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The Journal of Immunology, 2006, 177: 6730-6737.
Copyright © 2006 by The American Association of Immunologists, Inc.

Human Bone Marrow: A Reservoir for "Enhanced Effector Memory" CD8+ T Cells with Potent Recall Function1

Xiaoyu Zhang*, Haidong Dong{dagger}, Wei Lin*, Stephen Voss{ddagger}, Lucinda Hinkley{ddagger}, Melissa Westergren{ddagger}, Guoliang Tian§, Daniel Berry, David Lewellen, Richard G. Vile||, Lieping Chen#, Donna L. Farber** and Scott E. Strome2,*

* Department of Otorhinolaryngology–Head and Neck Surgery, University of Maryland School of Medicine, Baltimore, MD 21201; {dagger} Department of Immunology, Mayo Clinic College of Medicine, Rochester MN 55905; {ddagger} Department of Otorhinolaryngology–Head and Neck Surgery, Mayo Clinic College of Medicine, Rochester MN 55905; § Division of Biostatistics, University of Maryland Greenebaum Cancer Center, Baltimore, MD 21201; Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Rochester MN 55905; || Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester MN 55905; # Department of Dermatology, Johns Hopkins University, Baltimore, MD 21231; and ** Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of human bone marrow (BM) CD8+ T cells in the immune response to viral Ags is poorly defined. We report here the identification and characterization of a functionally enhanced effector memory CD8+ T cell population (TEM) in the BM of patients undergoing total joint replacement for osteoarthritis. These BM-derived TEM differ strikingly from correlate cells in peripheral blood (PB), expressing elevated levels of CD27, HLA-DR, CD38, CD69, and unique patterns of chemokine receptors. Interestingly, while BM TEM have low levels of resting perforin and granzyme B, these molecules evidence profound up-regulation in response to TCR stimulation resulting in enhanced cytotoxic potential. Moreover, compared with the TEM subset in PB, BM CD8+ TEM cells demonstrate a more vigorous recall response to pooled viral Ags. Our results reveal that human BM serves as a repository for viral Ag-specific TEM with great therapeutic potential in vaccine development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Memory T cells are defined by their capacity to mount a rapid response to secondary antigenic challenge (1), and their ability to maintain homeostatic proliferation in the absence of antigenic stimulation (2, 3). Recently, memory T cells have been categorized into effector memory (TEM),3 CD45RO+CD62LlowCCR7low, and central memory (TCM) CD45RO+CD62LhighCCR7high subsets based on both their homing characteristics and effector functions (4). Although TCM are primarily distributed in lymphoid tissue, TEM can traffic to and reside in diverse nonlymphoid sites, including lung, liver, and intestine (5).

Recent evidence suggests that residence in a particular anatomic compartment, e.g., bone marrow (BM) might confer distinct phenotypic or functional properties on the indigenous memory T cells. Early studies in tumor models found that the presence of live tumor cells in the BM was associated with systemic protection from tumor-specific challenge. In addition, tumor cells in BM were controlled in a dormant state by CD8+ T cells (6, 7). Correlate data from patients with breast cancer demonstrated that, following adoptive transfer, primed T cells from the BM, but not the peripheral blood (PB), could effectively treat autologous breast cancer xenografts in NOD/SCID mice (8, 9, 10). Similar findings have recently been described for pancreatic tumors (11), myeloma (12), and melanoma (13). Finally, in mouse viral infection models, the BM was found to harbor virus-specific memory CD8+ T cells that could mediate protection from lymphocytic choriomeningitis virus infection when adoptively transferred into naive SCID hosts (14), and virus-specific memory CD8+ T cells were also produced in BM in response to vesicular stomatitis virus infection (5).

