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The Journal of Immunology, 2004, 172: 4804-4811.
Copyright © 2004 by The American Association of Immunologists

CD40 Ligation In Vivo Induces Bystander Proliferation of Memory Phenotype CD8 T Cells1

Marie Koschella, David Voehringer2 and Hanspeter Pircher3

Institute of Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Injection of agonistic anti-CD40 Abs into mice has been shown to amplify weak CD8 T cell responses to poorly immunogenic compounds and to convert T cell tolerance to T cell priming. In this study we demonstrate that anti-CD40 treatment of C57BL/6 mice, without Ag delivery, led to a marked increase in the number of memory phenotype CD4 and CD8 T cells. Adoptive transfer experiments using CD40-deficient hosts further revealed that the proliferative response of memory T cells, induced by systemic CD40 signaling, was dependent on CD40 expression of host APCs. CD40 ligation in vivo induced vigorous cell division of both memory phenotype and bona fide virus-specific memory CD8 T cells in a partially IL-15-dependent manner. However, only memory phenotype, but not Ag-experienced memory CD8 T cells increased in cell number after anti-CD40 treatment in vivo. Taken together our data show that activation of APC via CD40 induces a marked bystander proliferation of memory phenotype T cells. In addition, we demonstrate that bona fide Ag-experienced memory CD8 T cells respond differently to anti-CD40-induced signals than memory phenotype CD8 T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD40, a member of the TNF receptor family, was first identified and functionally characterized on B cells (1). Beside B cells, dendritic cells (DC),4 activated macrophages, follicular DC, and endothelial cells also express CD40. CD40 ligand (CD40L) is expressed predominantly on activated CD4 T cells, but expression has also been reported on other leukocytes (2). The importance of CD40L-CD40 interaction in T cell-B cell collaboration has been demonstrated impressively in mice that lack these molecules and fail to initiate Ig class switching, somatic hypermutation, and germinal center formation (3, 4, 5, 6). Beside their function in B cell priming, CD40L-CD40 interactions play an important role in mediating CD4 helper functions for CD8 T cells (7, 8, 9). In this latter situation, DC present Ag to CD4 Th cells, which consecutively activate DC through CD40L. Besides further amplification of CD4 T cells, this activation allows DC to induce a potent CD8 T cell response. Further studies demonstrating conversion of T cell tolerance to T cell priming by injection of agonistic anti-CD40 mAb provided additional evidence for the concept that the activation state of APC determines primarily the outcome of a CD8 T cell response (10, 11, 12, 13, 14). However, this concept was challenged recently by the finding that CD8 T cells transiently express CD40 after activation and that they could receive CD4 help directly via CD40 (15). The latter study further implied that in the former studies injection of agonistic anti-CD40 mAb had a direct effect on CD8 T cells. To address this important issue, we examined the effect of anti-CD40 treatment on the turnover of memory CD8 T cells in vivo in the absence of Ag delivery. Strikingly, we observed a marked increase in the number of memory phenotype CD8 T cells after injection of agonistic anti-CD40 mAb into normal mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 (B6) mice were purchased from Harlan Winkelmann (Borchen, Germany). B6.PL-Thy-1a (B6.Thy1.1) and P14 TCR-transgenic (TCR-tg) mice (16) (line 318) specific for aa 33–41 of the lymphocytic choriomeningitis virus (LCMV) glycoprotein were bred at our colony. C57BL/6-IL15tm1Imx (17) (IL-15−/−) and B6.129P2-Tnfrsf5tm1Kik (6) (CD40−/−) mice were purchased from Taconic Farms (Germantown, NY) and The Jackson Laboratory (Bar Harbor, ME), respectively. IL-12p40−/− (18), IFN-{gamma}−/− (19), and IFN type I receptor−/− mice (20) were gifts from Dr. H. Mossmann (MPI, Freiburg, Germany), Dr. M. Kopf (ETH, Zurich, Switzerland), and Dr. R. Zinkernagel (University Hospital, Zurich, Switzerland), respectively. All mice had been backcrossed (more than eight times) to B6 mice, except the IFN type I receptor−/− mice, which were on a 129 background. Female or male mice were used at 8–16 wk of age. Mice were bred and kept in a conventional animal house facility.

