Cells Abrogates Tolerance Induced by Apoptotic Activation-Induced

The Journal of Immunology

Activation-Induced CD154 Expression Abrogates Tolerance Induced by Apoptotic Cells1

Prajwal Gurung,*† Tamara A. Kucaba,* Thomas A. Ferguson,‡ and Thomas S. Grifﬁth2*†

The decision to generate a productive immune response or tolerance often depends on the context in which T cells ﬁrst see Ag. Using a classical system of tolerance induction, we examined the immunological consequence of Ag encountered in the presence of naive or activated apoptotic cells. Naive apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154, as tolerance resulted after i.v. injection of either naive or activated apoptotic CD154؊/؊ T cells, while coinjection of an agonistic anti-CD40 mAb with naive apoptotic T cells induced robust immunity. Dendritic cells fed activated apoptotic T cells in vitro produced IL-12p40 in a CD154-dependent manner, and the use of IL-12p40؊/؊ mice or mAb-mediated neutralization of IL-12 revealed a link between CD154, IL-12, and the ability of activated apoptotic T cells to induce immunity rather than tolerance. Collectively, these results show that CD154 expression on apoptotic T cells can determine the outcome of an immune response to Ag recognized within the context of the apoptotic cells and suggest that the balance between naive and activated apoptotic T cells may dictate whether a productive immune response is encouraged. The Journal of Immunology, 2009, 183: 6114–6123.

ell death is important to homeostasis in the immune sys- generate tolerance that would prevent essential T cell-mediated tem and critical to maintaining important T cell popula- immunity from developing. The question then is what determines C tions. Developing thymocytes undergo apoptosis during whether T cell apoptosis leads to tolerance or a productive immune selection in the thymus, leading to the removal of self-reactive response? clones (1), while effector T cells undergo activated-induced apo- The role of apoptosis in the maintenance of immunological ho- ptotic cell death following an immune response, leaving behind a meostasis and tolerance has been inferred from several experimen- small memory pool of cells to respond to future challenges (2). tal findings, e.g., tolerance induction after injection of apoptotic Although the apoptosis occurring in these situations does not typ- cells and the capacity of APC to induce and maintain tolerance ically evoke an immunological response, the death of large num- after dead cell phagocytosis (4, 8, 9). Furthermore, the therapeutic bers of T cells in the periphery can induce a tolerogenic response use of apoptotic cells has been extensively explored in the trans- (3). In this scenario, apoptotic T cells are cross-presented to the plantation field (10–13). Interestingly, the combination of donor- ϩ immune system, resulting in the activation of CD8 regulatory T specific transfusion of apoptotic cells and blockade of the CD40- 3 cells (Treg) that kill effector T cell targets (4, 5). This form of CD154 pathway profoundly increases transplant survival (14–18). tolerance extends to naive T cells of the same specificity and sug- Although the use of apoptotic cells to prolong the survival of organ gests that this active immunoregulation is directed toward the Ag- transplants (13) and treat autoimmune diseases (19) is well docu- reactive T cells for the purpose of controlling any self-responses mented, the mechanism(s) by which tolerance vs immunity is in- that might be generated when large numbers of T cells die and duced to Ag presented within the context of dead cells remain(s) release potentially dangerous autoantigens and cytokines (3, 4). unclear. During certain infections, there is also the death of large numbers It has recently become evident that there are a number of of cells; however, in many cases, productive immunity can de- properties of apoptotic cells that can determine whether toler- velop. For example, large numbers of activated T cells can die by ance or immunity is induced. For example, apoptotic cells can apoptosis during the initial steps of the immune response to Lis- release immunosuppressive (20) or immunogenic molecules teria monocytogenes (6, 7), but in this case the dead cells do not (21) that modulate immunity. In addition, caspase activation and reactive oxygen species production during apoptosis are critical factors that determine whether immunity or tolerance is *Department of Urology and †Interdisciplinary Program in Immunology, University of Iowa, Iowa City, IA 52242; and ‡Department of Ophthalmology and Visual Sci- induced (22). These observations together suggest that apopto- ences, Washington University School of Medicine, St. Louis, MO 63110 tic cells have some intrinsic properties that can modulate im- Received for publication May 27, 2009. Accepted for publication September 7, 2009. munity. There has also been some data suggesting that the ac- The costs of publication of this article were defrayed in part by the payment of page tivation status of the cell undergoing apoptosis can play a role charges. This article must therefore be hereby marked advertisement in accordance in determining the type of immune response generated (23); with 18 U.S.C. Section 1734 solely to indicate this fact. however, the precise mechanism for this has not been deter- 1 This work was supported by National Institutes of Health Grants CA109446 and mined. In the present study, we examined the effect of T cell R56 AI077565 (to T.S.G.). apoptosis on the immune system with the hypothesis that the 2 Address correspondence and reprint requests to Dr. Thomas S. Griffith, Department of Urology, 3204 MERF, University of Iowa, 375 Newton Road, Iowa City, IA activation state of the apoptotic T cells will dictate the outcome 52242-1089. E-mail address: thomas-griffi[email protected] of the immune response. Using a well-established i.v. tolerance 3 Abbreviations used in this paper: Treg, regulatory T cell; TNP, trinitrophenyl; DC, model, where hapten-modified apoptotic cells are injected i.v. dendritic cell; DAMP, damage-associated molecular pattern; iNOS, inducible NO into mice before active immunization (24–27), we compared synthase; TNBS, 2,4,6-trinitrobenzene sulfonic acid. the tolerogenic nature of naive and activated apoptotic T cells. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 Our results demonstrate that, in contrast to naive apoptotic www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901676 The Journal of Immunology 6115