These results suggest that the BM may harbor a subset of memory CD8+ T cells that could be particularly useful in immunotherapy. However, the nature of these cells and their role in the immune response to viral infection in human remains poorly defined. To address these issues, we sought to investigate the phenotypic signature and effector function of memory T cells in human BM, isolated from a cohort of patients with degenerative joint disease. Our results define a distinct population of CD8+ TEM cells which exist in the BM of patients with OA, and maintain a unique phenotype, expressing high levels of CD27, CD28, CD38, CD69, and HLA-DR. These cells exhibit a profound recall response to viral Ags and display unique patterns of perforin and granzyme B regulation in response to TCR stimulation. Our findings provide a critical step for understanding the role of BM memory cells in the immune response to viral Ags and in maintaining memory T cell homeostasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Specimen procurement

Entry criteria for this study included a diagnosis of OA requiring a total joint arthroplasty, and the absence of known immunosuppression, autoimmune disease, and cancer (other than nonmelanoma skin cancer). The protocol was approved by both the Mayo Clinic College of Medicine Institutional Review Board and the University of Maryland, School of Medicine Institutional Review Board. All patients signed written informed consent. Approximately, 50 ml of PB and 10 ml of BM were obtained at the time of surgery, while the patients were under anesthesia. The BM was collected directly from the medullary canal of the femur during bone preparation for total hip arthroplasty. The marrow was collected with a suction, syringe, or receptacle as it was forced out of the canal with reaming or broaching instruments.

Cell preparation

Mononuclear cells from PB and BM were isolated by Ficoll-Paque (Amersham Biosciences). The cells were used directly for flow cytometry, cultured with Ags and mitogens, or cryopreserved for future experimental analysis. For isolation of CD8+ TEM and TCM cells, mononuclear cells from PB and BM were stained with mouse anti-human mAbs against CD8, CD45RO, and CD62L. CD8+ TEM cells and TCM cells were isolated using a FACSVantage SE with CellQuest Pro software (BD Biosciences). Purity was evaluated by post-sort flow cytometry. Sorted cells exhibited a purity of ≥98%.

Isolated CD8+ TEM and TCM cells were expanded using a rapid expansion protocol described previously (15). Briefly, T cells were cultured with 30 ng/ml anti-CD3 Ab (OrthoClone OKT3; Ortho Diagnostics) and 1000 Ug/ml IL-2 (Proleukin; Chiron) in the presence of irradiated (30 Gy), allogeneic PBMC as feeder cells at a concentration of 1 x 106/ml. After 14 days, cells were harvested and used for experiments or cryopreserved. In vitro-expanded TEM and TCM cells were only used in the Ab-redirected cytotoxicity assay. Fresh or frozen PBMCs and BM cells (BMCs) were used in the other experiments.

Synthetic CMV, EBV, and influenza virus (CEF) peptides

A panel of twenty-three 8- to 11-mer CEF peptides (16) was synthesized by Mayo Protein Core Facility. Purity was determined by HPLC and mass spectrophotometry. The peptides were dissolved in DMSO at 10 mg/ml and a peptide pool was made at a concentration of 100 µg/ml for each peptide.

HLA typing

Genomic DNA was extracted from whole blood of patients using QIAamp DNA blood mini kit (Qiagen). HLA class I typing was performed with Biotest HLA-A SSC kit (Biotest).

Abs and peptide MHC class I pentamer

PerCP-labeled anti-CD3, allophycocyanin-labeled anti-CD8, PE-, or FITC-labeled anti-CD45RO, PE-Cy5-labeled anti-CD62L, PE-labeled anti-CD25, CD38, CD69, HLA-DR, CCR5, CCR7, and CXCR1, FITC-labeled anti-CD45RA, CD57, and isotype control mAbs were purchased from BD Pharmingen. PE-labeled anti-CD27, CD28, and CXCR4 were obtained from eBioscience. PE-labeled anti-human IL-7R{alpha} and goat anti-human IL-15R{alpha} were purchased from R&D Systems. PE-labeled donkey anti-goat IgG were obtained from Jackson ImmunoResearch Laboratories. Allophycocyanin-labeled HLA-A0201/NLVPMVATV pentamer was purchased from Proimmune.

