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International Immunology Advance Access originally published online on April 18, 2006
International Immunology 2006 18(6):887-895; doi:10.1093/intimm/dxl025
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

Meiqing Shi and Jim Xiang

Research Unit, Saskatchewan Cancer Agency, Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, 20 Campus Drive, Saskatoon, Canada S7N 4H4

Correspondence to: J. Xiang; E-mail: jxiang{at}scf.sk.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.

Keywords: CD4+ T cells, CD8+ T cell memory, dentritic cells, flow cytometry


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
CD8+ CTLs play an important role in protection against virus, intracellular bacteria as well as tumors. The CD8+ T cell responses to antigens consist of the following phases: the proliferation of naive cells to produce large numbers of effector cells, the contraction of these effector populations into memory cells once antigen is cleared whereby only the effector cells which express IL-7R give rise to CD8+ memory T (Tm) cells (1, 2), and the long-term memory maintenance of these memory cells. Therefore, to achieve a rapid and efficacious response of CD8+ Tm cells after the second encounter with a pathogen or tumor antigen is the goal of many vaccination protocols.

Primary responses of CD8+ T cells to non-inflammatory immunogens are known to require CD4+ T cell help (3–5). CD4+ T cells express high levels of CD40L after activation, which can activate antigen-presenting cells (APCs) through CD40 signaling, licensing the APCs to stimulate CD8+ T cells (3–6) or directly activate CD8+ T cells through CD40–CD40L interaction between CD4+ and CD8+ T cells (7). By contrast, priming of naive CD8+ T cells to many viral and bacterial infections is usually (8–11), although not always (12), independent of CD4+ T cells. In these settings, instead of activation of APCs through CD40 signaling, pathogen-derived products, including CPG-containing DNA, lipopeptides and peptidoglycan, are directly recognized by Toll-like receptors (TLRs) on the APC surface, allowing CD40-independent APC activation (8, 13–15).

Development of CD8+ T cell memory has been extensively studied. However, the exact role of CD4+ T cells in the mounting of CD8+ T cell memory is still controversial (7, 9, 11, 12, 16–21). Increasing evidences have shown that CD4+ T cell help is essential for the development of a functional CD8+ T cell memory (7, 11, 19, 20). In particular, CD4+ T cells are required for the maintenance of CD8+ Tm cells following acute infection (21). More recently, it has been reported that CD8+ Tm cells, generated in the absence of CD4+ T cells, undergo apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand (18). However, in another study, it has appeared that CD8+ Tm cells generated without CD4+ T cell-mediated help during Listeria monocytogenes infection remain fully functional, although the frequency of CD8+ T cells during priming is significantly affected by the lack of help provided by CD4+ T cells (12). Also, mycobacterial vaccination induces protective immunity against pulmonary tuberculosis in the absence of CD4+ T cells through activation of CD8+ T cells (22). These data have led to the hypothesis that fully functional CD8+ T cell memory can be developed in the absence of CD4+ T cells under some circumstances as well, depending on the experimental system used including the infection agents and the maturation stage of APCs.