Flow cytometry

Lymphocytes (105–106 in 100 µl) were stained in PBS containing 2% FCS, 0.1% NaN3, and Ab diluted to the working concentration at 4°C for 20 min. For PBL, 10 U/ml liquemin (Roche, Basel, Switzerland) was added to the staining buffer, and RBCs were lysed before analysis using FACS lysing solution (BD PharMingen, San Diego, CA). The following mAb specific for CD4 (clone GK1.5), CD8 (clone 53-6.7), CD44 (clone IM7), CD90.1 (clone OX-7), CD122 (clone TM-{beta}-1), KLRG1 (2F1), TCR V{alpha}2 (clone B20.1), and TCR V{beta}8 (clone MR5-2) were used. All mAb were purchased from BD PharMingen. The mAb were directly labeled with FITC, PE, or allophycocyanin or were biotinylated. In the latter case, cells were stained in a second step with PE-streptavidin. H-2Db MHC class I tetramers complexed with streptavidin-PE and containing the LCMV gp33 and NP396 peptide were prepared as previously described (21). For intracellular IFN-{gamma} staining, spleen cells (106) were first stimulated for 5 h with PMA (10−8 M) and ionomycin (5 µg/ml) in the presence of GolgiStop (BD PharMingen) in 24-well plates. Afterward, cells were surface-stained with anti-CD8-FITC, washed, permeabilized, and stained with PE-conjugated rat anti-mouse IFN-{gamma} mAb (clone XMG1.2; BD PharMingen). Bromodeoxyuridine (BrdU) incorporation was determined using the BrdU flow kit (BD Biosciences, Mountain View, CA) according to the manufacturer’s instructions. Cells were analyzed on a FACSort or a FACSCalibur flow cytometer (BD Biosciences).

In vivo treatment with anti-CD40 mAb, poly(I:C), LPS, or CpG oligodeoxynucleotides

Unless indicated otherwise, anti-CD40 treatment was performed by i.p. injection of 30 µg of anti-CD40 mAb (clone FGK-45) (22) or rat IgG1, {kappa} (clone R3-34; BD PharMingen) as isotype control on days 0 and 2. FGK-45 mAb was derived from concentrated hybridoma supernatant using protein-free hybridoma medium (Life Technologies/Invitrogen, Karlsruhe, Germany). For purification, affinity chromatography over protein G-Sepharose (Amersham Pharmacia Biotech, Freiburg, Germany) was used. The indicated doses of poly(I:C) (Sigma-Aldrich, Taufkirchen, Germany) and LPS (Alexis, Lausen, Switzerland) were also given i.v. on days 0 and 2. The phosphorothioate-modified oligodeoxynucleotide 1668 (5'-TCC ATG ACG TTC CTG ATG CT-3') (23) containing a CpG motif was purchased from TIB Molbiol (Berlin, Germany) and was injected once (20 nmol = 120 µg) in a volume of 200 µl i.v. For BrdU incorporation experiments, mice were fed BrdU-containing water (0.8 mg/ml), which was prepared and changed every 36 h.

Generation of LCMV-specific memory T cells

P14 memory T cells were generated as previously described (24). Briefly, spleen cells from Thy1.1+ P14 TCR-tg mice containing 105 TCR-tg+ (V{alpha}2+, V{beta}8+) T cells were transferred i.v. into sex-matched B6 recipient mice, followed by infection with 200 PFU of LCMV-WE. After 5–8 wk, mice were sacrificed, CD8+ cells were purified with BD IMag Anti-Mouse CD8a Particles-DM (BD PharMingen) from the spleen, and labeled with CFSE (Molecular Probes, Leiden, The Netherlands), and 2 x 106 TCR-tg P14 cells were transferred into B6, CD40−/−, or IL-15−/− hosts. Recipients were treated with 30 µg of anti-CD40 mAb 24 and 72 h after cell transfer. Cell division was visualized on day 6 after cell transfer by dilution of CFSE on Thy1.1+ P14 memory T cells in the spleen. LCMV-immune B6 mice were generated by i.v. injection of 200 PFU of LCMV-WE and were used 4–6 wk after infection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systemic stimulation via CD40 leads to a strongly increased number of CD44high memory phenotype T cells

Adult B6 mice were injected i.p. with 30 µg of agonistic anti-CD40 mAb (clone FGK-45) on days 0 and 2. On day 7, the percentage of CD44high CD4 and CD8 T cells was determined. As shown in Fig. 1, A and B, anti-CD40 treatment induced a marked increase in CD44high CD8 T cells in PBL and spleen. The increase in CD44high CD4 T cells was less pronounced in PBL, but was clearly evident in the spleen. Importantly, absolute numbers of CD44high CD4 and CD8 T cells were also increased in spleen and lymph nodes, but not in bone marrow, of CD40-treated mice (Fig. 1C). Dose-response experiments showed that two injections of 30 µg of anti-CD40 mAb were sufficient to induce a 3-fold increase in CD44high CD8 T cells, whereas two injections of 3 µg of mAb showed only a moderate effect (Fig. 2A). Kinetic analysis further revealed that the number of CD44high CD8 T cells peaked on day 8 after anti-CD40 treatment (Fig. 2B). Thus, injection of agonistic anti-CD40 mAb into normal mice induced a marked increase in the frequency of memory phenotype CD44high T cells.



