50, member of the NF-B family of transcription factors displayed a similar impairment in cell survival as PKC؊/؊ CD8؉ T lymphocytes. This implicates c-Rel as a key target of PKC-mediated survival signals in CD8؉ T cells. In addition, both c-Rel؊/؊ and PKC؊/؊ T cells also displayed Downloaded from
impaired expression of the antiapoptotic Bcl-xL protein upon activation. Changes in Bcl-xL expression, however, did not correlate with the survival of CD4؉ or CD8؉ lymphocytes. The addition of protein kinase B-mediated survival signals could restore partially CD4؉ T cell viability, but did not dramatically influence CD8؉ survival. Active protein kinase B was also unable to restore ,proliferative responses in CD8؉ PKC؊/؊ T cells. The survival of CD4؉ and CD8؉ T cells deficient in either PKC or c-Rel however, was promoted by the addition of IL-2. Collectively, these data demonstrate that CD4؉ and CD8؉ T cell survival signals http://www.jimmunol.org/ are differentially programmed. The Journal of Immunology, 2007, 178: 2932–2939.
oth immature and mature T cells are destined to die upon the generation of lipid second messengers at the plasma mem- Ag-specific TCR engagement. In thymocytes, TCR-spe- brane. The conversion of phosphatidylinositol-3,4-bisphosphate B cific engagement with high-affinity ligands leads to neg- into phosphatidylinositol-3,4,5-trisphosphate by PI3K prompts the ative selection or clonal deletion (reviewed in Ref. 1). Similarly, in recruitment of PKB to phosphatidylinositol-3,4,5-trisphosphate via the absence of costimulatory and proinflammatory signals, mature its pleckstrin homology domain. Once recruited to the plasma T cells also undergo clonal deletion upon engagement with high- membrane, PKB is subsequently phosphorylated and fully acti-
affinity ligands (2). Therefore, one fundamental issue in T cell vated (9). Active PKB has been demonstrated to promote survival by guest on September 27, 2021 immunity is to understand the nature of survival signals and the due to its ability to phosphorylate, and thereby inhibit, the pro- integration of these signals during T cell activation. Although a apoptotic BH3-only protein BAD (10–12). As well, the forkhead number of molecules, such as Bcl-xL and FLIP, have been shown transcription factors are also direct substrates of PKB (13–15); to play a role in T cell survival (3–7), the signaling pathways that phosphorylation of the forkhead transcription factors FOXO by mediate T cell survival remain unclear. PKB prevents the transcription of the proapoptotic BH3-only pro- 3 The serine/threonine kinase protein kinase B (PKB)␣ (Akt1) tein Bim (16), as well as the transcription of Fas ligand (13). Many has been shown to promote survival in multiple cell lineages, in- other molecules such as GSK-3, Cot, and p27 have been shown to cluding T cells (PKB reviewed in Ref. 8). PKB is activated after be downstream targets of PKB␣ (17–20). In addition to its direct substrates, PKB also promotes cell survival through the activation *Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Can- of the NF- B family of transcription factors (21–24). ada; †Department of Immunology, University of Toronto, Toronto, Ontario, Canada; In mammals, the NF-B transcription factors are composed of ‡Institute for Breast Cancer Research, Ontario Cancer Institute, University of Toronto, § homo- or heterodimers of five conserved subunits: p65/RelA, c- Toronto, Ontario, Canada; Abramson Family Cancer Research Institute, Philadel- phia, PA 19104; and ¶Walter and Eliza Hall Institute of Medical Research, Parkville, Rel, RelB, NF- B1/p50, and NF- B2/p52 (NF- B reviewed in Australia Ref. 25). These dimeric transcription factors are essential for T cell Received for publication February 8, 2006. Accepted for publication December activation and regulate genes associated with proliferation, cyto- 21, 2006. kine production, as well as protection against apoptosis. In T cells, The costs of publication of this article were defrayed in part by the payment of page NF-B signaling has been linked not only to the induction of an- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tiapoptotic members of the Bcl-2 family of proteins, such as 1 This work was supported by the Canadian Institutes for Health Research and Na- Bcl-xL (26–28), but also to protection from Fas-mediated apopto- tional Cancer Institute of Canada, with funds from the Canadian Cancer Society. sis (29–31). Initiation of NF-B-mediated transcription requires S.D.S. is supported by a Canadian Institutes for Health Research MD/PhD award. the activation of the IB kinase (IKK) complex. This complex P.S.O. holds a Canada Research Chair. consists of two kinase subunits, IKK␣ and IKK, as well as a 2 Address correspondence and reprint requests to Dr. Pamela S. Ohashi, The Campbell ␥ Institute for Breast Cancer Research, University of Toronto, 610 University Avenue, regulatory subunit IKK /NF- B essential modulator. Activation Room 706, Toronto, Ontario, Canada M5G 2C1. E-mail address: pohashi@uhnres. of the kinases in this complex results in the phosphorylation and utoronto.ca subsequent proteosomal degradation of the IB protein. The IB 3 Abbreviations used in this paper: PKB, protein kinase B; 7AAD, 7-aminoactino- protein functions by sequestering NF-B transcription factors in mycin D; IKK, IB kinase; MFI, mean fluorescence intensity; PKC, protein kinase C. the cytosol. Degradation of IB allows the nuclear translocation of Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 the NF-B transcription factor and gene transcription.