Flow cytometry

PBMCs and BMCs were incubated with the mAbs to cell surface molecules for 30 min at 4°C, washed in PBS with 0.5% BSA (pH 7.0), and fixed in PBS with 2% paraformaldehyde. For IL-15R{alpha}, cells were first stained with specific Ab, washed, and treated with PE-labeled secondary Ab. Subsequently, IL-15R{alpha}-stained cells were stained with anti-CD8, anti-CD45RO, and anti-CD62L mAbs. Labeled cells were analyzed on a FACSCalibur with CellQuest software or on a LSRII with FACSDiva Software (BD Biosciences). Peptide HLA pentamer staining was performed according to the manufacturer’s protocol. A total of 1 x 106 cells was incubated with 10 µl of allophycocyanin-labeled pentamer for 45 min at 4°C, washed in PBS for twice, followed by incubation with FITC-labeled anti-CD8 mAb for 30 min. At least 5 x 105 events were collected for each sample.

CD107a and intracellular IFN-{gamma} staining

CD107a staining was performed as recently described with a few modifications (17). Lymphocytes were stimulated in vitro with 2 µg/ml CEF peptides or mitogens in the presence of monensin A (Sigma-Aldrich) and FITC-conjugated mAbs for CD107a or isotype control (BD Pharmingen) for 5 h. Cells were then harvested, washed, and stained for other surface molecules or fixed and permeabilized and stained for intracellular IFN-{gamma}. No addition of peptides was included as a negative control for spontaneous CD107a expression and/or cytokine production.

To assess the production of IFN-{gamma}, Ag or mitogen-stimulated lymphocytes were harvested and stained with mAbs to cell surface Ags, and subsequently fixed and permeabilized in 250 µl of BD Cytofix/Cytoperm solution for 20 min at 4°C. Cells were then incubated in 50 µl of BD Perm/Wash solution containing PE-conjugated anti-IFN-{gamma} Ab or appropriate isotype control for 30 min at 4°C, washed twice, and fixed in paraformaldehyde.

Cytotoxicity assay

CD8+ TEM and TCM cells were used for an Ab-redirected cytotoxicity assay 14 days after anti-CD3 stimulation. Serial dilutions of T cells were incubated in triplicate with 1 x 104 FcR-expressing P815 target cells in the presence of 0.5 µg/ml anti-CD3 and 100 U/ml IL-2. After 4 h, plates were centrifuged and supernatants were collected. The levels of lactate dehydrogenase were determined using a Roche cytotoxicity detection kit according to the manufacturer’s instructions.

Statistical analysis

To assess differences in phenotype, IFN-{gamma} production and CD107a expression between BM and PB-derived CD8+ TEM and TCM subsets, we first performed QQ plots. These plots showed that the normality assumptions were violated so that the paired t test could not be used. Therefore, the Wilcoxon matched pairs signed rank sum test was used to calculate the p values. The significance level was set at 5%. Calculations were implemented with S-PLUS (Insightful; Academic Site Edition version 6.2.1). The Student’s t test was used to assess the statistical significance in the cytotoxicity assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Demographic Information

We obtained written informed consent from 22 patients with a diagnosis of osteoarthritis (OA) who were scheduled to undergo total joint replacement. All participants were Caucasian with no known diagnosis of cancer (other than nonmelanoma skin cancer), nor a history of autoimmune disease. Participants ranged in age from 47 to 75 with an average age of 62.6. Twelve of these patients were male and 10 were female. Not all samples were used in each experiment.

The T cell fraction in BM contains a high percentage of CD8+ cells

To investigate the nature and distribution of viral-specific memory CD8+ T cells, we first defined the cellular composition of paired BM and PB lymphocytes from patients with OA. CD3+ T cells comprised of 36.55% of lymphocytes in BM, which was smaller than the 63.98% observed in PB. In BM, 48.16% of CD3+ cells were CD8+, compared with only 26.03% in PB. Thus, the ratio of CD4+/CD8+ was decreased from 2.94 in PB to 1.11 in BM (Table I). The expression of CD45RO and CD45RA were similar in both the CD4 and CD8 subset in PB and BM. In comparison to paired PB samples, the proportion of NK cells was decreased in the BM lymphocyte population, while the percentage of CD19+ cells was increased. These data indicate that the T cell fraction in BM is composed of a high percentage of CD8+ cells which express CD45RA and CD45RO at levels similar to correlate cells in the PB.