ϩ cells, activated apoptotic CD4 T cells did not induce tolerance ␥-irradiated to induce apoptosis and injected i.v. as described above, but because they express CD154. CD154 induces IL-12 production, the mice were challenged 5 days later with the footpad injection of TNBS. which also promotes immunity rather than tolerance. Thus, the CD8ϩ T cell, CD4ϩ T cell, and CD11cϩ dendritic cell (DC) outcome of an encounter between apoptotic T cells and the isolation immune system can depend on the activation state of the apoptotic T cells, implying that T cell death during an active T cell CD8ϩ T cells were purified from splenocyte single-cell suspensions by ϩ response may function to further promote immunity. negative selection using a CD8 T cell isolation kit (Miltenyi Biotec) per the manufacturer’s instruction. CD4ϩ T cells were purified from the spleens of C57BL/6 or OT-II mice by negative selection using a CD4ϩ T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instruc- Materials and Methods ϩ ϩ ϩ Animals and reagents tion. Enriched CD8 T cells, CD4 T cells, or CD4 OT-II T cells were adoptively transferred i.v. via the retro-orbital plexus. CD11cϩ DC were C57BL/6 and BALB/c mice were purchased from The National Cancer isolated from the spleens of C57BL/6 mice injected with Ad5-Flt3L (30) Institute. CD154Ϫ/Ϫ mice were obtained from Dr. T. Ratliff (Purdue Uni- by positive selection using CD11c (N418) MicroBeads (Miltenyi Biotec). ϩ versity, West Lafayette, IN), CD40Ϫ/Ϫ mice were obtained from Dr. G. Purified CD11c DC (0.5 ϫ 106) were cultured with naive or activated Bishop (University of Iowa, Iowa City, IA), KbϪ/Ϫ act-mOVA mice (28) apoptotic cells (106) in complete RPMI 1640 (HyClone) in a total volume ϩ were obtained from Dr. J. Harty (University of Iowa), and OT-II mice were of 1 ml for 48 h. As a negative or positive control, CD11c DC were either obtained from Dr. Y. Luo (University of Iowa). CD154Ϫ/Ϫ, CD40Ϫ/Ϫ, cultured alone or with 1 ␮g of LPS, respectively. After culture, superna- KbϪ/Ϫ act-mOVA, and OT-II mice were backcrossed onto the C57BL/6 tants were collected and analyzed for the presence of IL-12p40 by ELISA background Ͼ10 generations. IL-12p40Ϫ/Ϫ mice were obtained from Dr. (eBioscience). In some cases, CD80 and CD86 expression was also mea- K. Legge (University of Iowa) and were backcrossed onto the BALB/c sured on the DC by flow cytometry. Purity of the isolated populations was background Ͼ10 generations. Anti-CD4 (GK1.5) and anti-CD40 (FGK45) determined by flow cytometry using anti-CD4, anti-CD8, and anti-CD11c mAb were purified from hybridoma supernatants. Neutralizing anti- mAb and was Ͼ90%. IL-12 mAb (15.1.2 and 15.6.7) (29) were obtained from Dr. R. Fairchild (The Cleveland Clinic, Cleveland OH). Neutralizing anti-IL-23, PE- In vivo proliferation conjugated anti-CD80, and PE-conjugated anti-CD86 mAb were purchased from eBioscience. Rat IgG isotype control was obtained from To measure the ability of Ag derived from naive or activated apoptotic cells to stimulate T cell proliferation, C57BL/6 mice were injected with 106 Dr. T. Waldschmidt (University of Iowa). All animal procedures were 7 performed according to National Institutes of Health guidelines and CFSE-labeled OT-II T cells i.v. 24 h before receiving 10 naive or acti- approved by the University of Iowa Institutional Animal Care and Use vated apoptotic act-mOVA spleen cells i.v. After 5 days, splenocytes from Committee. In all in vivo experiments, groups consisted of four or more the recipient mice were isolated, and cell proliferation, as measured by animals and experiments were repeated at least two times with similar CFSE dilution, was determined by flow cytometry. results before reporting. ␥ OVA323–339 restimulation and intracellular IFN- staining Activation and trinitrophenyl (TNP) coupling of spleen cells C57BL/6 mice were injected with 106 OT-II T cells i.v. 24 h before receiving 107 naive or PMA/ionomycin-activated apoptotic (induced by Spleen cells were isolated and activated with either PMA/ionomycin (20 ␥ ng/ml/200 ng/ml; Sigma-Aldrich) or plate-bound anti-CD3/anti-CD28 -irradiation) act-mOVA spleen cells i.v. After 5 days, splenocytes ␮ from the recipient mice were isolated and 2 ϫ 106 cells were cultured mAb (1 g/ml; eBioscience) for 16 h. Splenocyte activation was confirmed ␮ in vitro alone or restimulated with OVA323–339 (3 g/ml) for5hinthe by measuring CD25 and CD69 up-regulation using FITC-conjugated spe- ␮ cific mAb (eBioscience) and flow cytometry. CD154 expression on naive presence of brefeldin A (3 g/ml). The cells were collected and pro- ϩ cessed for IFN-␥ intracellular staining using a PE-conjugated anti- and activated CD4 T cells was measured by adding either biotinylated ␥ ␥ anti-CD154 (MR1. 