www.jimmunol.org The Journal of Immunology 2933
Protein kinase C (PKC), a member of the calcium-independent annexin V FITC and 7AAD. Samples were acquired on a FACScan in- subfamily of PKCs (PKC reviewed in Ref. 32), has been dem- strument (BD Biosciences) and were analyzed using CellQuest software ␥ (BD Biosciences). For intracellular Bcl-xL staining, Fc RIII were first onstrated to be an important mediator of NF- B signaling. T lym- ϫ 6 blocked with an Ab. A total of 1 10 cells was then fixed and perme- phocytes from mice deficient for PKC demonstrate not only im- abilized using BD Cytofix and BD Cytoperm (BD Biosciences) as per the paired activation of NF- B, but also reduced activation of the manufacturer’s instructions. Cells were then stained with anti-Bcl-xL FITC AP-1 transcription factor (33, 34). The functional consequence of Ab or an isotype control Ab. Samples were then acquired and analyzed. this impaired signaling is that PKCϪ/Ϫ T cells displayed impaired Western blot analysis proliferation (35) and reduced production of IL-2 (33) upon acti- vation. Recently, a role for PKC in the promotion of survival of Single-cell suspensions were lysed by incubation on ice in Gentle Soft activated T cells has been described (36). Moreover, it has been Buffer (10 mM NaCl, 20 mM PIPES (pH 7.4), 0.5% Nonidet P-40, 5 mM suggested previously that PKB and PKC molecules physically EDTA, 5 g/ml leupeptin, 1 mM benzamidine, aprotinin, and 100 M Na3VO4) for 20 min. The lysates were then cleared by centrifugation, and associate (37, 38). Together, these reports suggest interplay be- the supernatants were normalized for total protein (Bio-Rad). Protein was tween the PKB and PKC signaling pathways in the promotion of resolved by 4–20% SDS-PAGE and then electroblotted onto polyvinyli- survival of activated T cells. Therefore, we investigated the roles dene difluoride membrane (Costar). The membranes were blocked in 5% of PKC, PKB, and members of the NF-B family in controlling nonfat milk in TBST and probed with primary Abs for 12 h at 4°C. Anti- Bcl-xL was purchased from Cell Signaling Technology, and anti-actin was the survival of activated primary murine T cells. Our results dem- purchased from Sigma-Aldrich. onstrate that CD4ϩ and CD8ϩ T cell survival are programmed differentially. PKC signaling events are important for both CD4ϩ Proliferation assay ϩ Downloaded from and CD8 T cell survival, which can be modulated by PKB or Splenocytes from P14 PKCϪ/Ϫ, P14 PKCϪ/Ϫ PKB, and P14 PKCϩ/Ϫ IL-2. PKC- and c-Rel-mediated signals, however, appear to be mice (2 ϫ 105 cells/well) were incubated 72 h at 37°C in 96-well flat- more important for CD8ϩ T cell survival. bottom plates with increasing concentrations of gp33 peptide. Proliferation was measured by the incorporation of an overnight pulse of 1 Ci of 3 Materials and Methods [ H]thymidine after 72 h of incubation. Mice Results Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ http://www.jimmunol.org/ The PKC / (33), nf-b1 / (p50 / ) (39), and the c-rel / (40) mouse The PKC signals are critical for T cell survival in vitro lines have all been described previously, as have the human CD2-gag-PKB transgenic (B6/PKB) (24), and the P14 TCR transgenic (41). The PKB/ To examine whether PKC conveys survival signals in activated T PKCϪ/Ϫ line of mice was generated by breeding B6/PKB mice to PKC- cells, T lymphocytes from PKCϪ/Ϫ, littermate PKCϩ/Ϫ, and deficient mice. These mice were subsequently crossed to the P14 TCR control C57BL/6 mice were stimulated with plate-bound anti-CD3 transgenic mice. C57BL/6J mice were obtained from The Jackson Labo- ratory. All mice were maintained in a specific pathogen-free environment and anti-CD28 Abs. After stimulation, cells were incubated for at the Ontario Cancer Institute, according to institutional guidelines. 20 h without stimulation, and then viability was assessed by flow cytometry using annexin V and 7AAD. Cell survival was moni- Reagents and Abs tored in both the CD4ϩ and the CD8ϩ T cell subsets and was Murine rIL-2 was purchased from PeproTech. Purified anti-CD3 (2C11) normalized in each culture to the percentage of viable cells after by guest on September 27, 2021 and anti-CD28 (37.51) were purchased from eBioscience. Anti-Bcl-x L removal from the stimulation (viability at t0). As compared with FITC Ab was purchased from Southern Biotechnology Associates. Purified the C57BL/6 and littermate heterozygous control cells, which dis- anti-Fc␥RIII/II (2.4G2) was purchased from BD Pharmingen. The follow- ϩ ϩ played similar viability in both the CD4 and the CD8 T cell ing Abs purchased from BD Pharmingen were used for flow cytometry (PE ϩ conjugated): anti-CD4 and anti-CD8. Th annexin V FITC was also pur- compartments, the