In the current study, we focused on the contribution of CD4+ T cells to the maintenance and expansion of CD8+ Tm cells derived from dendritic cell (DC) activation in vitro. We established a model system, in which CD8+ T cells derived from ovalbumin peptide (OVA)-specific TCR transgenic OT I mice (23), after priming with OVA protein-pulsed DC in vitro, were adoptively transferred into mice. Subsequently, the maintenance and expansion of the CD8+ Tm cells in the presence or absence of CD4+ T cells were monitored. In contrast to the previous report showing that CD4+ T cells are required for the maintenance (21) and expansion (12) of CD8+ Tm cells after acute infections, the results in our model system indicate that the maintenance and expansion of CD8+ Tm cells derived from in vitro DC activation are CD4+ T cell independent.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents, antibodies, cell lines and animals
OVA protein was obtained from sigma (St Louis, MO, USA). OVA I (SIINFEKL) was synthesized by Multiple Peptide Systems (University of Calgary, Calgary, AB). GolgiStop was purchased from BD-Biosciences (San Diego, CA, USA). The following mAbs were purchased from BD-Biosciences: FITC-labeled rat anti-mouse CD44 (Clone: IM7), FITC-labeled rat IgG2b (Clone: A95-1), biotin-conjugated anti-mouse CD45.1 (Clone: A20), biotin-conjugated mouse IgG2a (G155-178), PE-labeled rat anti-mouse IFN-{gamma} (Clone: XMG1.2), PE-labeled rat IgG1 (R3-34) and FITC-labeled rat IgG2a (R35-95). PE-labeled H-2Kb/OVA257–264 tetramer, FITC-labeled rat anti-mouse CD8 (Clone: KT15) and streptavidin-conjugated ECD (PE–Texas Red-X) were obtained from Beckman Coulter (San Diego, CA, USA). The highly lung metastatic C57BL/6 mouse melanoma BL6-10 and OVA-transfected BL6-10 (BL6-10OVA) cell lines were generated in our own laboratory (23). Female C57BL/6 mice were obtained from Charles River Laboratories (St Laurent, Quebec, Canada). The OVA-specific TCR transgenic OT I mice and CD4–/– mice on C57BL/6 background and C57BL/6.1 mice were purchased from the Jackson Laboratory (Bar Harbot, MA, USA). Homozygous OT I/B6.1 mice were generated by back-crossing the C57BL/6.1 mice onto the OT I mice on C57BL/6 background for three generations; homozygosity was confirmed by polymerase chain reaction according to Jackson laboratory's protocols. All mice were housed in the animal facility at the Saskatoon Cancer Center and treated according to Animal Care Committee guidelines of University of Saskatchewan. The mice were used at 8–10 weeks of age.

DCs
Bone marrow (BM)-derived DCs were generated as described previously (23). Briefly, BM cells were collected from the femorae and tibiae of normal or designated gene-deleted C57BL/6 mice. The BM cells were depleted of RBCs with 0.84% ammonium chloride and plated in DC culture medium [DMEM plus 10% FCS, granulocyte macrophage colony-stimulating factor (20 ng ml–1) and IL-4 (20 ng ml–1)]. On day 3, the non-adherent granulocytes and T and B cells were gently removed and fresh media was added. Two days later, the loosely adherent proliferating DC aggregates were dislodged and replanted. On day 6, the non-adherent DC cells were harvested. The DCs generated in this manner were mature DCs and displayed (i) typical morphologic features of DCs and (ii) expression of MHC class I (H-2Kb) and II (I-Ab) antigens, co-stimulatory molecules (CD40, CD80 and CD86) and adhesion molecules (ICAM-1, CD11b, and CD11c) (data not shown). These DCs were pulsed with 0.5 mg ml–1 OVA protein overnight at 37°C in the presence of LPS (1 mg ml–1), then washed extensively (23) and referred to as DCOVA (ovalbumin protein-pulsed bone marrow-derived dendritic cells).

OT I CD8+ T cells
Spleens were removed from OT I C57BL/6 mice and mechanically disrupted to obtain a single-cell suspension. The erythrocytes were lysed using 0.84% ammonium chloride. Naive T cells were enriched by passage through nylon wool columns. Naive OVA-specific CD8+ T cells were then purified by negative selection using anti-mouse CD4 (L3T4) paramagnetic beads (DYNAL Inc., Lake Success, NY, USA). To generate OVA-specific active CD8+ T cells, naive CD8+ T cells (2 x 105 cells ml–1) from OT I mice were stimulated for 3 days with irradiated (4000 rads) DCOVA (1 x 105 cells ml–1). After co-culture with DCOVA, the OVA-specific CD8+ T cells displayed CD25 and CD69, indicating they were highly activated (data not shown). These in vitro activated CD8+ T cells were separated by Ficoll-Paque (Sigma) density gradient centrifugation and further purified using CD8 microbeads (Milttenyi Biotec, Auburn, CA, USA).