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  		Table I. Cellular composition of mononuclear cells in PB and BMa

 
BM contains an increased population of CD8+ TEM cells vs PB

Our analysis of the total component of memory T cells in the BM and PB revealed significantly higher levels of CD38 and CD69 expression in BM CD8 T cells, with reduced levels of CD62L (data not shown). Because CD62L alone can distinguish CD8+ TEM and TCM (18, 19, 20), the low expression of CD62L suggested that these activated cells might be from the TEM subset. Therefore, we evaluated the TCM and TEM components of memory T cells in paired BM and PB samples. Although both CD8+ TCM and TEM subsets were present in the PB, the predominant subset represented in BM-derived memory CD8+ T cells was TEM (Fig. 1).


Figure 1
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  		FIGURE 1. BM contains an increased population of CD8+ TEM cells vs PB. PBMCs and BMCs were isolated from seven patients with OA and CD8+ T cells were analyzed for the expression of CD45RO and CD62L by four-color flow cytometry. A, A representative sample is shown as a plot of CD45RO expression vs CD62L expression within the CD8+ T cell population. B, Percentage of TCM and TEM from BM and PB.

 
BM and PB CD8+ TEM cells are phenotypically distinct

To assess the phenotypic characteristics of CD8+ TEM cells in BM, we stained freshly isolated PBMCs and BMCs from seven patients with a panel of T cell differentiation and activation-associated markers using four-color flow cytometric analysis (Fig. 2). In comparison to cells from PB, the CD8+ TEM cell subset in BM had increased expression of the CD27 and CD28 costimulatory receptors (p < 0.05). Similarly, CD38, CD69, and HLA-DR activation markers were significantly up-regulated in BM TEM (all p values <0.05) (Fig. 2A), whereas CD25 expression was not different between TEM in BM and PB (data not shown). Interestingly, CD57, a marker associated with lymphocyte senescence, was down-regulated in BM CD8+ TEM cells. These data suggest that, in comparison to PB TEM, BM CD8+ TEM maintain an "activated" phenotype characterized by high expression levels of select costimulatory and activation markers.


Figure 2
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  		FIGURE 2. The phenotype of CD8+ TEM cells from BM. A, Cell surface expression of costimulatory and activation associated molecules on TEM and TCM of PB and BM were analyzed by four-color flow cytometry. TEM and TCM subsets were gated based on the expression of CD62L on CD8+CD45RO+cells. Results are shown as mean ± SD (n = 9). *, p < 0.05. B, A representative FACS analyses from one patient is shown as plot of CD62L expression vs costimulatory and activation associated markers within a gated population of CD8+ cells. The values in the upper and lower right quadrants represent the percentage of positive cells within the CD62L+ and CD62L– cell populations.

 
To determine whether the phenotypic signature of BM-derived CD8+ TEM was also reflected in the TCM subset, we performed matched pair analysis of TCM. As was observed in the TEM subset of BM, BM-derived CD8+ TCM cells also expressed elevated levels of CD38, CD69, and HLA-DR (p < 0.05) (Fig. 2B). However, the relative expression of these markers in the TCM subset was generally lower than that in corresponding TEM. Additionally, differences in the increased expression of the CD27 and CD28 costimulatory receptors, observed between BM and PB CD8+ TEM, were not reflected in TCM. In fact, CD27 and CD28 were expressed at high levels in both BM and PB CD8+ TCM. These data demonstrate that the activation phenotype of BM CD8+ TEM is subset specific.

BM CD8+ TEM have increased cytotoxic potential

Having demonstrated that BM-derived TEM cells possess a unique activation phenotype, we sought to initially define their function by evaluating cytolytic capability. Interestingly, in comparison to PB TEM, flow cytometric analysis revealed lower expression levels of both perforin and granzyme B in the TEM subset of BM. However, when CD8 T cells from BM were activated with anti-CD3 mAb, to mimic TCR stimulation, they evidenced profound up-regulation of perforin, granzyme B, and FasL (Fig. 3, A and B). To define the functional import of the up-regulation of perforin, granzyme B, and Fas L, we compared in vitro cytotoxicity of cultured TEM cells from BM with that of PB in an anti-CD3-redirected cytolysis assay (Fig. 3C), and found that BM-derived TEM cells had increased cytotoxicity in comparison to PB TEM cells. Analogous studies using TCM cells from both BM and PB revealed no site-specific differences. As expected, T cells failed to kill P815 targets when anti-CD3 mAb was not anchored on the target cells (data not shown). Taken together, our results indicate that: 1) despite low levels of perforin and granzyme B in the resting state, upon TCR stimulation, BM CD8 T cells rapidly accumulate these molecules and 2) BM CD8+ TEM cells kill targets more effectively than TEM in PB.