1 ␮g/ml; Miltenyi Biotec) or biotinylated isotype mAb IFN- mAb (eBioscience). The percentage of IFN- -producing OT-II T (hamster IgG; eBioscience) to the cells while they were in culture being cells (as determined by PE-Cy5-conjugated anti-CD90.1 mAb (eBio- science) staining) in response to OVA323–339 restimulation (after sub- activated with PMA/ionomycin. After 16 h, the cells were washed, stained ␥ϩ with FITC-conjugated anti-CD4 mAb (eBioscience), fixed with 2% tracting the percentage of IFN- OT-II T cell from unstimulated cul- paraformaldehyde, permeabilized with 0.05% saponin buffer, and incu- tures) was then reported. bated with streptavidin-PE. CD154 expression on CD4ϩ T cells was then determined by flow cytometry. Single-cell suspensions of freshly Statistical analysis isolated naive or activated spleen cells were coupled with TNP as pre- 8 Significant differences between groups were evaluated using a two-tailed viously described (4). Briefly, 10 cells were incubated in 0.5 ml of PBS Student’s t test ( p Ͻ 0.05). and 0.5 ml of 10 mM 2,4,6-trinitrobenzene sulfonic acid (TNBS) for 5 min at room temperature. After incubation, the cells were washed three times with PBS before use. Naive or activated TNP-coupled spleen cells Results were then ␥-irradiated (3000 rad) to induce apoptosis. Necrotic cells Activated apoptotic cells prime for immunity and do not induce were prepared by alternating five freeze/thaw cycles using liquid nitro- tolerance gen/37°C water bath. TNP-coupled apoptotic (107) or equivalent necrotic cells were injected i.v. via the retro-orbital plexus. The activation state of immune cells undergoing apoptotic death can dramatically influence immune function (23), and activated Measurement of immune response: tolerogenicity and apoptotic cells, but not naive apoptotic cells, can mature and ac- immunogenicity tivate human DC in vitro (31). We therefore explored the role of In the i.v. tolerance system used herein, the tolerogenicity and immuno- activated apoptotic cells in a classical tolerance model (4, 5, 22), genicity of i.v. administered apoptotic cells was tested by slightly modi- where hapten-modified apoptotic cells are injected i.v. to induce fying the experimental design (22). To measure the tolerogenicity of the tolerance or measure immunity (supplemental Fig. 1).4 Animals i.v. administered apoptotic cells, apoptosis was first induced in haptenated (TNP-coupled) splenocytes by ␥-irradiation and then the cells were imme- were given either haptenated (TNP-coupled) naive or activated diately injected i.v. After 48 h, animals were immunized with 0.1 ml of 10 apoptotic cells (supplemental Fig. 2) i.v. 48 h before s.c. immu- mM TNBS s.c. After another 4 days, the immune response was examined nization. Data in Fig. 1A show that naive apoptotic cells were by challenging the animals with 33 ␮l of 10 mM TNBS in the right footpad tolerogenic, which is consistent with previous results (5), but the ␮ and 33 l of PBS in the left footpad. The delayed-type hypersensitivity activated apoptotic cells (either PMA/ionomycin or anti-CD3/anti- response to TNBS (TNP) was measured 24 h later by a masked observer. Values are expressed in ␮m Ϯ SE and represent the difference between the CD28 activated) were unable to induce tolerance (Fig. 1A). Thus, TNBS-challenged right footpad and PBS-challenged left footpad of the T cell activation before the induction of apoptosis abrogates the same mouse. Background values represent the difference between the challenged and unchallenged footpad in naive mice. To measure the immunogenicity of the i.v. administered apoptotic cells, the haptenated cells were 4 The online version of this article contains supplemental material. 6116 CD154 EXPRESSION PREVENTS i.v. TOLERANCE