Adoptive transfer, immunization and tumor cell challenge
Wild-type or CD4-deficient C57BL/6 mice were injected in tail veins with 5 x 106 in vitro activated OVA-specific CD8+ T cells diluted in PBS. In some cases, mice were challenged with DCOVA or tumor cells (BL6-10 or BL6-10OVA) later. Mice were monitored daily for survival after tumor cell challenge. In another set of experiment, C57BL/6 mice were injected in tail veins with DCOVA and then challenged with DCOVA later.

Tetramer staining
Blood was taken from the tail of mice. Spleens were removed from mice, and spleen cells were separated and depleted of erythrocytes. The blood samples or spleen cells were incubated with 10 µl PE-conjugated H-2Kb/OVA257–264 tetramer and 1 µl FITC-conjugated anti-CD8 mAb or FITC-conjugated anti-CD44 mAb for 30 min at room temperature. In some cases, the cells were additionally stained with biotin-conjugated anti-CD45.1, followed by washes and staining with streptavidin-conjugated ECD. The erythrocytes were then lysed using lysis/fixed buffer (Becoman counter). The cells were washed and analyzed by flow cytometry.

Intracellular cytokine staining
Cytokine expression was examined ex vivo in freshly isolated spleen cells. Spleens were removed from mice, and spleen cells were harvested and depleted of erythrocytes. The spleen cells were cultured for 5 h with 2 µM monensin (GolgiStop) in the presence of 2 µM OVA I. After culture, cells were stained with FITC–anti-CD8. In some cases, the cells were additionally stained with biotin-conjugated anti-CD45.1, followed by washes and staining with streptavidin-conjugated ECD. The cells were then fixed, and cell membranes were permeabilized in Cytofix/Cytoperm solution (BD-Biosciences) and stained with PE-labeled anti-IFN-{gamma}. Cells were then washed and analyzed using a FACSCaliber.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
CD8+ T cells activated by protein-pulsed DCs can become long-lived memory cells
We first investigated whether CD8+ T cells activated by protein-pulsed DCs can become long-lived memory cells. We established an in vitro model in which T cells from spleens of OT I mice were enriched by passage through nylon wool columns and then OVA-specific CD8+ T cells were purified from T cells by negative selection using anti-mouse CD4 (L3T4) paramagnetic beads. The OVA-specific CD8+ T cells were incubated with DCOVA for 3 days. After co-culture, the OVA-specific CD8+ T cells expressed CD25 and CD69, indicating they were highly activated (data not shown). The active CD8+ T cells were purified using CD8 microbeads and then adoptively transferred into naive mice. Six months later, OVA-specific CD8+ T cells could be still detected in the blood of mice by Kb/OVA tetramer staining (Fig. 1A). The tetramer-positive cells were shown to be CD44 positive (Fig. 1A), suggesting that they are CD8+ Tm cells (24, 25), rather than naive CD8+ T cells. To determine the ability of protein-pulsed DCs to induce CD8+ Tm cells in vivo, we immunized mice with BM-derived DCOVA and then challenged the mice 3 months later with DCOVA. As a control, naive mice were injected with DCOVA. Significant numbers of OVA-specific CD8+ T cells were detected in the peripheral blood of immunized mice rather than naive mice on day 3 after DCOVA challenge (Fig. 1B), indicating that a recall response occurred in the immunized mice after the challenge. Thus, CD8+ T cells activated in vivo by protein-pulsed DCs have the potential to become long-lived functional memory cells as well.


Figure 1
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  		Fig. 1 Effector CD8+ T cells activated in vitro or in vivo by protein-pulsed DCs can become long-lived memory cells. (A) OVA-specific CD8+ T cells (5 x 106) activated by DCOVA were adoptively transferred to C57BL/6 mice. Six months later, blood was taken from the mice and double staining for Kb/OVA tetramer and CD8 or CD44 was done to determine the percentage of OVA-specific cells in the total CD8+ population or CD44+ population (top numbers), as well as in the total PBMCs (bottom numbers in parentheses). (B) Naive C57BL/6 mice (nB6) or mice (iB6) immunized with 1 x 106 of DCOVA 3 months previously were challenged with 1 x 106 DCOVA, and double staining for Kb/OVA tetramer and CD8 was performed on day 3 after the challenge to determine the percentage of OVA-specific CD8+ T cells in the total CD8+ population (top numbers) or in the total PBMCs (bottom numbers in parentheses). The results presented are representative of two separate experiments with three mice per group.