Figure 3
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  		FIGURE 3. Cytotoxicity of BM-derived CD8+ TEM cells. A, The expression of perforin, granzyme B, and FasL were compared between PB and BM CD8+ TEM cells. Data represent the mean (±SD) from seven patients. *, p < 0.05. A representative FACS analyses is shown as plot of CD62L vs perforin, granzyme B, and FasL within gated CD8+ cells. The values in the upper right and lower right quadrants represent the percentage of positive cells within CD62L+ and CD62L– cell populations. B, Comparison of perforin, granzyme B, and Fas L expression on anti-CD3-activated PB and BM CD8+ T cells. PBMCs and BMCs were stimulated with 0.5 µg/ml coated anti-CD3 mAb for 3 days. Cells were then stained for intracellular perforin, granzyme, and surface Fas L and other surface markers, and analyzed by flow cytometry. One of three similar experiments is shown. C, Direct killing activity of TEM and TCM of PB and BM was compared using T cell lines generated from three patients in an anti-CD3-redirected cytolysis assay. Representative data from one patient is shown. Statistical analysis of the pooled data confirmed that cytotoxicity was significantly higher in the BM TEM subset.

 
The TEM component contributes to the augmented Ag-specific CD8+ recall response to viral Ag in the BM

The increased CD8+ TEM cell component in BM, and its distinct phenotype and cytotoxic potential, prompted us to evaluate the role of this particular subset of cells in the response to viral recall Ags. We used five paired BM and PB samples to determine granule exocytosis in TEM and TCM subsets in response to stimulation with a mixture of CMV, EBV, and Flu peptides. BM and PB samples from four patients showed specific degranulation responses to Ag. In both BM and PB, the majority of cells expressing the CD107a degranulation marker were TEM (Fig. 4, A and B). Importantly, however, in comparison to PB TEM, BM TEM evidenced increased Ag-specific expression of CD107a. These data demonstrate that BM CD8+ TEM cells have enhanced granule exocytosis in response to viral Ags in comparison to correlate cells in the PB.


Figure 4
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  		FIGURE 4. The TEM component contributes to the augmented Ag-specific CD8 response to recall Ag in the BM. A, PBMCs and BMCs from five donors were incubated with 2 µg/ml CEF peptides for 5 h, and then analyzed for CD107a mobilization. B, A representative sample is shown as a FACS plot. Lymphocytes were identified by forward scatter and side scatter. Lymphocytes were further gated on CD8+CD45RO+ and CD107a expression was plotted against CD62L expression. The values in the upper left and upper right corners represent the frequencies of CD107a-positive cells within CD62L– and CD62L+ memory CD8+ T cell populations. C, Mononuclear cells from PB and BM (n = 6) were stimulated with 25 ng/ml PMA plus 1 µg/ml ionomycin for 5 h in the presence of CD107a mAb and monensin. Samples were then stained for CD3, CD8, and intracellular IFN-{gamma}, and analyzed by flow cytometry. The results are shown as the percentage of CD107a or IFN-{gamma}-positive cells within CD8+ T cell gate. D, A representative FACS analysis from one patient is shown. Plots are of gated CD8+ T cells.