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  		FIGURE 1. Increase in memory phenotype CD44high T cells after anti-CD40 treatment. A, B6 mice were injected with 30 µg of anti-CD40 mAb on days 0 and 2. PBL were analyzed immediately before (left) and 7 days after anti-CD40 treatment (right). B, Percentages of CD44high cells in CD4 and CD8 T cell subsets in PBL and spleen from untreated and anti-CD40-treated B6 mice. C, Absolute cell numbers of CD44high T cells in spleen, inguinal lymph nodes, and bone marrow of mice treated with anti-CD40 mAb (•) or with an isotype control mAb ({circ}) 7 days after treatment. Dots indicate values from individual mice.

 


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  		FIGURE 2. Dose response and kinetics of CD44high CD8 T cells after anti-CD40 treatment. A, B6 mice were injected on days 0 and 2 with the amount of anti-CD40 mAb indicated. The percentages of CD44high cells of CD8 T cells in PBL before and 7 days after treatment were determined. Dots indicate values from individual mice. B, Percentage CD44high cells of CD8 T cells in PBL on the days indicated after anti-CD40 treatment. Dots indicate values from individual mice.

 
Increase in KLRG1+ memory T cells after anti-CD40 mAb injection

We have shown previously that expression of the killer cell lectin-like receptor G1 (KLRG1) serves to identify Ag-experienced CD4 and CD8 T cells in mice (25) and humans (26). Therefore, KLRG1 was used as an additional marker to trace memory T cells. As shown in Fig. 3, injection of anti-CD40 mAb into B6 mice increased the frequency of KLRG1+ cells in the CD8 T cell compartment from 5–10 to ~40% in PBL and spleen. Similar to the analysis with CD44 as a memory marker, an increase in KLRG1+ cells in the CD4 T cell subset was observed, but again it was less prominent compared with CD8 T cells. Anti-CD40 treatment increased both the relative frequency and the absolute numbers of KLRG1+ T cells in spleen and lymph node, but not in bone marrow (Fig. 3C). Thus, anti-CD40 treatment also increased the frequency of memory phenotype T cells, defined by expression of KLRG1.



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  		FIGURE 3. Increase in KLRG1+ memory T cells after anti-CD40 treatment. A, PBL from B6 mice were analyzed immediately before (left) and 7 days after (right) injection of anti-CD40 mAb. B, Percentages of KLRG1+ cells in CD4 and CD8 T cell subsets of PBL and spleen from untreated and anti-CD40-treated B6 mice (day 7). C, Absolute cell numbers of KLRG1+ T cells in spleen, inguinal lymph nodes, and bone marrow of mice treated with anti-CD40 mAb (•) or with an isotype control mAb ({circ}) 7 days after treatment. Dots indicate values from individual mice.

 
Increase in IFN-{gamma}-producing CD8 T cells after anti-CD40 mAb injection

Rapid production of IFN-{gamma} is a hallmark of memory CD8 T cells (27). It was of interest to examine whether systemic CD40 triggering also increased the frequency of IFN-{gamma}-producing CD8 T cells. IFN-{gamma} production was determined by intracellular staining of spleen cells from anti-CD40-treated and untreated mice after a short stimulation (5 h) with PMA and ionomycin in vitro. Indeed, Fig. 4 illustrates that injection of anti-CD40 mAb into B6 mice increased the frequency of IFN-{gamma}-producing cells in the CD8 T cell compartment from ~20% in controls to ~50% in anti-CD40-treated animals. A similar conclusion was reached when absolute numbers of IFN-{gamma}+ CD8 T cells in anti-CD40-treated and control mice were compared (Fig. 4C). Thus, anti-CD40 treatment increased the frequency of memory CD8 T cells, defined by their phenotype as well as by their function to rapidly secrete cytokines.



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  		FIGURE 4. Increase in IFN-{gamma}-producing CD8 T cells after anti-CD40 treatment. A, Spleen cells from untreated and anti-CD40-treated B6 mice (day 7) were stimulated for 5 h with PMA and ionomycin. Afterward, cells were surface-stained with anti-CD8 mAb, followed by intracellular IFN-{gamma} staining. B, Percentages of IFN-{gamma}+ cells of CD8+ spleen cells from untreated and anti-CD40-treated B6 mice. C, Absolute cell numbers of IFN-{gamma}-producing CD8 T cells in spleen of anti-CD40-treated and untreated control mice 7 days after treatment. Dots indicate values from individual mice.