CD4 T cells lacking PKC displayed a de- chased from BD Pharmingen, whereas the 7-aminoactinomycin D (7AAD) crease in cell viability after 20 h of culture (Fig. 1A). The average was purchased from Sigma-Aldrich. The mouse IgG FITC isotype control viability of the PKC-deficient CD4ϩ T cells was 36.7% as com- Ab was purchased from Jackson ImmunoResearch Laboratories. pared with 52.1% for the C57BL/6 cells and 55.2% for the ϩ Ϫ In vitro cell stimulation PKC / control cells. This difference in viability between the C57BL/6 and knockout CD4ϩ T cells is statically significant ( p Ͻ To assay Bcl-xL expression, splenic T cells were purified using the Pan T Cell Isolation Kit on the AutoMACS (Miltenyi Biotec), as per the manu- 0.0003 by two-tailed Student’s t test). The impaired survival in Ϫ/Ϫ ϩ facturer’s instructions. Purified T cells were plated at 1–2 ϫ 106 cells/ml in PKC T cells, however, was much more drastic in the CD8 24-well flat-bottom plates coated with either purified anti-CD3 or com- compartment, because the PKC-deficient cells averaged only bined anti-CD3 and anti-CD28. After the indicated times, cells were har- 12.8% viable cells as compared with 59.2% viability for the vested for analysis. PKCϩ/Ϫ CD8ϩ T cells and 64.8% for the C57BL/6 control cells. Cell viability assay These results are consistent with a recent report that suggests PKC is important for CD8ϩ T cell survival (36). Interestingly, we A total of 107 splenocytes was plated at 2 ϫ 106 cells/ml in six-well Ϫ/Ϫ ϩ ϩ flat-bottom plates coated with 2 g/ml anti-CD3 and 2 g/ml anti-CD28. found no enhanced cell death in PKC CD4 or CD8 T cells Cells were stimulated for 72 h in RPMI 1640 medium supplemented with that were subjected to activation-induced cell death via TCR re- 10% heat-inactivated FCS (Invitrogen Life Technologies), 50 M 2-ME, 2 stimulation (data not shown). Thus, PKC may not be required to mM glutamine, and 0.1% penicillin/streptomycin. After 72 h of stimula- protect against all forms of cell death. Our data, however, dem- tion, cells were washed and cell viability was assessed in the CD4 and CD8 onstrate that PKC is required to maintain the survival of activated compartments from a triplicate of aliquots using flow cytometry (viability ϩ ϫ 5 T lymphocytes, particularly those of the CD8 lineage. at t0; t stands for time). A total of 2 10 cells was then plated in triplicate wells in 96-well flat-bottom plates. After 20 h of incubation, cell viability In the next experiments, we sought to ascertain the molecules was assessed again in the CD4 and CD8 compartments using flow cytom- downstream of PKC that are responsible for promoting cell sur- etry. Cell viability is reported as a percentage of the viability of each ϭ vival. PKC is known to activate NF- B (33, 34, 42, 43). We culture at t0, using the formula: percentage viability ((viability after ϫ investigated whether T cells lacking only p50 or c-Rel displayed a 20 h)/(viability at t0)) 100. similar phenotype as T cells lacking PKC. When cell viability Flow cytometry was assessed in CD4ϩ T cells lacking either p50 or c-Rel, no For cell viability assays, 2 ϫ 105 cells were first stained with either anti- impairment in survival was noted relative to wild-type controls CD4 or anti-CD8 at 4°C. Cell viability was then assessed by staining with (Fig. 1B). This suggests that there might be redundancy between 2934 DIFFERENTIAL PROGRAMMING OF CD4ϩ AND CD8ϩ T CELL SURVIVAL
Ϫ/Ϫ ϩ FIGURE 2. Impaired expression of Bcl-xL in PKC CD4 and ϩ 6 FIGURE 1. PKC and c-Rel are required for the survival of activated T CD8 T lymphocytes. Purified splenic T cells (1 ϫ 10 cells) from Ϫ Ϫ ϩ Ϫ ϫ 7 Ϫ/Ϫ ϩ/Ϫ PKC / , PKC / , and C57BL/6 mice were stimulated with 2 g/ml
cells. Splenocytes (1 10 cells) from A, PKC , littermate PKC , Downloaded from Ϫ/Ϫ Ϫ/Ϫ and C57BL/6 mice, or B, c-Rel , p50 , and C57BL/6 mice were stim- anti-CD3 combined with 2 g/ml anti-CD28 Ab for 24 h. Bcl-xL expres- ϩ ϩ ulated with anti-CD3 and anti-CD28 Abs (2 g/ml of each) for 72 h. Cell sion in the CD4 and CD8 T cells was then determined by flow cytom- viability was then assessed by flow cytometry by staining with annexin V etry. Numbers in the histograms indicate the MFIs of staining for Bcl-xL. Specific staining for Bcl-x was confirmed using an isotype control Ab. and 7AAD to determine background death (viability at t0). The average L These data are representative of three independent experiments. viability at t0 ranged from 68 to 85% for the cells in A and 60 to 89% for the cells in B. Cells were then washed and recultured (2 ϫ 105 cells) in
triplicate without stimulation. After 20 h of reculture, cell viability was http://www.jimmunol.org/