 
CD8+ Tm cells derived from in vitro DC activation are fully functional
To ascertain whether the CD8+ Tm cells derived from in vitro BM-derived DC activation are functional, the mice were challenged with DCOVA or injected with PBS only as a control 3 months after adoptive transfer of in vitro activated OVA-specific CD8+ T cells, followed by analysis of proliferation and IFN-{gamma} secretion by CD8+ T cells 3 days later. As shown in Fig. 2(A), the frequency and absolute number of OVA-specific CD8+ T cells were significantly elevated following the challenge with DCOVA, indicating that CD8+ Tm derived from in vitro DC activation are functional (Fig. 2A).


Figure 2
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  		Fig. 2 Memory CD8+ T cells derived from in vitro DC activation are fully functional. (A) C57BL/6 mice were injected with 5 x 106 of OVA-specific CD8+ T cells activated by DCOVA, and then challenged 3 months later with 1 x 106 of DCOVA or PBS as control. OVA-specific CD8+ T cells in peripheral blood (upper panel) were examined by double staining for tetramer and CD8 on day 3 after the challenge. The frequency (lower left and middle panels) and absolute number (lower right panel) of IFN-{gamma}-producing CD8+ T cells were also enumerated ex vivo in spleen cultures on day 3 after the challenge. Double-positive cells are presented as the percentage in total CD8+ population (top numbers) and in total PBMCs or total spleen cells (bottom numbers in parentheses). (B) OVA-specific B6.1 mice-derived CD8+ T cells (5 x 106) activated by DCOVA were adoptively transferred to B6 mice. Three months later, mice were challenge with 1 x 106 of DCOVA. A triple staining for CD8, CD45.1 and tetramer or intracellular IFN-{gamma} was performed on day 3 after the challenge to detect OVA-specific CD8+ T cells in peripheral blood (upper panel) or spleen culture (lower panel). (C) Naive C57BL/6 mice (nB6/BL6-10OVA) or mice (tB6/BL6-10OVA) injected 3 months previously with 5 x 106 of OVA-specific CD8+ T cells activated by DCOVA were challenged with 0.5 x 106 of BL6-10OVA tumor cells. As control, mice (tB6/BL6-10) injected 3 months previously with 5 x 106 of OVA-specific CD8+ T cells activated by DCOVA were also challenged with 0.5 x 106 of BL6-10 tumor cells. Survival was monitored daily. The results presented are representative of two separate experiments with four mice (A and B) or five mice (C) per group.

 
To further confirm the fact that the increased frequency or absolute number of tetramer- or IFN-{gamma}-positive CD8+ T cells after DCOVA challenge was due to the expansion of CD8+ Tm cells rather than naive CD8+ T cells, C57BL/6 mice were adoptively transferred with active OVA-specific CD8+ T cells generated by incubation of CD8+ T cells from OT I mice in C57BL/6.1 background with DCOVA and challenged 3 months later with DCOVA. A triple staining for CD8, CD45.1 and tetramer or IFN-{gamma} showed that the tetramer- or IFN-{gamma}-positive CD8+ T cells display CD45.1 (Fig. 2B), indicating that the expansion of memory CD8+, but not naive CD8+ T cells, accounted for the elevated frequency or absolute number of OVA-specific CD8+ T cells in mice on day 3 after injection with DCOVA.

Next, we assessed the capacity of CD8+ Tm cells derived from in vitro DC activation to confer protective immunity. Mice were injected with in vitro activated OVA-specific CD8+ T cells, and then challenged 3 months later with OVA-expressing BL6-10OVA tumor cells. As a positive control, naive mice (without injection of active OVA-specific CD8+ T cells) were injected with BL6-10OVA. As expected, the control mice died within 30 days (Fig. 2C). In contrast, mice injected previously with activated OVA-specific CD8+ T cells were resistant to the challenge with BL6-10OVA and survived for >120 days. The specificity of the protection was confirmed by the observation that OVA-specific CD8+ Tm cells did not protect against BL6-10 tumor cells that did not express OVA, with all mice succumbing to the tumor cells within 30 days (Fig. 2C). These results strongly suggested that CD8+ Tm cells derived from in vitro DC activation have the ability to confer protective immunity.