 
CD44low T cells do not differentiate into CD44high T cells upon anti-CD40 treatment

To determine whether memory-phenotype CD44high CD8 T cells could be derived from naive CD44low cells that up-regulate CD44 expression, P14 TCR-tg mice (line 318) were used. This transgenic line expresses a TCR specific for the gp33 epitope of LCMV on about half their CD8 T cells, whereas the remaining CD8 T cells express endogenous polyclonal Ag receptors. In P14 mice that had not undergone LCMV immunization, P14 TCR+ CD8 T cells, identified by gp33-Db tetramer staining, were naive and exhibited a CD44low phenotype. Upon injection with anti-CD40 mAb, CD44 expression of P14 TCR+ cells remained low, and the frequency of these cells decreased ~2-fold, whereas the percentage of memory phenotype CD44high CD8 T cells present in the compartment that express endogenous TCRs, increased ~5-fold (Fig. 5A). In absolute cell numbers, gp33-tetramer-negative cells markedly increased after anti-CD40 treatment, whereas the number of gp33-tetramer-positive cells remained constant (Fig. 5B). This result showed that injection of anti-CD40 mAb failed to induce CD44 up-regulation of naive CD44low cells. Furthermore, it indicated that the increase in CD44high CD8 T cells after Ab injection was due to a proliferative response of these cells.



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  		FIGURE 5. CD44low T cells do not differentiate into CD44high T cells upon anti-CD40 treatment. A, P14 TCR-tg mice specific for the gp33 epitope of LCMV were injected with anti-CD40 mAb (right) or with an isotype control mAb (left). The dot plots are gated on splenic CD8+ and show CD44 expression of cells bearing transgenic (Db-GP33+) and endogenous TCRs (Db-GP33−) determined by gp33-tetramer staining 7 days after treatment. B, Absolute cell numbers of gp33 tetramer+ and gp33 tetramer− cells in the spleen of P14 TCR-tg mice treated with anti-CD40 mAb ({blacksquare}) or with isotype control mAb ({square}) 7 days after treatment. Mean values (±SD) from three mice per group are shown.

 
Indirect stimulation of CD44high CD8 T cells by anti-CD40 treatment

A recent report demonstrated that CD8 T cells could receive CD4 help directly through CD40L-CD40 interactions (15). We failed, however, to detect CD40 expression on ex vivo-isolated CD44high CD8 T cells by flow cytometry (data not shown). Nonetheless, it was important to test whether the injected anti-CD40 mAb provided a direct stimulatory signal to memory CD8 T cells. Therefore, CD8+ spleen cells from B6.Thy1.1 mice were adoptively transferred to B6 or CD40-deficient hosts (both Thy1.2), followed by injection of anti-CD40 mAb. In anti-CD40-treated B6 recipient mice, a marked increase in both host (Thy1.1−) and donor (Thy1.1+) CD44high CD8 T cells was observed (Fig. 6, top). In contrast, the same treatment of CD40-deficient recipients failed to increase the frequency of CD44high CD8 T cells from B6.Thy1.1 donor mice (Fig. 6, bottom). As expected, the frequency of host CD44high CD8 T cells in CD40-deficient mice was not increased upon anti-CD40 treatment. Thus, this experiment indicated that the increase in memory phenotype CD8 T cells after anti-CD40 mAb injection was not due to direct stimulation, but was mediated by activation of host APC via CD40.



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  		FIGURE 6. CD44high CD8 T cells are not stimulated directly by anti-CD40 mAb. Purified CD8 T cells (4 x 106) from a B6.Thy1.1 spleen were adoptively transferred into B6 or CD40-deficient mice (both Thy1.2). The dot plots are gated on CD8+ T cells and show CD44 expression of host (Thy1.1−) and donor (Thy1.1+) cells before and 7 days after anti-CD40 treatment.

 
CD40-induced bystander proliferation of CD44high CD8 T cells is more vigorous than stimulation via poly(I:C), LPS, or CpG DNA