FIGURE 1. Activated (act.) apoptotic (apop.) cells prime for immunity and do not induce tolerance when injected i.v. A, Tolerogenicity: C57BL/6 mice were injected i.v. with naive or activated (PMA/ionomycin (P/I)- or anti-CD3/CD28-stimulated) apoptotic TNP-coupled splenocytes i.v. 48 h before TNBS immunization. After 4 days, mice were challenged with TNBS in the right and PBS in the left footpad. B, Immunogenicity: C57BL/6 mice were injected i.v. with necrotic, naive apoptotic, or activated apoptotic TNP-coupled splenocytes. Four days later, mice were challenged with TNBS in the right and PBS in the left footpad. C, Tolerogenicity: C57BL/6 mice were injected i.v. with naive or activated apoptotic TNP-coupled splenocytes i.v. 48 h before TNBS immunization. The activated populations included bulk splenocytes, purified CD4ϩ T cells, CD4-depleted (depl.) splenocytes, purified CD8ϩ T cells, or CD8-depleted splenocytes. After 4 days, mice were challenged with TNBS in the right and PBS in the left footpad. A–C, Measurements (␮m Ϯ SE) were taken 24 h later and represent the difference between the right and left footpads. Immune control p Ͻ 0.05 vs ,ء .groups were injected with TNBS 4 days before footpad challenge, and background (BKG) groups were only footpad challenged immune control. DTH, Delayed-type hypersensitivity. tolerogenic effect of the apoptotic cells. Administration of apopto- KbϪ/Ϫ act-mOVA splenocytes (28). After 5 days, splenocytes tic cells i.v. does not directly prime a CD4ϩ T cell-mediated im- from the recipient mice were isolated and OT-II T cell prolifera- mune response, while necrotic cells can stimulate potent CD4ϩ T tion was assessed by measuring CFSE dilution. There was robust cell-mediated immunity (5). Similar to necrotic cells, i.v. admin- OT-II proliferation when the mice were given activated apoptotic istered activated apoptotic cells were also strongly immunogenic act-mOVA cells (comparable to that seen in mice immunized with (Fig. 1B). Since we used agents that specifically activate T cells, CFA/OVA), but the proliferation was much reduced when naive we then tested whether there was a specific subpopulation within apoptotic cells were administered (Fig. 2B). We also took spleno- the activated splenocytes responsible for overcoming tolerance. cytes from the recipient mice and cultured them in vitro for 48 h ϩ ϩ Purified CD4 T cells, purified CD8 T cells, and the correspond- alone or with OVA323–339. The cells were processed to detect ingly T cell-depleted splenocytes were then activated with PMA/ OT-II T cell production of IFN-␥ by flow cytometry, which re- ionomycin. The activated cells were haptenated, irradiated, and vealed that the mice primed with naive apoptotic cells contained injected i.v. 48 h before s.c. immunization. The results show that significantly less IFN-␥-producing OT-II T cells after in vitro re- tolerance was observed when activated apoptotic splenocytes were stimulation compared with mice primed with activated apoptotic depleted of CD4ϩ cells or when purified activated apoptotic CD8ϩ cells (Fig. 2C). Similar to the experiments with haptenated spleno- T cells were injected i.v. (Fig. 1C). However, activated apoptotic cytes, the i.v. administration of naive apoptotic act-mOVA spleno- CD8-depleted splenocytes and purified activated apoptotic CD4ϩ cytes to C57BL/6 mice induced tolerance to OVA, but the delivery T cells were able to abrogate tolerance as well as bulk splenocytes. of activated apoptotic act-mOVA splenocytes lead to immunity These results indicate that it is the activated apoptotic CD4ϩ T (supplemental Fig. 3). Thus, the tolerance observed after i.v. ad- cells that prevent tolerance induction. ministration of naive apoptotic cells relates to the failure of the Apoptotic cells induce tolerance following i.v. injection by be- naive apoptotic cells to stimulate CD4ϩ T cell proliferation and ing engulfed by splenic DC and cross-presented to the immune attain full effector function (i.e., inability to produce proinflamma- system in a tolerogenic form. Recent work by Ren et al. (32) found tory cytokines (IFN-␥)). Collectively, the results in Figs. 1 and 2 that DC exposed to apoptotic cells (dexamethasone-treated spleno- show that the activation status of cells before inducing apoptosis cytes) inhibited T cell expansion. Thus, one potential difference has dramatically different consequences on the response of the between naive and activate apoptotic cells could be that DC that immune system. have eaten the naive apoptotic cells are unable to stimulate CD4ϩ T cell proliferation, whereas DC that have engulfed activated ap- CD154 expression abolishes the ability of activated apoptotic optotic cells become properly licensed to induce a proliferative cells to induce tolerance response. We tested this possibility by feeding CD11cϩ DC either Naive and activated T cells differ in their expression of cell surface naive or activated splenocytes and then measured CD80 and CD86 markers; specifically, activated cells have increased levels of co- expression on the DC 48 h later. DC-fed activated apoptotic cells stimulatory molecules and their ligands (33, 34), prompting us to up-regulated CD80 and CD86 expression, whereas DC-fed naive test whether this could account for the different immunological apoptotic cells expressed these molecules at a similar level as DC outcome following injection of naive and activated apoptotic cells. cultured alone (Fig. 2A). One important costimulatory protein expressed early after T cell We then tested the ability of the i.v. administered naive or ac- activation is CD154 (35–37), which is readily up-regulated on tivated apoptotic cells to prime CD4ϩ T cells in vivo. To do this, CD4ϩ T cells within PMA/ionomycin-activated splenocytes (Fig. CFSE-labeled OT-II T cells were adoptively transferred into 3A). CD154 is involved in the activation and licensing of DC C57BL/6 mice 24 h before injecting naive or activated apoptotic through CD40 ligation (38, 39), and we have previously shown The Journal of Immunology 6117