Maintenance of CD8+ Tm cells derived from in vitro DC activation is independent of CD4+ T cells
As recent data suggest that CD4+ T cells are required for the maintenance of CD8+ Tm cells developed during acute infection (21), a question is raised regarding whether CD4+ T cells are essential for the maintenance of CD8+ Tm cells after priming with non-infectious antigens. The preceding results have demonstrated that CD8+ Tm cells derived from in vitro DC activation are fully functional. Thus, we set out to use this model system to examine the contribution of CD4+ T cells to the maintenance of CD8+ Tm cells derived from in vitro DC activation. To this end, equal numbers of OVA-specific CD8+ T cells activated by DCOVA were adoptively transferred into CD4-deficient or wild-type C57BL/6 mice, and Kb/OVA tetramer-positive cells were enumerated at various time points. Comparable numbers of CD8+ Tm cells were detected in the peripheral blood (frequency) or spleen (absolute number) of CD4-deficient and wild-type mice up to 90 days after the adoptive transfer (Fig. 3A), indicating that CD8+ Tm cells derived from in vitro DC activation can survive in the absence of CD4+ T cells.


Figure 3
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  		Fig. 3 CD4+ T cells are not required for the maintenance of functional memory CD8+ T cells derived from in vitro DC activation. OVA-specific CD8+ T cells (5 x 106) activated by DCOVA in vitro were adoptively transferred into wild-type or CD4-deficient C57BL/6 mice. The frequency (A: left panel) and absolute number (A: right panel) of OVA-specific CD8+ T cells in peripheral blood or spleen were enumerated at various time points. In another set of experiments, mice were injected with 5 x 106 of OVA-specific CD8+ T cells activated by DCOVA in vitro, and then challenged 3 months later with 1 x 106 of DCOVA. Double staining for tetramer and CD8 were performed to detect OVA-specific CD8+ T cells in peripheral blood on day 0 (B) and 3 (C: upper panel) after the challenge. The frequency (C: lower left and middle) and absolute number (C: lower right) of IFN-{gamma}-producing CD8+ T cells were also quantitated ex vivo in spleen culture on day 3 after the challenge. Double-positive cells are presented as the percentage in total CD8+ population (top numbers) and in total PBMCs or total spleen cells (bottom numbers in parentheses). The results presented are representative of two separate experiments with five mice per group.

 
It is necessary to examine whether the CD8+ Tm cells maintained in CD4-deficient mice are functional. Therefore, CD4-deficient or wild-type mice were injected with active OVA-specific CD8+ T cells, and then challenged 3 months later with DCOVA. The recall response of the CD8+ Tm cells maintained either in the absence or presence of CD4+ T cells was quite similar, as evidenced by the fact that both of them proliferate and secrete IFN-{gamma} and that almost equal frequency as well as absolute number of OVA-specific CD8+ T cells were detected in CD4-deficient or wild-type mice (Fig. 3B and C). These results suggest that the CD8+ Tm cells maintained in the absence of CD4+ T cells are fully functional.

CD4+ T cells are not essential for the expansion of CD8+ Tm cells
We next determined whether CD8+ Tm cells can expand without CD4+ T cell help. In vitro activated OVA-specific CD8+ T cells were adoptively transferred into C57BL/6 mice. Three months later, the mice were challenge with DCOVA. To deplete CD4+ T cells, mice were treated on days –6, –3 and 0 with 0.5 mg of anti-CD4 mAb GK1.5 or control IgG. FACS analysis demonstrated that >99% of CD4+ T cells in the mice treated with anti-CD4 mAb were eliminated (data not shown). As shown in Fig. 4, the CD8+ Tm cells expanded equivalently after treatment with either anti-CD4 mAb or control IgG, as assessed by tetramer staining and intracellular IFN-{gamma} staining showing comparable frequency and absolute number of OVA-specific CD8+ T cells. These results demonstrated that CD4+ T cells are not essential for the expansion of CD8+ Tm cells.