Previous studies have shown that agents that stimulate the innate immune system, such as poly(I:C) (28), LPS (29), and CpG DNA (30), are capable of inducing bystander proliferation of memory phenotype CD44high CD8 T cells. To compare the magnitude of the CD40-induced response to these stimuli, B6 mice were injected with anti-CD40 mAb, poly(I:C), LPS, or CpG oligodeoxynucleotides, and 7 days after treatment the percentage of CD44high CD8 T cells was determined. As shown in Fig. 7A, anti-CD40 treatment (two doses of 30 µg) of B6 mice raised the frequency of CD44high cells in the CD8 T cell compartment from ~20 to ~70%, whereas injection of poly(I:C) (two doses of 300 µg), LPS (two doses of 30 µg), or CpG DNA (one dose of 120 µg) increased these values only up to ~30%. A similar result was seen when proliferation of memory CD8 T cells was determined by BrdU incorporation. In these experiments B6 mice were injected with anti-CD40 mAb or with poly(I:C) and were given water containing BrdU for 3 days. The results showed that ~60% of total CD8 T cells incorporated BrdU after anti-CD40 treatment compared with ~20% after poly(I:C) injection. In addition, the data revealed that BrdU incorporation was restricted exclusively to the CD44high subset after stimulation with anti-CD40 mAb or with poly(I:C) (Fig. 7B).



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  		FIGURE 7. Potent proliferation of memory phenotype CD44high T cells by anti-CD40 treatment. A, B6 mice were injected with the indicated dose of anti-CD40 mAb, poly(I:C), LPS, or CpG oligodeoxynucleotides or were left untreated. Seven days after treatment, the percentage of CD44high cells of CD8+ PBL was determined. Mean values (±SD) for two to four mice per group are shown. B, B6 mice were injected with anti-CD40 mAb or poly(I:C) on days 0 and 2. Between days 4 and 7, mice were given BrdU in the drinking water. Spleen cells were harvested on day 8 and stained for CD8, CD44, and incorporated BrdU. The dot plots are gated on CD8 T cells.

 
Cytokine requirement of CD40-induced proliferation of CD44high CD8 T cells

Bystander proliferation of CD44high CD8 T cells, induced by injection of poly(I:C), LPS, or CpG DNA, has been shown to require the release of type I IFN, which then induces IL-15 synthesis that functions as a final effector cytokine (31). To determine the cytokine requirement of CD40-induced bystander proliferation, mice deficient in IL-12, IL-15, IFN-{gamma}, or type I IFN receptor were injected with anti-CD40 mAb, and the numbers of CD44high CD8 T cells were determined. Surprisingly, relative frequencies and absolute cell numbers of CD44high CD8 T cells were increased in all lines analyzed (Fig. 8).



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  		FIGURE 8. CD40-induced proliferation of memory phenotype CD44high CD8 T cells is independent of IL-12, IL-15, IFN-{gamma}, and type I IFN receptor. Mice deficient in the molecules indicated were injected with anti-CD40 mAb. A, The percentage of CD44high cells of CD8+ PBL before and 7 days after treatment is shown. Each line with the corresponding dots represents an individual mouse immediately before and after treatment. B, Absolute cell numbers of CD44high CD8 T cells in spleen of anti-CD40-treated and untreated control mice 7 days after treatment. Dots indicate values from individual mice.

 
The IL-2R {beta}-chain (CD122) is an important component of IL-2 and IL-15 cytokine receptors. In B6 mice, memory phenotype CD44high CD8 T cells consist of two subsets expressing CD122 at a high and a low level, whereas the CD122high subset is missing in IL-15−/− mice (32). Therefore, it was of interest to determine whether anti-CD40-induced proliferation involved both CD122high and CD122low memory cell subsets. Fig. 9, bottom, illustrates that injection of anti-CD40 mAb into B6 mice induced a similar increase in memory phenotype cells in both subsets. Moreover, anti-CD40 treatment of IL-15−/− mice induced CD122highCD44high cells that were missing almost completely in untreated mice (Fig. 9, top).



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  		FIGURE 9. Anti-CD40 treatment leads to an increase in both CD122high and CD122low memory phenotype CD44high CD8 T cells in B6 and IL-15-deficient mice. PBL from CD40-treated (left) and untreated (right) B6 and IL-15-deficient mice were analyzed. The dot plots display CD44 vs CD122 expression, gated on CD8 T cells.

 
The increase in CD44high CD8 T cells in IL-15−/− mice upon anti-CD40 treatment was unexpected when considering previous work about the role of IL-15 in bystander proliferation of memory phenotype CD8 T cells (32). Therefore, the IL-15 dependency was further examined in sensitive CFSE assays by adoptively transferring CFSE-labeled T cells to IL-15−/− and IL-15+/+ hosts. Anti-CD40-induced proliferation, manifested by a stepwise loss of CFSE, was restricted to CD44high T cells and was prominent in IL-15+/+ hosts. In IL-15-deficient hosts, cell division of CD44high T cells after anti-CD40 treatment was still evident compared with that in untreated controls, but it was reduced significantly compared with IL-15+/+ hosts (Fig. 10). Thus, these data indicated that CD40-induced bystander proliferation of CD44high memory phenotype CD8 T cells was partially dependent on IL-15.