assessed by flow cytometry by staining with annexin V and 7AAD. The as significantly (4, 47). All of these findings implicate Bcl-xL as an data are expressed as the percentage of viable cells after 20 h of reculture attractive candidate for a prosurvival molecule regulated by PKC divided by the percentage of viable cells at t (after stimulation). The data 0 and c-Rel. To investigate this possibility, the expression of Bcl-xL are shown as an average value of triplicate wells with error bars showing was assessed in T cells lacking PKC. SD. These data are representative of three independent experiments. Purified splenic T cells were stimulated by plate-bound Abs to CD3 and CD28 for 24 h and then stained with either anti-CD4 or
anti-CD8 as well as for intracellular Bcl-xL. As illustrated in Fig. the various subunits of NF-B that the absence of one is insuffi- 2, CD4ϩ T cells lacking PKC displayed reduced expression of ϩ ϩ Ϫ / by guest on September 27, 2021 cient to impair the survival of CD4 T cells. This does not, how- Bcl-xL relative to both the C57BL/6 and the littermate PKC ever, appear to be the case for CD8ϩ T cells. The CD8ϩ T cells controls. PKCϪ/Ϫ CD8ϩ lymphocytes showed a minor reduction ϩ ϩ ϩ lacking c-Rel displayed a similar phenotype to CD8 T lympho- in Bcl-xL levels. These results demonstrate that CD4 and CD8 ϩ Ϫ/Ϫ cytes lacking PKC . The CD8 c-Rel T cells averaged only T cells lacking PKC display impaired expression of Bcl-xL during 24.8% viability as compared with 71.7% viability for the C57BL/6 the process of T cell activation. cells. These findings implicate the PKC to c-Rel signaling axis as The kinetics of Bcl-xL expression during T cell activation was a major survival signaling pathway in CTL. also assessed by flow cytometry in both CD4ϩ and CD8ϩ T cells. The specific requirement for the c-Rel subunit of NF-B down- As can be seen in Fig. 3, stimulation of T lymphocytes from stream of PKC for CD8ϩ T cell survival was demonstrated by the C57BL/6 mice with anti-CD3 Ab alone resulted in ϳ2-fold in- Ϫ/Ϫ lack of impaired survival in the T cells from the p50 mice. The crease in expression of Bcl-xL after 16 h of stimulation. The level ϩ CD8 T cells from these mice displayed a similar profile as the T of Bcl-xL expression increased further after 24 h of stimulation lymphocytes from the C57BL/6 mice (Fig. 1B). The CD8ϩ p50Ϫ/Ϫ with anti-CD3 in both the CD4ϩ and CD8ϩ T cells. Combined T cells had a marginally impaired average survival relative to the anti-CD3 and anti-CD28 Ab treatment of the wild-type T cells
control cells (59.4 vs 71.7%). This difference, however, was not enhanced the expression of Bcl-xL at all time points assayed in statistically significant ( p Ͼ 0.1 by two-tailed Student’s t test). both the CD4ϩ and CD8ϩ T lymphocytes. These data are consis- These results demonstrate that, unlike c-Rel, the p50 subunit of tent with previous studies that demonstrated that signals through NF- B is dispensable for the PKC -mediated survival signaling CD28 augment the expression of Bcl-xL (4, 48, 49). In contrast to pathway in activated CD8ϩ T lymphocytes. the control cells, both CD4ϩ and CD8ϩ T cells from the PKCϪ/Ϫ mice showed a reduced proportion of cells expressing Bcl-x at L PKC -mediated signals enhance the expression of Bcl-xL upon T ϩ ϩ high levels when stimulated through the Ag receptor alone or with cell activation in both CD4 and CD8 T lymphocytes anti-CD3 and anti-CD28 Abs. This difference was most prominent There exists strong evidence connecting NF-B with the expres- after 24 h. These findings suggest that PKC is required down-
sion of Bcl-xL. Mapping of the bcl-x promoter in mouse revealed stream of the Ag receptor for optimal expression of Bcl-xL upon T multiple B consensus binding sites (44, 45). As well, studies in T cell activation. cell lines (26) and primary human T cells (27) demonstrated a requirement for NF-B in the expression of Bcl-x . Furthermore, c-Rel deficiency results in diminished expression of Bcl-xL upon L T cell activation the expression of pattern of Bcl-xL supports a role in the survival of activated T cells. Naive T cells express relatively high levels of Our results from Fig. 1 suggest that signals downstream of PKC
Bcl-2 (46) and almost undetectable levels of Bcl-xL (47). Upon and c-Rel, but not p50, are important for the survival of activated ϩ activation, however, the expression of Bcl-xL is induced rapidly in CD8 T cells. If Bcl-xL is indeed a key survival effector down- T lymphocytes, whereas the expression of Bcl-2 does not change stream of c-Rel, one prediction would be that the expression of The Journal of Immunology 2935
FIGURE 3. Kinetics of induction Ϫ/Ϫ of Bcl-xL in PKC T cells. Puri- fied splenic T cells (1 ϫ 106 cells) from PKCϪ/Ϫ (black histogram) and C57BL/6 (gray histogram) mice were stimulated with plate-bound Ab. Cells were stimulated with either 2 g/ml anti-CD3 Ab or 2 g/ml anti-CD3 combined with 2 g/ml anti-CD28