Figure 4
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  		Fig. 4 Expansion of memory CD8+ T cells is independent of CD4+ T cells. Mice were injected with 5 x 106 of OVA-specific CD8+ T cells activated by DCOVA in vitro, and then challenged 3 months later with 1 x 106 of DCOVA. To deplete CD4+ T cells, mice were treated on days –6, –3 and 0 with 0.5 mg of anti-CD4 mAb (GK1.5) or control IgG. Recall responses of memory CD8+ T cells were assessed by tetramer staining for OVA-specific CD8+ T cells in peripheral blood (upper panel) and intracellular staining for IFN-{gamma}-producing CD8+ T cells in spleen cultures (lower panel). OVA-specific CD8+ T cells are presented as the percentage in total CD8+ population (upper panel: top numbers) or in total PBMCs (upper panel: bottom numbers in parentheses). IFN-{gamma}+CD8+ T cells are presented as the percentage in total CD8+ population (lower panel: top numbers), in total spleen cells (lower panel: bottom numbers in parentheses) as well as the absolute number in the whole spleen (lower right panel). The results presented are representative of two separate experiments with three mice per group.

 
CD8+ Tm cells maintained in the absence of CD4+ T cells have the capacity to confer protective immunity
The preceding results have indicated that CD8+ T cells, after activation by BM-derived DCs in vitro, can give rise to functional long-lived memory cells in the CD4+ T cell-absent environments. The question was raised regarding whether CD8+ Tm cells maintained in the absence of CD4+ T cells are able to confer protective immunity. To this end, CD4-deficient as well as wild-type C57BL/6 mice were injected with equal number (5 x 106) of OVA-specific CD8+ T cells activated by BM-derived DCs in vitro, and then challenged 3 months later with OVA-expressing BL6-10OVA tumor cells. As a positive control, naive CD4-deficient and wild-type C57BL/6 mice that did not received transferred CD8+ T cells were inoculated with BL6-10OVA. Also, wild-type mice injected with active OVA-specific CD8+ T cells were challenged with BL6-10 tumor cells. We enumerated the OVA-specific CD8+ T cells on day 0 and 4 following the tumor cell challenge and monitored daily the survival of mice. As expected, comparable numbers of OVA-specific CD8+ T cells at day 0 (before challenge) were detected in CD4-deficient or wild-type mice injected with active CD8+ T cells 3 months previously (Fig. 5A), indicating that the mice started with equal number of OVA-specific CD8+ T cells before tumor cell challenge. Actually, we have shown above that both frequency and absolute number of CD8+ Tm cells in wild-type and CD4–/– mice up to 3 months after adoptive transfer are comparable (Fig. 3A and B). The frequency of OVA-specific CD8+ T cells are significantly enhanced after tumor cell challenge in both wild-type and CD4-deficient mice on day 4 (Fig. 5A), further supporting the notion that DC activation-derived CD8+ Tm cells maintained in the absence of CD4+ T cells can be fully expanded. As anticipated, the naive mice succumbed to the tumor cells within 30 days (Fig. 5B). Of interest, the mice that had been injected with active CD8+ T cells 3 months previously, no matter whether they have CD4+ T cells or not, exhibited protection and long-term survival for >120 days after tumor challenge (Fig. 5B). The specificity of the protection was confirmed by the observation that the wild-type mice injected previously with active OVA-specific CD8+ T cells succumbed to BL6-10 tumor cells that did not express OVA within 30 days (Fig. 5B). We also found that all mice succumbed to tumor challenge have lung metastatic tumor colonies (data not shown). In contrast, neither wild-type nor CD4-deficient mice previously receiving transferred CD8+ T cells developed any lung metastatic tumor colonies during the observation period (data not shown). In addition, we also performed a titration experiment and found that injection of the OVA-specific CD8+ T cells as low as 0.05 x 106 cells still can protect both wild-type and CD4–/– mice from the tumor cell challenge 3 months later (data not shown). Collectively, these results provide evidence that CD8+ Tm cells maintained in the absence of CD4+ T cells are able to confer fully protective immunity against tumor cell challenge.