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  		FIGURE 10. Cell division of adoptively transferred CD44high memory phenotype CD8 T cells from B6 mice in IL-15+/+ and IL-15−/− hosts. CFSE-labeled, purified CD8 T cells (2 x 106) from B6 mice were transferred into the hosts indicated. Recipient mice were left untreated or were injected with anti-CD40 mAb on days 1 and 3. Mice were sacrificed on day 6. The dot plots show CD44 expression vs CFSE gated on splenic CD8 T cells. The values indicated in the dot plots refer to the percentage of donor CD8 T cells that had divided one or more times.

 
Anti-CD40 treatment induces cell division of P14 TCR-tg memory T cells, but does not lead to an increase in cell numbers

To date, all experiments were performed with CD44high memory phenotype CD8 T cells. To extend our analysis to bona fide memory CD8 T cells with defined Ag specificity, P14 TCR-tg memory cells were generated in vivo by adoptive transfer of P14 cells into B6 mice, followed by LCMV infection (24). Bystander proliferation was examined by adoptively transferring CFSE-labeled P14 memory cells (Thy1.1) into uninfected hosts (all Thy1.2), followed by anti-CD40 mAb treatment. Cell division of P14 memory was visualized on day 6 after cell transfer in the spleen. Similar to memory phenotype CD8 T cells from B6 mice, anti-CD40 treatment of B6 hosts induced considerable cell division of P14 memory cells, manifested by a stepwise dilution of the CFSE label (Fig. 11, A and B, left). CD40-induced cell division of P14 memory cells was dependent on expression of CD40 by host APC (Fig. 11, A and B, middle) and was reduced in IL-15-deficient hosts (Fig. 11, A and B, right). In contrast to polyclonal CD44high memory phenotype CD8 T cells, anti-CD40 treatment did not lead to an accumulation of P14 memory T cells, and even fewer P14 memory T cells were recovered from the spleen of anti-CD40-treated B6 hosts (Fig. 11C). The decrease in P14 memory T cells in the spleen was unlikely to be due to redistribution, as only a few P14 memory T cells were recovered from liver (Fig. 11C, left, {circ}) or lung (data not shown).



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  		FIGURE 11. Cell division of P14 TCR-tg memory T cells induced by anti-CD40 treatment. A, Purified CD8+ P14 memory cells (Thy1.1+, 2 x 106) were labeled with CFSE and transferred to B6, CD40-deficient, or IL-15-deficient hosts (all Thy1.2+). Recipient mice were left untreated or were injected with anti-CD40 mAb on days 1 and 3. Cell division was measured on day 6 by dilution of CFSE on Thy1.1+ CFSE-labeled P14 T cells in the spleen. Values indicated in the dot plots refer to the percentage of P14 memory cells that had divided one or more times. B, The data indicate the percentage of P14 memory T cells that underwent one or more rounds of division (% cell division) in the indicated hosts, after injection of anti-CD40 mAb. Mean values (±SD) for two to five mice per group are shown. C, Absolute cell numbers of P14 memory T cells recovered from the spleen (•) or liver ({circ}) of anti-CD40-treated and untreated hosts 6 days after treatment. Dots indicate values from individual mice.

 
To extend these findings to a non-TCR-tg system, the effect of anti-CD40 treatment on bona fide CD8 memory T cells specific for the two immunodominant epitopes of LCMV, gp33 and NP396, was determined in LCMV-immune B6 mice. Nearly all gp33 and NP396 tetramer-positive memory CD8 T cells expressed CD44 at a high level (Fig. 12A). Similar to the adoptive transfer experiments with P14 TCR-tg memory T cells, anti-CD40 treatment decreased the relative frequency of gp33- and NP396-specific memory cells while increasing the portion of CD44high memory phenotype CD8 T cells (Fig. 12B). In absolute numbers, gp33- and NP396-specific memory T cells also decreased slightly in the spleen after anti-CD40 treatment, while the number of CD44high memory phenotype CD8 T increased ~2- to 3-fold. Taken together these data show that CD40 ligation in vivo induced cell division of both CD44high memory phenotype and bona fide LCMV-specific memory CD8 T cells. However, only CD44high memory phenotype, not LCMV-specific memory CD8 T cells, increased in absolute cell number in anti-CD40-treated mice.