FIGURE 2. Activated (Act.) apoptotic (apop/apop.) cells, but not naive apoptotic cells, license DC to prime CD4ϩ T cells. A, CD11cϩ DC were isolated and cultured with naive or activated (PMA/ionomycin) apoptotic cells for 48 h. CD80 and CD86 expression was then measured by flow cytometry. B, C57BL/6 mice received 106 CFSE-labeled OT-II T cells 24 h before receiving 107 naive or activated apoptotic KbϪ/Ϫ act-mOVA splenocytes (spl.) i.v. After 5 days, splenocytes from the recipient mice were isolated, and cell proliferation, as measured by CFSE dilution, was determined by flow cytometry. The percentage of cells that underwent five divisions is indicated for each group (n ϭ 3). C, Alternatively, splenocytes from the recipient mice were isolated ␮ ␮ and cultured in vitro alone or restimulated with OVA323–339 (3 g/ml) for5hinthepresence of brefeldin A (3 g/ml). The cells were collected and ␥ ␥ processed for IFN- intracellular staining. The percentage of IFN- -producing OT-II T cells in response to OVA323–339 restimulation (after subtracting the percentage of IFN-␥ϩ OT-II T cell from unstimulated (Unstim) cultures) is depicted.

that the combination of agonistic anti-CD40 mAb with naive ap- Activated apoptotic T cells stimulate immunity by inducing IL-12 optotic cells primes for immunity (4). This observation is verified from DC in Fig. 3B, where coinjection of naive apoptotic cells and anti- Data in Fig. 1 demonstrate that naive and activated apoptotic cells CD40 mAb induced immunity instead of tolerance. We then tested stimulated CD4ϩ T cell proliferation through the DC; however, the role of CD40-CD154 interactions in the present system by only activated apoptotic cells induced effector cytokine production ϩ/ϩ Ϫ/Ϫ using splenocytes from CD154 and CD154 mice. Injection (IFN-␥). These results suggest that activated apoptotic cells might ϩ/ϩ Ϫ/Ϫ of naive CD154 and CD154 apoptotic cells before immu- stimulate DC function to promote immunity. One DC-derived cy- nization induced tolerance, whereas the injection of the activated tokine important for stimulating CD4ϩ T cell function is IL-12 ϩ/ϩ (CD154 ) apoptotic cells did not (Fig. 3B). Interestingly, acti- (41–43); therefore, we initially examined the ability of naive or Ϫ/Ϫ vated apoptotic CD154 splenocytes (supplemental Fig. 4) ad- activated apoptotic cells to stimulate DC to produce this cytokine. ministered i.v. into C57BL/6 recipients induced tolerance rather Naive or activated apoptotic spleen cells were added to DC for than immunity. The relevant CD154 expression was on the acti- 48 h and the culture supernatants were analyzed for IL-12p40. vated apoptotic cells and not in the recipient mice because i.v. When the DC were cultured with CD154ϩ/ϩ activated apoptotic Ϫ/Ϫ treatment of CD154 recipients with naive or activated apopto- cells, significant IL-12p40 was produced (Fig. 5A). In contrast, ϩ/ϩ tic CD154 cells led to similar results (Fig. 3C). Importantly, DC cultured with naive apoptotic cells (either CD154ϩ/ϩ or Ϫ/Ϫ the CD154 activated apoptotic cells were unable to prime for CD154Ϫ/Ϫ) or DC cultured with CD154Ϫ/Ϫ activated apoptotic immunity when injected i.v. into either C57BL/6 (Fig. 3D)or cells did not produce significant IL-12p40 above background lev- Ϫ/Ϫ CD154 recipients (Fig. 3E), suggesting the essential role of els (DC plus media). These results demonstrate that CD154 ex- CD154 expression on the activated apoptotic cells for promoting pression on the activated apoptotic cells induce DC production of immunity. Collectively, the data in Fig. 3 demonstrate that activa- IL-12p40. tion-induced expression of CD154 on apoptotic lymphocytes is We further examined the role of activated apoptotic cell-induced instrumental in instructing the immune system’s response to Ag IL-12 using two complementary in vivo methods. In the first ap- associated with the apoptotic cells. proach, naive and activated apoptotic cells were injected i.v. into Because CD40 is the only receptor known to bind to CD154 BALB/c or IL-12p40Ϫ/Ϫ mice and the mice were immunized 48 h (40), it was predicted that CD154-expressing, activated apopto- later. When the delayed-type hypersensitivity response was Ϫ Ϫ tic cells would be tolerogenic when injected into CD40 / re- measured 4 days later, we found that both naive and activated cipient mice before immunization and would also be unable to apoptotic cells induced tolerance (Fig. 5B). Since IL-12p40 can directly prime for immunity. This proved to be the case because form a heterodimer with either IL-12p35 or IL-23p19 to form both naive and activated C57BL/6 apoptotic splenocytes in- bioactive IL-12 or IL-23, respectively (44), our second ap- Ϫ Ϫ jected i.v. into CD40 / recipients before immunization in- proach used neutralizing mAb to specifically assess the impor- duced tolerance (Fig. 4A), and neither apoptotic cell population tance of these cytokines. C57BL/6 mice were given naive or directly primed for immunity (Fig. 4B). Together with the data activated apoptotic cells 48 h before immunization. In addition, in Fig. 3, these results suggest that the tolerogenic or immuno- some of the mice that received activated apoptotic cells also genic properties of apoptotic cells can be determined by CD154 received either neutralizing anti-IL-12 (29) or anti-IL-23 mAb. expression, which can simulate CD40-expressing cells in the Specific neutralization of IL-12, but not IL-23, restored toler- recipient. ance in mice given activated apoptotic cells (Fig. 5C). Together, 6118 CD154 EXPRESSION PREVENTS i.v. TOLERANCE