Ab for the indicated times. Bcl-xL ex- pression in the CD4ϩ and CD8ϩ T cells was then determined by flow cy- tometry. Numbers in the histograms indicate the MFIs of staining for Ϫ/Ϫ Bcl-xL for PKC (black) and C57BL/6 (gray) T cells. Specific
staining for Bcl-xL was confirmed us- ing an isotype control Ab. These data are representative of three indepen- dent experiments. Downloaded from
Ϫ/Ϫ Ϫ/Ϫ Bcl-xL should also be impaired in the c-Rel , but not the p50 PKC to affect Bcl-xL expression. Our laboratory has previously T cells. Accordingly, the expression of Bcl-xL in T cells from described a transgenic model that expresses active PKB in T cells c-RelϪ/Ϫ and p50Ϫ/Ϫ mice was assessed by flow cytometry. In- using the CD2 promoter. We have characterized T cells in these
tracellular Bcl-xL staining confirmed the specific role of c-Rel in mice and have shown that they have increased survival to multiple http://www.jimmunol.org/ the expression of Bcl-xL (Fig. 4). The mean fluorescence intensity stimuli, enhanced NF- B activation, and increased levels of Bcl-xL of Bcl-xL staining after 24 h of stimulation with anti-CD3 Ab was (24). To examine whether PKB influences Bcl-xL expression in the 16.4 for the C57BL/6 T cells, 14.86 for the p50Ϫ/Ϫ T cells, and absence of PKC, the human CD2-gag-PKB␣ transgenic mice Ϫ/Ϫ only 8.9 for the c-Rel T lymphocytes. The MFI of Bcl-xL stain- were bred onto the PKC -null background. The expression of ing after stimulation through CD3 and CD28 further confirmed Bcl-xL in activated T cells was then assessed by Western blot these findings, because the MFI of Bcl-xL staining was 26.72 for analysis. Ϫ/Ϫ the wild-type T cells, 27.31 for the p50 T cells, and 10.86 for Active PKB rescued the expression of Bcl-xL in the absence of Ϫ/Ϫ the c-Rel T cells. Thus, our data obtained by intracellular stain- PKC . As can be seen in Fig. 5A, the expression of Bcl-xL in Ϫ/Ϫ
ing indicate that the c-Rel, but not the p50 subunit of NF- B plays activated PKC PKB T cells was higher than that of the by guest on September 27, 2021 Ϫ/Ϫ a nondispensable role in the up-regulation of Bcl-xL in activated T PKC T cells after both 16 and 24 h of stimulation. The level lymphocytes.
Active PKB can rescue Bcl-xL expression in the absence of PKC in CD8ϩ T cells Multiple studies (21–24, 50) have demonstrated that PKB can en- hance the activation of NF-B. Studies by Kane et al. (23, 50) have suggested that PKB exerts a greater affect upon the transcription of NF-B-dependent genes, such as IL-2, when it is activated in con- junction with other signaling pathways, particularly PKC. Ac- cordingly, we wanted to establish whether transgenic expression of
active PKB could rescue the expression of Bcl-xL in T cells lack- ing PKC, or whether alternatively, PKB requires the presence of
FIGURE 5. Transgenic expression of PKB can rescue the expression of ϩ Ϫ/Ϫ ϫ 6 Bcl-xL in CD8 PKC T cells. A, Purified splenic T cells (2 10 cells) from PKCϪ/Ϫ (), PKCϪ/Ϫ PKB (P), and C57BL/6 (B) mice were stimulated with plate-bound Ab (1 g/ml anti-CD3 combined with 1 g/ml anti-CD28 Ab) for the indicated times. Bcl-xL expression was de- termined by SDS-PAGE and Western blot analysis. B, Purified splenic T Ϫ/Ϫ ϫ 6 Ϫ/Ϫ Ϫ/Ϫ FIGURE 4. Impaired induction of Bcl-xL in c-Rel T cells. Purified cells (1 10 cells) from PKC , littermate PKC PKB, and splenic T cells (1 ϫ 106 cells) from c-RelϪ/Ϫ (red histogram), p50Ϫ/Ϫ (blue C57BL/6 mice were stimulated with plate-bound Ab. Cells were stimulated histogram), and C57BL/6 (black histogram) mice were stimulated with with 2 g/ml anti-CD3 Ab combined with 2 g/ml anti-CD28 Ab for 24 h. ϩ ϩ plate-bound Ab. Cells were stimulated with 2 g/ml anti-CD3 Ab or 2 Bcl-xL expression in CD4 and CD8 T cells was then determined by flow Ϯ g/ml anti-CD3 combined with 2 g/ml anti-CD28 Ab for 24 h. Bcl-xL cytometry. The average MFI of Bcl-xL staining of triplicate samples SDs expression was then determined by flow cytometry. Specific staining was are plotted. Specific staining for Bcl-xL was confirmed using an isotype confirmed using an isotype control Ab (dotted histogram). These data are control Ab (data not shown). These data are representative of three inde- representative of three independent experiments. pendent experiments. 2936 DIFFERENTIAL PROGRAMMING OF CD4ϩ AND CD8ϩ T CELL SURVIVAL
Ϫ/Ϫ of Bcl-xL expression in the PKC PKB T cells, however, was
slightly reduced as compared with the amount of Bcl-xL expressed by the C57BL/6 control cells. Thus, although transgenic expres-
sion of PKB could rescue the expression of Bcl-xL in cells lacking PKC, this rescue did not appear to be complete.