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  		FIGURE 12. Anti-CD40 treatment decreases the frequency of LCMV-specific CD8 memory T cells in LCMV-immune B6 mice. A, LCMV-immune B6 mice were injected with 30 µg of anti-CD40 mAb on days 0 and 2. Dot plots show CD44 expression vs gp33- and NP396-tetramer staining gated on CD8 T cells of PBL analyzed immediately before (left) and 7 days after anti-CD40 treatment. B, Percentages of gp33 tetramer+, NP396 tetramer+, and CD44high cells of splenic CD8 T cells from untreated and anti-CD40-treated B6 LCMV-immune mice. Bars indicate mean values (±SD) from four mice per group. C, Absolute cell numbers of gp33 tetramer+, NP396 tetramer+, and CD44high T cells in spleen of anti-CD40-treated and untreated control mice 7 days after treatment. Bars indicate mean values (±SD) from four mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies have shown that agonistic anti-CD40 mAb function as a powerful adjuvant to amplify weak CD8 T cell responses to poorly immunogenic compounds. This was first demonstrated by three pioneering studies, which revealed that activation of APC via CD40 could replace the requirement for CD4 help in vivo (7, 8, 9). Subsequent studies built on this concept demonstrated that systemic CD40 activation could induce tumor eradication (33) and prevented reactivation of latent herpes virus (34) in mice. Other studies reported that CD40 signaling during a response to Ag (35) or superantigen (36) enhanced in vivo proliferation and delayed the clonal decline in Ag-specific T cells. Besides boosting weak CD8 T cell responses, anti-CD40 treatment has also been shown to convert T cell tolerance to T cell priming in several models (10, 11, 12, 13, 14). The debate, however, about whether anti-CD40 treatment provides a strong adjuvant effect, rather than mimicking CD4 T cell help, is not yet settled.

The present report differs from these studies in that the consequence of anti-CD40 treatment was examined in the absence of Ag delivery. Nonetheless, the possibility that the marked cell division of memory phenotype CD8 T cells upon anti-CD40 treatment was due to an Ag-driven response to environmental Ags needs to be considered. However, the finding that anti-CD40 treatment also induced cell division of P14 memory cells specific for the gp33 epitope of LCMV after transfer into noninfected hosts would argue against this idea. Thus, CD40 activation in the absence of deliberate Ag injection is likely to induce bystander, not Ag-driven, proliferation of memory T cells.

By using adoptive transfers of HY-TCR-tg cells into T cell-deficient hosts, Bourgeois et al. (15) demonstrated that CD8 T cells could receive CD40 help directly through CD40. This latter study further suggested that the adjuvant effect of agonistic anti-CD40 mAb could be due to direct CD40 stimulation of activated CD8 T cells expressing CD40. Our results demonstrate that CD40-induced proliferation of memory phenotype CD8 T cells was dependent on expression of CD40 by APC. Similarly, the boosting effect of anti-CD40 mAb on adoptively transferred OT-1 CD8 T cells induced by soluble OVA Ag has been shown to require CD40 expression on host APC (35). A recent study further demonstrated that CD40-deficient CD8 memory T cells specific for influenza develop and function normally in a CD40-sufficient environment (37). Thus, activation of CD8 T cells by CD40 signaling is indirect for bystander and also for certain Ag-induced responses. Further work will be required to determine the contributions of direct vs indirect CD4 help for CD8 T cells in other models.

Cytokine-driven bystander proliferation of memory phenotype CD8 T cells has been extensively studied by Sprent and colleagues (38). Their work showed that injection of either IFN-{alpha}{beta} or inducers of IFN-{alpha}{beta}, such as synthetic dsRNA poly(I:C) (28), LPS (29), or CpG DNA (30), caused increased turnover of CD44high CD8 T cells. IL-15, induced by IFN-{alpha}{beta}, was further identified as the final effector cytokine that mediates this effect (31). The effect of injection of anti-CD40 mAb differs from that seen with these stimuli in two repects. Firstly, the degree of memory cell proliferation induced, as determined by BrdU incorporation or the increase in CD44high CD8 T cells, was more vigorous. Secondly, IL-15 deficiency reduced, but did not completely abolish, cell division of both memory phenotype and bona fide Ag-specific memory T cells induced by CD40 ligation. The discrepancy between the partial IL-15 dependence in the CFSE experiments compared with the marked increase in CD44high memory phenotype T cells in IL-15−/− mice is most likely explained by the two different assays used. CFSE assays may be more suitable for revealing small differences in cycling capacity compared with simple determination of expanded cell numbers. CD44high CD8 T cells from B6 mice have been shown to consist of two subsets, expressing CD122 at either a high or a low level, and IL-15−/− mice were reported to lack the CD122high subset (32). Our data revealed that both CD122low and CD122high memory cell subsets were increased in IL-15−/− mice upon anti-CD40 treatment. This finding is different from poly(I:C) or LPS-induced memory cell proliferation, where only CD122high memory cells were reported to respond specifically to these stimuli.