FIGURE 3. Tolerogenicity of i.v. injected apoptotic cells (apop/apop.) is determined by CD154 expression. A, C57BL/6 splenocytes were activated (Act./act.) for 16 h with PMA/ionomycin (P/I) before measuring CD154 expression on CD4ϩ T cells. B, Tolerogenicity: C57BL/6 mice were injected i.v. with CD154ϩ/ϩ or CD154Ϫ/Ϫ naive or activated apoptotic TNP-coupled splenocytes i.v. 48 h before TNBS immunization. Some of the mice were injected with naive apoptotic TNP-coupled splenocytes and agonistic anti-CD40 mAb (FGK45; 300 ␮g). After 4 days, mice were challenged with TNBS in the right and PBS in the left footpad. C, Tolerogenicity: C57BL/6 or CD154Ϫ/Ϫ mice were injected i.v. with CD154ϩ/ϩ naive or activated apoptotic TNP-coupled splenocytes i.v. 48 h before TNBS immunization. After 4 days, mice were challenged with TNBS in the right and PBS in the left footpad. D and E, Immunogenicity: C57BL/6 (D) and CD154Ϫ/Ϫ (E) mice were injected i.v. with CD154ϩ/ϩ or CD154Ϫ/Ϫ naive or activated apoptotic TNP-coupled spleen cells. Four days later, mice were challenged with TNBS in the right and PBS in the left footpad. B–E, Measurements (␮m Ϯ SE) were taken 24 h after footpad challenge and represent the difference between the right and left footpads. In all cases, immune control groups were injected with TNBS 4 days ,p Ͻ 0.05 vs immune control. SSC, side scatter; DTH ,ء .before footpad challenge, and background (BKG) groups were only footpad challenged delayed-type hypersensitivity. The Journal of Immunology 6119

hypothesis, naive or activated apoptotic cells were injected into intact or CD4ϩ T cell-depleted mice. After 7 days, CD8ϩ T cells were puriﬁed from the spleens of these mice and transferred into naive recipients. These mice were immediately immunized, challenged after 4 days, and immunity was measured 24 h later. Consistent with our previous reports, CD8ϩ T cells from mice previously given naive, apoptotic cells transferred tolerance to naive mice (4, 5); however, CD8ϩ T cells from mice previously given activated, apoptotic cells did not (Fig. 6). Interestingly, CD8ϩ T cells from CD4ϩ T cell-depleted mice given activated apoptotic cells now transferred tolerance to naive recipients. Although the removal of CD4ϩ T cells did not alter generation ϩ of the CD8 Treg following naive apoptotic cell delivery, the depletion of CD4ϩ helper T cells led to the conversion of the immunogenic CD154-expressing activated apoptotic T cells into a tolerizing signal capable of stimulating the development ϩ of CD8 Treg. Thus, the tolerogenic or immunogenic nature of naive or activated apoptotic cells correlates with the activation of CD4ϩ T cells.

Activated apoptotic cells are dominant over naive apoptotic cells in instructing the immune system The data presented demonstrate that naive apoptotic cells induce immune tolerance when administered i.v.; in sharp contrast, activated apoptotic cells are potent stimulators of immunity when given by the same route. Because we have shown that both populations of apoptotic cells interact with DC to stimulate their re- spective effects, the question then arose as to what would be the result if both populations of apoptotic cells (i.e., naive and activated) were simultaneously presented to the immune system. This was addressed with the experiment in Fig. 7. Mice received 107 apoptotic cells i.v. 48 h before immunization, but with each injec- FIGURE 4. CD40Ϫ/Ϫ mice are tolerized by naive and activated (act.) tion the ratio of naive:activated apoptotic cells was varied. The apoptotic (apop.) cells. A, Tolerogenicity: CD40Ϫ/Ϫ mice were injected i.v. results show that with ratios of 5:1 and 10:1 naive:activated apo- with CD154ϩ/ϩ naive or activated (PMA/ionomycin (P/I)) apoptotic TNP- ptotic cells, the effect of activated apoptotic cells was dominant coupled splenocytes i.v. 48 h before TNBS immunization. After 4 days, (i.e., no tolerance). Only when the naive:activated apoptotic cells mice were challenged with TNBS in the right and PBS in the left footpad. ratio was 20:1 or higher was tolerance observed. Thus, activated Ϫ Ϫ B, Immunogenicity: CD40 / mice were injected i.v. with naive or acti- apoptotic cells show a dominant effect on the outcome of an im- vated apoptotic TNP-coupled spleen cells. Four days later, mice were chal- mune response taking place in the presence of dead cells. lenged with TNBS in the right and PBS in the left footpad. A and B, Measurements (␮m Ϯ SE) were taken 24 h later and represent the difference between the right and left footpads. Immune control groups were Discussion injected with TNBS 4 days before footpad challenge, and background The concept that physiological cell death (i.e., apoptosis) was -p Ͻ 0.05 vs immune con- tolerogenic and pathological cell death (i.e., necrosis) was im ,ء .BKG) groups were only footpad challenged) trol. DTH, delayed-type hypersensitivity. munogenic was historically based on the nature of the death process and the molecular signals produced by the dying cells. As investigation of cell death became more sophisticated, it was these results show that IL-12 production is required for acti- concluded that the differences between tolerogenic and immu- vated apoptotic cells to induce immunity, and suggest a link nogenic cell death extended well beyond the distinct molecular among CD154, IL-12, and the ability of activated apoptotic differences of apoptotic and necrotic cells. A number of molec- cells to stimulate immunity. ular mechanisms have been suggested to explain how apoptotic cells actively participate in determining whether tolerance or Eliminating CD4ϩ T cell help restores infectious tolerance ϩ immunity ensues, such as the intrinsic antigenicity of the cells mediated by CD8 T cells in mice given activated apoptotic (21), the nature of the cell death stimulus (45), the cell death cells pathway engaged (21, 22, 46), the production of reactive oxy- We have previously shown that CD8ϩ T cells from mice given gen species (22), and the release of immunosuppressive cyto- naive apoptotic cells can transfer tolerance to naive recipients (“in- kines such as IL-10 (20) and TGF-␤ (47). In addition, apoptotic ϩ fectious tolerance”) (5). In addition, we know that these CD8 Treg cells express phosphatidylserine, which signals them for phago- are induced because of the lack of CD4ϩ T cell-mediated “help,” cytosis shortly after the apoptotic process has begun (48), en- as demonstrated by depletion of CD4ϩ T cells converting immu- suring that the apoptotic cells are rapidly cleared before dam- nogenic necrotic cells into a tolerogen (5). One prediction would age-associated molecular patterns (DAMPs) (49, 50) can be ϩ be that activated apoptotic cells would not induce CD8 Treg (be- released. In line with this idea, we have observed that the cause they stimulate CD4ϩ T cell help) and that removal of CD4ϩ tolerogenic ability of naive apoptotic splenocytes can be elim- ϩ T cells would again reveal a CD8 Treg population. To test this inated if the apoptotic cells are cultured for 18 h before i.v. 6120 CD154 EXPRESSION PREVENTS i.v. TOLERANCE