Figure 5
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  		Fig. 5 Memory CD8+ T cells maintained in the absence of CD4+ T cells confer protective immunity against challenge of tumor cells. OVA-specific CD8+ T cells (5 x 106) activated by DCOVA in vitro were adoptively transferred in wild-type or CD4-deficient mice. Three months after transfer, the wild-type (tWT/BL6-10OVA) and CD4-deficient (tCD4KO/BL6-10OVA) mice as well as naive wild-type (nWT/BL6-10OVA) or naive CD4-deficient (nCD4KO/BL6-10OVA) mice were challenged with 0.5 x 106 of BL6-10OVA tumor cells. As control, wild-type mice (tWT/BL6-10) receiving adoptively transferred CD8+ T cells 3 months previously were also challenged with BL6-10 tumor cells. OVA-specific CD8+ T cells in peripheral blood were quantitated on day 0 and 4 after challenge by tetramer staining (A). Mice were also monitored daily for survival (B). The results presented are representative of two separate experiments with five mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
CD8+ T cells are critically involved in protection against virus, intracellular bacteria and tumors. The understanding of the interplay among APCs, CD4+ Th cells and CD8+ T cells leading to the activation and differentiation of CD8+ T cells will assist the development of future vaccination strategies. In the absence of external stimuli, APCs exist in a resting state in which they have only a limited ability to prime naive CD8+ T cells. Thus, maturation and activation of APCs is one of the prerequisites for priming of CD8+ T cells by APCs. It is generally accepted that APCs can be activated by at least two different pathways (3–6, 8–11). In the CD8+ T cell response to non-infectious antigens such as the minor histocompatibility, peptide-pulsed DCs and tumor antigens, CD4+ T cells are required to activate or ‘license’ APCs through CD40 ligation so that the APCs are capable of priming naive CD8+ T cells (3–6). In contrast, during many viral or bacterial infections, including the systemic response to Listeria, APC activation is independent of CD40–CD40L interaction (8–11). Instead, APCs become activated through TLR recognition of pathogen-derived products such as flagellin, lipopeptides, peptidoglycan and CpG DNA (8, 13–15). Therefore, the primary response of CD8+ T cells can be characterized as being either dependent or independent of CD4+ T cells, depending on the type of antigens delivered. In contrast to priming, experimental data from several groups have led to the conclusion that CD4+ T cell-mediated help are required for mounting of stable, protective CD8 memory, particularly during acute infections (11, 19, 20). However, it had been poorly understood whether CD4+ T cells are required during priming or afterward for mounting fully functional CD8 memory until recent findings demonstrated that CD4+ T cells are essential for the maintenance, not programming, of memory CD8+ T cells after acute infection (21). In this study, we have examined whether CD4+ T cells are essential for the maintenance and expansion of functional CD8+ Tm cells derived from in vitro BM-derived DC activation.