It is well known that viral infections induce massive T cell proliferation. Even though the frequency of Ag-specific CD8 T cells can be strikingly high in certain virus models (21), extensive bystander proliferation of memory CD8 T cells is nonetheless observed (28). This effect has been largely attributed to the release of IFN-{alpha}{beta}, leading to IL-15-dependent proliferation of memory CD8 T cells (31). In this study we provide evidence that activation of APC via CD40 also induces bystander proliferation of memory phenotype CD8 T cells. Anti-CD40-induced bystander proliferation still occurred in the absence of IL-15, albeit at a reduced level. The types of APC that could mediate this effect are not yet defined. Besides B cells, DC, macrophages, parenchymal microglial, and endothelial/epithelial cells express CD40 (2). In the light of their well-established role for activation of T cells, CD40-stimulated DCs are likely candidates (39). CD40 ligation of DC is known to up-regulate costimulatory molecules and to induce various cytokines (40, 41, 42, 43). Interestingly, bystander proliferation of CD44high CD8 T cells can also be induced by injection of {alpha}-galactosylceramide, which activates NK T cells (44). Furthermore, treatment of mice with {alpha}-galactosylceramide has recently been shown to lead to NK T cell-mediated activation of DC (45), and this process has further been demonstrated to require CD40L/CD40 interactions (46). Thus, it is likely that bystander proliferation of memory CD8 T cells induced by {alpha}-galactosylceramide injection is also mediated by CD40-stimulated APCs.

Our results revealed that CD40 ligation induced cell division of both memory phenotype and bona fide memory CD8 T cells. However, only memory phenotype T cells increased in cell number after anti-CD40 treatment, whereas the number of bona fide memory cells remained constant or even decreased in the P14 transfer experiments. How can this finding be rationalized? CD44 has been commonly used as a marker to define Ag-experienced memory T cells. However, during homeostatic or lymphopenia-induced proliferation, naive CD8 T cells can also acquire a memory phenotype, as shown by up-regulation of CD44 and KLRG1, and by their ability to secrete cytokines (47, 48, 49, 50). CD44high memory phenotype CD8 T cells in normal naive mice have also been shown to exhibit characteristics similar to thymus-independent CD8 T cells (51). Thus, many memory phenotype CD8 T cells may not be true Ag-experienced memory cells. Along these lines, one may argue that only Ag-inexperienced memory phenotype T cells would have the ability to respond to CD40-activated APC by cell proliferation, whereas Ag-experienced memory T cells would undergo abortive cell division only. The idea that foreign Ag-experienced memory T cells respond differently to stimuli than CD44high memory phenotype T cells is also supported by a recent report by Welsh and colleagues (52) published during revision of this manuscript. This latter study demonstrated a decrease in the frequency of virus-specific memory CD8 T cells during reconstitution of lymphopenic environments. These data taken together with the present report caution the exclusive use of markers to define foreign Ag-experienced memory CD8 T cells.

In conclusion, our data show that activation of APC via CD40 induces a marked proliferative response of memory phenotype CD8 T cells in the absence of Ag delivery. Under physiological conditions, CD4 T cells induced by ongoing infections stimulate APC via CD40L-CD40 interaction. This stimulation amplifies the Ag-specific T cell response and, as the present report indicates, also induces bystander proliferation of memory phenotype CD8 T cells, which may have been generated during previous lymphopenias. This mechanism may help to maintain a diverse pool of CD8 T cells capable of responding to new pathogens.


   Acknowledgments
 
We thank Stephen Batsford and Peter Aichele for comments on the manuscript; Anton Rolink for the FGK-45 hybridoma; Horst Mossmann, Manfred Kopf, and Rolf Zinkernagel for providing knockout mice; and Theresa Treuer, Sonja Wagenknecht, Rainer Bronner, and Thomas Imhof for animal husbandry.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft (SFB620, Teilprojekt B2). Back

2 Current address: Division of Infectious Diseases, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143-0654. Back

3 Address correspondence and reprint requests to Dr. Hanspeter Pircher, Department of Immunology, Institute of Medical Microbiology and Hygiene, Hermann Herder Strasse 11, University of Freiburg, D-79104 Freiburg, Germany. E-mail address: pircher{at}UKL.uni-freiburg.de Back

4 Abbreviations used in this paper: DC, dendritic cell; BrdU, bromodeoxyuridine; CD40L, CD40 ligand; LCMV, lymphocytic choriomeningitis virus; SEA, staphylococcal enterotoxin A; tg, transgenic. Back

Received for publication June 11, 2003. Accepted for publication February 5, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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