Given that we had found a difference in the expression of Bcl-xL in CD4ϩ and CD8ϩ cells (Fig. 2), we next examined whether PKB ϩ ϩ had a differential ability to restore Bcl-xL in CD4 and CD8 T cells that lacked PKC. Interestingly, as can be seen in Fig. 5B, ϩ active PKB was unable to rescue the expression of Bcl-xL in CD4
T cells, because the average MFI of Bcl-xL staining in the PKCϪ/Ϫ PKB CD4ϩ T lymphocytes was comparable to that of Ϫ/Ϫ the PKC T cells. Conversely, the average MFI of Bcl-xL staining in the PKCϪ/Ϫ PKB CD8ϩ T cells was 15.6 as compared with 10.0 for the PKCϪ/Ϫ CD8ϩ T lymphocytes. This difference is statistically significant ( p Ͻ 0.0006 by two-tailed Student’s t
test) and demonstrates that active PKB can rescue Bcl-xL expres- ϩ sion in CD8 T cells lacking PKC . This rescue of Bcl-xL ex- Downloaded from pression, however, is not total, because the MFI of Bcl-xL staining in the control C57BL/6 CD8ϩ T was 22.4. These data demonstrate that in CD8ϩ T cells, PKB can still promote the expression of Bcl-xL in the absence of PKC . This suggests that there exists a
signaling pathway that links PKB to Bcl-xL expression that does not require PKC in CTL. http://www.jimmunol.org/
PKB partially restores cell viability in CD4ϩ PKCϪ/Ϫ T cells Next, we examined whether PKB expression was sufficient to res- cue cell survival. Like in the previous survival experiments, the viability of in vitro-activated T cells from C57BL/6, PKCϪ/Ϫ, and PKCϪ/Ϫ PKB mice was assessed by flow cytometry using annexin V and 7AAD staining 20 h after their removal from plate-
bound Ab stimulation. Fig. 6A shows the percentage of viable by guest on September 27, 2021 CD4ϩ and CD8ϩ cells in each culture after their removal from
stimulation (viability at t0) as well as after 20 h of reculture. These data show that PKB was able to rescue partially the survival of PKCϪ/Ϫ CD4ϩ T cells. When the survival in each culture was FIGURE 6. Expression of active PKB partially restores CD4ϩ T cell survival. Splenocytes (1 ϫ 107 cells) from PKCϪ/Ϫ, PKCϪ/Ϫ PKB, and normalized to the viability at t0 (data not shown), the average viability of the PKCϪ/Ϫ CD4ϩ T cells was 30.0 vs 49.1% for the C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 Abs (2 PKCϪ/Ϫ PKB CD4ϩ T cells. This difference is statistically sig- g/ml of each) for 72 h. Cell viability was assessed by flow cytometry by Ͻ staining with annexin V and 7AAD to determine background death (via- nificant ( p 0.003 by two-tailed Student’s t test) and demon- ϫ 5 bility at t0). Cells were then washed and recultured (2 10 cells) in strates that the PKB transgene can affect cell viability. The effect triplicate without stimulation. After 20 h of reculture, cell viability was is through a pathway that does not alter Bcl-xL expression, because assessed by flow cytometry by staining with annexin V and 7AAD. FACS we demonstrated active PKB does not restore Bcl-xL expression in plots at each time point with the nonadjusted percentage of viable cells CD4ϩ T cells (Fig. 5). This rescue of cell survival, however, is not (percentage of annexin VϪ7AADϪ cells) are shown in A. Splenocytes from Ϫ Ϫ Ϫ Ϫ ϩ Ϫ complete. The PKCϪ/Ϫ PKB CD4ϩ T cells were not as viable P14 PKC / , P14 PKC / PKB, and P14 PKC / mice (2 ϫ 105 cells/ after 20 h as the C57BL/6 CD4ϩ T cells, which had an average of well) were incubated with increasing concentrations of p33 peptide. Pro- 3 69.5% live cells in culture relative to their viability at t . Thus, liferation measured by the incorporation of [ H]thymidine after 72 h is 0 shown in B. These data are representative of four independent experiments. active PKB expression only partially restored T cell survival in Ϫ/Ϫ ϩ PKC CD4 T cells through a Bcl-xL-independent pathway. In CD8ϩ cells, the PKB transgene had a reduced effect on cell Ϫ/Ϫ viability. The PKCϪ/Ϫ CD8ϩ T cells averaged 4.2% living cells PKB-mediated signals do not rescue proliferation in PKC in culture vs 10.6% for the PKCϪ/Ϫ PKB CD8ϩ T cells. Al- T cells though this difference is statistically significant ( p Ͻ 0.02 by two- T cells lacking PKC display poor proliferative responses in vitro Ϫ Ϫ tailed Student’s t test), the average viability of the PKC / PKB when stimulated with Abs (33, 34) or cognate peptide Ag (35). We ϩ CD8 T cells was still reduced greatly as compared with the also wanted to determine whether active PKB could provide ap- ϩ C57BL/6 CD8 T cell cultures. The control cell cultures had an propriate signals to restore the proliferative defect in vitro. To do average of 61.9% viable cells after 20 h relative to the viability at these experiments, P14 transgenic mice expressing a TCR specific