First of all, we developed a model system in which OVA-specific CD8+ T cells were activated by OVA protein-pulsed DCs in vitro in the absence of OVA-specific CD4+ T cells. As CD8+ Tm cells derived from in vitro DC activation are fully functional, we then used this model system to assess the importance of CD4+ T cells for the maintenance of CD8+ Tm cells. Our results demonstrated that CD4+ T cells were dispensable for the maintenance of CD8+ Tm cells derived from in vitro DC activation as evidenced by the fact that neither frequency nor absolute number of OVA-specific CD8+ T cells before or after DCOVA challenge showed significant difference between CD4-deficient mice and wild-type mice until 90 days after adoptive transfer of OVA-specific CD8+ T cells primed in vitro. These results have been further confirmed using MHC II-deficient mice (data not shown). Thus, an important issue arising from this finding is why CD4+ T cells are required for the maintenance of CD8+ Tm cells after acute infections (21), whereas maintenance of CD8+ Tm cells generated by stimulation of protein-pulsed DCs is independent of CD4+ T cells. It has been reported that the duration of interaction between T cells and DCs is relatively short in vivo (26, 27). Thus, one possible explanation for the difference is that the ‘communication’ or ‘signaling’ of CD8+ T cells with DCs has been intensified through enhanced opportunity of the direct contact between CD8+ T cells and DCs, as well as the increased duration of contact between CD8+ T cells and DCs in our in vitro model system, perhaps allowing the memory CD8+ T cell precursors to obtain much stronger signals from DCs as they acquired during acute infection. Indeed, it has been reported that limiting the infectious period diminishes CD8+ T cell memory development, although the size of primary CD8+ T cell responses was not affected (28). Second, during acute infections, pathogen-derived products induce inflammation and trigger secretion of a great amount of cytokines, which might have the potential to influence the signal transduction between APCs and CD8+ T cells during the priming. Alternatively, some pathogens develop immune evasion mechanisms by suppressing maturation and activation of DCs (29, 30), which might undermine the signaling between APCs and CD8+ T cells. In any case, this suggests that the intrinsic cellular differences must have existed between the CD8+ Tm cell precursors formed during in vivo acute infections and those generated by stimulation with in vitro protein-pulsed DCs. Actually, recent findings have suggested that effector CD8+ T cells primed by peptide-pulsed DCs become memory cells in a very short period (within 4–6 days) as compared with those primed during acute infection (31). Also the selective expression of IL-7R identifies effector CD8 T cells that give rise to memory cells after priming with L. monocytogenes (1), rather than after priming by peptide-pulsed DCs (32). These findings support the notion that a difference exists between CD8+ Tm cell precursors generated after DC stimulation and those induced during acute infection. Then, the questions are raised regarding what is the difference between CD8+ Tm cell precursors formed during in vivo acute infections and those generated by stimulation with in vitro protein-pulsed DCs, and what are the survival factors for memory CD8+ T cells generated by stimulation with protein-pulsed DCs. It is of interest to note that IL-7 and IL-15 have been suggested to play a role in the maintenance of naive and Tm cells (33–37). Whether these cytokines contribute to the maintenance of the CD8+ Tm cells generated by in vitro simulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed DCs remains to be clarified.

Our results showing that CD4+ T cell help is not required for recall responses of CD8+ Tm cells are consistent with earlier studies using DC immunization (17) or bacterial infection (11), but contrast a recent study related to bacterial infections (12), which demonstrated that CD4+ T cells and CD40L are required for gaining an optimal recall response of CD8+ Tm cells. The discrepancy among these studies probably attributes the antigen density presented by DCs in different models used. It was reported that CD8+ Tm cells proliferated at lower antigen concentration than did naive CD8+ T cells, and were less dependent on co-stimulatory molecules (38–40). Previous data (40) and our unpublished results have demonstrated that co-stimulatory molecules are required to gain an optimal recall response of CD8+ Tm cells when antigen concentration is low, although the recall response is not affected by the absence of co-stimulatory molecules when the antigen concentration reaches a high plateau. Therefore, it is conceivable that, in some experimental model, an interaction between CD4+ T cells and APCs is essential to enhance expression of co-stimulatory molecules by DCs when the antigen density presented by APCs is not high enough.

Taken together, we demonstrated that CD8+ Tm cell precursors induced by in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed DCs are able to become fully functional CD8+ Tm cells. The maintenance and expansion of these CD8+ Tm cells are independent of CD4+ T cells. Thus, our findings identify a new model in which CD8+ Tm cells can be maintained and expanded in the absence of CD4+ T cells, implying that individuals with defective function of CD4+ T cells such as patients with HIV still might be vaccinated using properly designed protocols.


   Acknowledgements
 
This work was supported by a research grant (MOP 67230) from the Canadian Institute of Health Research to J.X. We are grateful to Mark Boyd for his assistance with the FACS analyses. The authors have no conflicting financial interest.


   Abbreviations
 
APC, antigen-presenting cell
BM, bone marrow
DCOVA, ovalbumin protein-pulsed bone marrow-derived dendritic cell
OVA, ovalbumin
TLR, Toll-like receptor
Tm cell, memory T cell

   Notes
 
Transmitting editor: T. Hirano

Received 23 November 2005, accepted 13 March 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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