FIGURE 5. The inability of activated (act.) apoptotic (apop.) cells to induce tolerance relates to IL-12 production. A, Splenic CD11cϩ DC were isolated (iso) and cultured alone, with naive or activated apoptotic CD154ϩ/ϩ splenocytes, with naive or activated apoptotic CD154Ϫ/Ϫ splenocytes, or with an agonistic anti-CD40 mAb (FGK45) or isotype control mAb. After 48 h, the amount of IL-12p40 in the culture supernatant was measured by ELISA. B, Tolerogenicity: BALB/c or IL-12p40Ϫ/Ϫ mice were injected i.v. with naive or activated (PMA/ionomycin (P/I)) apoptotic TNP-coupled splenocytes i.v. 48 h before TNBS immunization. After 4 days, mice were challenged with TNBS in the right and PBS in the left footpad. C, Tolerogenicity: C57BL/6 were injected i.v. with naive or activated apoptotic TNP-coupled splenocytes i.v. 48 h before TNBS immunization. Some of the mice were injected with activated apoptotic TNP-coupled splenocytes and anti-IL-12 mAb (200 ␮g each of 15.1.2 and 15.6.7), anti-IL-23 mAb (10 ␮g), or isotype control mAb (400 ␮g). After 4 days, mice were challenged with TNBS in the right and PBS in the left footpad. A and B, Measurements (␮m Ϯ SE) were taken 24 h after footpad challenge and represent the difference between the right and left footpads. Immune control p Ͻ 0.05 vs ,ء .groups were injected with TNBS 4 days before footpad challenge, and background (BKG) groups were only footpad challenged immune control. DTH, delayed-type sensitivity. injection (P. Gurung, T. S. Grifﬁth, and T. A. Ferguson, unpub- immunogenic because of the uncontrolled release of a number lished data), suggesting that these late apoptotic/secondary ne- of DAMPs. For example, necrotic cell expression and/or release crotic cells are now releasing DAMPs that override their nor- of heat shock proteins or HMGB1 can strongly stimulate the mally tolerogenic nature. Necrosis was believed to be largely immune system through the interaction with a number of pattern recognition receptors (21, 51, 52). HMGB1 can also be released from apoptotic cells (53, 54); however, the HMGB1 is oxidized in a caspase-dependent manner that maintains the tolerogenic effect of the apoptotic cells (22). In addition, necrosis can activate the NLRP3 inﬂammasome, resulting in

FIGURE 6. Activated (act.) apoptotic cells induce immunity by stimulating CD4-mediated help. C57BL/6 mice or C57BL/6 mice depleted of CD4ϩ cells (anti-CD4 GK1.5 (100 ␮g/day/mouse for 3 days) were FIGURE 7. Activated (act.) apoptotic (apop.) cells are dominant over injected i.v. with naive or activated (PMA/ionomycin) apoptotic TNP- naive apoptotic cells in the induction of immunity vs tolerance. C57BL/6 coupled splenocytes (spl). Seven days later, spleens were removed and mice were injected i.v. with naive or activated (PMA/ionomycin (P/I)) CD8ϩ T cells were isolated by negative selection. Puriﬁed CD8ϩ T apoptotic TNP-coupled splenocytes i.v. 48 h before TNBS immunization. cells were then transferred to naive C57BL/6 mice, which were imme- Some groups of mice received a mixture of naive and activated apoptotic diately immunized with TNBS. Four days later, mice were challenged TNP-coupled splenocytes (107 cells total) at the indicated ratios. After 4 with TNBS in the right and PBS in the left footpad. Measurements days, mice were challenged with TNBS in the right and PBS in the left (␮m Ϯ SE) were taken 24 h later and represent the difference between footpad. Measurements (␮m Ϯ SE) were taken 24 h later and represent the the right and left footpads. Immune control groups were injected with difference between the right and left footpads. Immune control groups were TNBS 4 days before footpad challenge, and background (BKG) groups injected with TNBS 4 days before footpad challenge, and background -p Ͻ 0.05 vs immune con ,ء .p Ͻ 0.05 vs immune control. DTH, (BKG) groups were only footpad challenged ,ء .were only footpad challenged delayed-type sensitivity. trol. DTH, delayed type sensitivity. The Journal of Immunology 6121