t0. Thus, although PKB could rescue Bcl-xL expression in for the lymphocytic choriomeningitis virus glycoprotein peptide, PKCϪ/Ϫ CD8ϩ T cells (Fig. 5), it was unable to markedly im- gp33, presented in the context of H-2Db were bred with PKCϪ/Ϫ prove the cell survival of activated PKCϪ/Ϫ CD8ϩ T cells in and PKCϪ/Ϫ PKB mice. Spleen cells from P14 PKCϩ/Ϫ, P14 vitro. These findings further emphasize the different mechanisms PKCϪ/Ϫ, and P14 PKCϪ/Ϫ PKB mice were then cultured with that modulate CD4ϩ and CD8ϩ T cell survival. varying concentrations of the gp33 peptide and proliferation was The Journal of Immunology 2937
T cells as compared with activation with Abs alone (data not shown). These data suggest that the survival-promoting effects of exogenous IL-2 we observed in the c-RelϪ/Ϫ and PKCϪ/Ϫ T
lymphocytes were independent of Bcl-xL expression.
Discussion Signaling molecules promoting T cell survival The signaling pathways that govern activated T cell survival have FIGURE 7. Exogenous IL-2 rescues the survival of activated PKCϪ/Ϫ yet to be elucidated fully. This is the first study that directly com- ϩ and c-RelϪ/Ϫ T cells. Splenocytes (1 ϫ 107 cells) from PKCϪ/Ϫ, pares the role of PKC, c-Rel, and PKB in the survival of CD4 ϩ ϩ c-RelϪ/Ϫ, and C57BL/6 mice were stimulated with anti-CD3 and anti- and CD8 T cells. Our study has shown that both CD4 and CD28 Abs (2 g/ml of each) for 72 h. Cell viability was assessed by flow CD8ϩ T lymphocytes require PKC-derived signals to survive cytometry by staining with annexin V and 7AAD to determine background (Fig. 1A). In the absence of PKC, however, CD4ϩ T cell survival death (viability at t0). The average viability at t0 of the cells ranged from can be restored partially by PKB (Fig. 6), whereas PKB does not ϫ 5 65 to 84%. Cells were then washed and recultured (2 10 cells) in have a major impact upon the survival of CD8ϩ T cells. These data triplicate without stimulation in the presence or absence of 10 U/ml IL-2. demonstrate a different requirement for PKB-mediated survival After 20 h of reculture, cell viability was assessed by flow cytometry by ϩ ϩ staining with annexin V and 7AAD. The data are expressed as the per- signals in CD4 and CD8 T cells. These findings also indicate that PKC and PKB signal independently of each other to promote Downloaded from centage of viable cells after 20 h of reculture divided by the percentage of ϩ viable cells at t (after stimulation). The data are shown as an average value CD4 T cell survival. The importance of active PKB for the sur- 0 ϩ of triplicate wells with error bars showing SD. These data are representa- vival of CD4 T lymphocytes is supported by studies by Song tive of five independent experiments. et al. (54). These authors demonstrated that signals activating PKB are important in maintaining the long-term survival of CD4ϩ T cells in vivo (55). 3
measured by [ H]thymidine incorporation at different time points. Although our results indicate the PKC is required for the sur- http://www.jimmunol.org/ Fig. 6B shows that the PKB transgene could not rescue the pro- vival of both CD4ϩ and CD8ϩ T lymphocytes, our data also in- Ϫ Ϫ liferation of the PKC / T cells to a level comparable to the dicate that PKC delivers a more vital survival signal in activated ϩ Ϫ PKC / after 72 h of stimulation. These findings demonstrate CD8ϩ T lymphocytes than in CD4ϩ T cells. CD8ϩ cells lacking
that neither the restoration of Bcl-xL expression nor the potential PKC display a much more striking impairment in survival than do alternative signals provided by PKB is sufficient to restore the CD4ϩ PKCϪ/Ϫ T cells (Fig. 1A). The importance of PKC in Ϫ Ϫ proliferative response to Ag in PKC / T cells in vitro. CD8ϩ T cell survival in vivo has been illustrated previously by work by Barouch-Bentov et al. (36). Our data confirm the findings Ϫ/Ϫ Ϫ/Ϫ IL-2 rescues the survival of PKC and c-Rel T cells of Barouch-Bentov et al. (36) and further suggest that c-Rel is Because the expression of IL-2 has been linked to PKC signals required to relay PKC-mediated survival signals in activated by guest on