IL-1␤ and Il-18 release (55). Although many of the original importance of CD4ϩ T cell priming and TRAIL-expressing CD8ϩ parameters thought to distinguish tolerogenic/apoptotic/physi- Treg in i.v. tolerance (5). We found that these TRAIL-expressing ϩ ϩ ological cell death from immunogenic/necrotic/pathological CD8 Treg were similar to the “helpless” CD8 T cells generated cell death are still valid, it is important to keep in mind that in the absence of CD4ϩ T cell help (69), and the delivery of ne- ϩ immune response to dead cells can be influenced by many more crotic cells i.v. failed to induce tolerance and the CD8 Treg be- molecular differences than initially thought. cause CD4ϩ T cell priming occurred. Likewise, the present data The experiments described in this report were designed to show that CD8ϩ T cells from mice given activated apoptotic cells investigate the impact of the cellular activation state of apopto- were unable to transfer tolerance, suggesting that these mice re- tic lymphocytes on the immune system, with the intention of ceived the necessary CD4ϩ T cell help after priming with the elucidating the mechanism that instructs the immune system to CD154-expressing activated apoptotic cells for immunity. In line either ignore or respond to Ag recognized in the context of these with these results, we have examined splenic CD8ϩ T cells from apoptotic cells. Our results demonstrate the critical role of mice given either naive or activated apoptotic cells for TRAIL CD154 expression in determining the tolerogenic/immunogenic expression and found that only the CD8ϩ T cells from mice given nature of the apoptotic cells administered. Consequently, our naive apoptotic cells, but not activated apoptotic cells, had in- data are the first, to our knowledge, to show that i.v. adminis- creased TRAIL expression (supplemental Fig. 5). Although it is tered naive apoptotic cells induce tolerance because they lack clear from our previous and current data that the administration of CD154 expression, which is needed to stimulate IL-12 produc- either necrotic or activated apoptotic Ag-coupled cells induces im- tion from DC. Although CD154 expression has been reported munity instead of tolerance, the molecular cues displayed by the on multiple cells of the immune system, including CD8ϩ T necrotic or activated apoptotic cells that stimulate immunity are cells, B cells, granulocytes, and platelets, CD154 expression is most likely different. the best characterized on CD4ϩ T cells (38–40, 56–58). Why or when would it be beneficial for an apoptotic T cell to CD154-expressing CD4ϩ T cells play critical roles in activating express CD154 as it is dying in vivo? One possible scenario would DC (38, 57, 59), as DC maturation via CD40 ligation results in be during an acute infection, where the high number of CD154- costimulatory molecule expression and activates the DC to pro- expressing dead/dying T cells may help maintain a high threshold duce IL-12 (41, 42, 60, 61), an important cytokine needed to of inflammation required to clear the pathogen. This environment prime T cells during infection. The use of agonistic anti-CD40 would also facilitate the priming of “helped” CD8ϩ T cells and any Ab with apoptotic cells (62) or CD154ϩ apoptotic cell lines subsequent memory responses (66, 70, 71). Then, as CD154 ex- (63) to mature DC and abrogate the tolerogenic potential of the pression decreases in relation with the contraction of the effector apoptotic cells has also been previously reported. Recent stud- response, the tolerogenic effects of the apoptotic cells should re- ies with human PBMC demonstrated that bone marrow-derived emerge. The generation of helpless CD8ϩ T cells would be fa- DC cultured with naive apoptotic cells did not activate these vored at this stage of the immune response, which may be essential DC, as measured by the lack of CD80, CD86, and MHC class in the maintenance of tolerance (5, 69). By contrast, during chronic II expression and production of different proinflammatory cy- infections, persistent cross-presentation of Ag from CD154-ex- tokines (31). In contrast, DC activation readily occurred after pressing apoptotic T cells may participate in the maintenance of incubation with PBMC that had been activated before apoptosis the chronic low-level inflammation and may even facilitate the induction. Although broadly consistent with our data, this re- emergence of autoimmune reactions. Indeed, overexpression of port failed to define the molecular mechanism behind the stim- CD154 has been correlated with a number of autoimmune dis- ulation of human DC with activated apoptotic PBMC. eases, including systemic lupus erythematosus, rheumatoid arthri- Although our data describe the importance of CD40-CD154 tis, and inflammatory bowel disease (72). Thus, we may want to interactions between activated apoptotic cells and DC to abolish think of the apoptotic death of activated lymphocytes (those asso- the tolerogenic effects of the apoptotic cells, it is important to ciated with Ag/pathogen) as “inflammatory apoptosis” in compar- note that the presence or absence of this ligand-receptor inter- ison to the “silent apoptosis” of naive lymphocytes (those without action is probably not the only mechanism governing the im- Ag) (73). munogenic or tolerogenic ability of the apoptotic cells. For example, inhibition of inducible NO synthase (iNOS) by the Acknowledgments G arginine analog, N -monomethyl-L-arginine, can reverse the We thank Erik Brincks and Lyse Norian for helpful discussions. ability of DC that have eaten apoptotic cells to inhibit T cell proliferation (32). It was further determined that the iNOS pro- Disclosures ␥ ␥ duction by the DC was tied to IFN- responsiveness. 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