September 27, 2021 (33) and to the c-Rel transcription factor (40, 51), we tested CD8ϩ T cells, but not in CD4ϩ T cells (Fig. 1B). This finding is whether the addition of exogenous IL-2 was able to promote the consistent with a previous report that has linked PKC to the mo- survival of PKCϪ/Ϫ and c-RelϪ/Ϫ T cells. To do this, in vitro- bilization of c-Rel (56). It is also consistent with studies that have activated splenic T cells from PKCϪ/Ϫ, c-RelϪ/Ϫ, and C57BL/6 demonstrated a more profound impact upon activated CD8ϩ T cell mice were cultured for 20 h in the presence or absence of 10 U/ml survival than activated CD4ϩ T cell survival in mice with impaired IL-2. Cell viability was then assessed by flow cytometry using ability to activate NF-B (30, 57). The target genes of c-Rel that annexin V and 7AAD staining. As can be seen in Fig. 7, the ad- are important for the survival of CD8ϩ T lymphocytes remain to dition of exogenous IL-2 was able to restore the survival of PKC- be elucidated fully. ϩ Ϫ/Ϫ deficient CD4 T cells. The average survival of the PKC A multitude of studies have suggested that Bcl-xL is the key CD4ϩ T cells in the presence of IL-2 was 72.0%. Although this prosurvival molecule of activated T cells (4, 47, 58). Given that ϩ was slightly lower than the average survival of the control CD4 our results indicate that the expression of Bcl-xL is affected by both T cells, it represents a much more substantial rescue of cell via- PKC (Figs. 2 and 3) and c-Rel (Fig. 4), we suspected that the bility than was provided by the PKB transgene (Fig. 6A). Simi- diminished survival of CD8ϩ T cells we observed (Fig. 1) was due ϩ larly, in the case of the CD8 lymphocytes, IL-2 had a profound to an impaired ability to express Bcl-xL. Thus, we were surprised
effect upon survival. The addition of IL-2 was able to rescue fully to find that rescuing Bcl-xL expression with the PKB transgene in the survival defect in both the c-RelϪ/Ϫ and PKCϪ/Ϫ CD8ϩ T cytotoxic lymphocytes did not significantly alter their survival Ϫ/Ϫ cells. The average survival of the c-Rel T lymphocytes was (Fig. 6). These data question the importance of Bcl-xL expression actually slightly enhanced compared with the average survival of downstream of PKC and c-Rel for the survival of activated CD8ϩ the wild-type CD8ϩ T cells cultured with IL-2. Collectively, these T lymphocytes. Consistent with these findings, Zhang and He (59)
data demonstrate that unlike PKB, IL-2 can restore fully the sur- recently reported that Bcl-xL is dispensable for the survival of vival of T cells lacking PKC or c-Rel. CD8ϩ T cells. Our results would agree with these data and suggest Because signaling through the IL-2R has been linked to the that perhaps IL-2 is the important c-Rel target gene downstream of ϩ expression of Bcl-xL (52, 53), we wanted to ensure that the addi- PKC for the survival of CD8 T lymphocytes (Fig. 7). The im- tion of exogenous IL-2 was not altering the expression of Bcl-xL portance of IL-2 gene expression downstream of PKC in sup- in activated c-RelϪ/Ϫ and PKCϪ/Ϫ T cells. To do this, purified porting the survival of CD8ϩ T cells, however, may be dependent splenic T cells from c-RelϪ/Ϫ and PKCϪ/Ϫ mice were stimulated upon the context of T cell activation. Work in our own laboratory for 24 h with plate-bound Ab in the presence or absence of IL-2. (35) and that of others (60) has demonstrated that the absence of ϩ After 24 h, the expression of Bcl-xL was assessed using flow cy- the entire PKC signaling pathway in CD8 T cells can be over- tometry. The addition of either 10 or 50 U/ml IL-2 did not alter the come via the provision of strong pathogen-derived signals in vivo. Ϫ/Ϫ Ϫ/Ϫ expression of Bcl-xL in either the activated c-Rel or PKC PKC -mediated survival signals, however, may be more important 2938 DIFFERENTIAL PROGRAMMING OF CD4ϩ AND CD8ϩ T CELL SURVIVAL when pathogen-mediated signals are absent, such as in the context Acknowledgments of autoimmunity or during an antitumor response. We thank Rosa Pileggi and Sandra McGugan for administrative help. Bcl-x expression is not sufficient to restore proliferation L Disclosures ϩ Previous work by Zheng et al. (61) demonstrated that CD4 T The authors have no financial conflict of interest. cells lacking both p50 and c-Rel did not proliferate when stimu- lated. 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