 
Because these recall experiments used whole cell populations, we next sought to determine whether the increase in memory T cell function was dependent on Ag presentation through the TCR. Specifically, we compared the ability of TCR-dependent (CEF) and TCR-independent stimuli (PMA/ionomycin) to activate BM vs PB memory CD8+ T cells. Similar to our findings in TEM, after stimulation with CEF peptides in vitro, CD8+ memory T cells in both the BM and PB produced IFN-{gamma} and expressed CD107a. However, BM memory CD8+ T cells were significantly more potent effectors. The frequency of IFN-{gamma}-producing memory CD8+ T cells among total CD8+ T cells from PB varied from ~0.03 to 1.84% vs 0.06 to 5.48% in BM (p = 0.0547). The frequency of CD107a-expressing cells varied from 0.00 to 1.72% in PB CD8+ T cells and 0.09 to 5.94% in BM (p < 0.0015). Compared with CD8+ T cells from the PB, the frequency of IFN-{gamma} and CD107a-producing cells in the BM increased 2.7- and 3.3-fold, respectively. In addition, CD8+ T cells in BM showed a more vigorous IFN-{gamma} and CD107a response to PMA/ionomycin than correlate cells from paired PB. The frequency of IFN-{gamma} and CD107a-expressing cells in BM varied between 10.92 and 73.27% and 18.21 and 60%, respectively, while levels in PB were 0.65–16.01% and 2.71–26.93% respectively (Fig. 4, C and D). These data demonstrate that BM CD8 T cells have enhanced ability to respond to both TCR-dependent and -independent stimuli.

Frequencies of viral recall Ag-specific CD8+ T cells in BM

To assess whether the increased response of BM CD8+ T cells to CEF peptides was secondary to a higher percentage of Ag-specific T cells, we used HLA-A2 CMV (NLVPMVATV) pentameric complexes to quantify the proportion of CMV-specific T cells. Five patients had pentamer staining CD8+ T cells (Fig. 5). The CD8+ T cells specific for CMV pp65 (495–503) represented 0.31–4.35% of total CD8+ T cells in BM and 0.43–4.29% in the PB. Similar to the total population of CD8 T cells, CMV-specific CD8 T cells in BM demonstrated a TEM phenotype.


Figure 5
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  		FIGURE 5. Identification of CMV-specific CD8+ T cells by pentamer staining from PBMCs and BMCs. A, Lymphocytes from BM and PB were stained with HLA-A2/CMV pentamer and cell surface markers. Results are from five patients with detectable pentamer staining and one patient with undetectable staining, as a negative control. Cells were gated on CD3+CD8+ lymphocytes. B and C, A representative patient sample is shown. Gated populations are plotted as CD8 vs pentamer staining. The values are the percentage of CD8 T cells that are pentamer positive (B). The expression of CD45RO and CD62L in pentamer-positive cells (C).

 
Expression of chemokine receptors on CD8+ TEM cells in BM

Finally, we queried why this unique population of TEM cells is preferentially located in BM. Because chemokine receptors are involved in T cell homing and differentiation (21), we investigated the expression of the CCR5, CXCR4, and CXCR1 chemokine receptors on CD8+ T cells isolated from BM and PB. These receptors were chosen for study based on the reported high levels of their cognate ligands within the BM microenvironment (22, 23, 24). Flow cytometric analysis of paired samples revealed that the percentage of CCR5-expressing cells was increased in BM TEM. Although the majority of both PB and BM CD8 TEM cells were CXCR4 positive (data not shown), the BM cells exhibited increased staining intensity. Furthermore, compared with correlate cells in the PB, CXCR1 was down-regulated in the TEM, but not the TCM subset of BM CD8+ T cells. Interestingly, despite their recognized import for the proliferation and survival of CD8+ memory T cells, we did not identify any difference in the expression of the IL-7R{alpha} or IL-15R{alpha} in BM vs PB TEM (Fig. 6) (25, 26, 27, 28, 29). These data demonstrate that BM TEM have unique patterns of chemokine receptor expression which may contribute to their accumulation within this compartment.


Figure 6
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  		FIGURE 6. The expression of the receptors for chemokines and IL-7 and IL-15 by BM CD8+ TEM cells. The surface expression of chemokine receptors, CXCR4, CCR5, and CXCR1, and {alpha}-chain of the IL-7R and IL-15R were determined by four-color flow cytometric analysis of six patients. Results are shown as mean ± SD. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies in cancer patients have defined the human BM as a harbor for tumor Ag-specific memory CD8+ T cells with potent effector function (8, 11, 12, 13, 30, 31). However, the phenotype and functional recall response of BM CD8+ memory T cells to viral Ags in humans remains poorly understood. In this study, we characterize a population of effector memory CD8+ T cells in the BM of patients with OA, which demonstrate a unique effector phenotype characterized by high levels of CD27, CD38, CD69, and HLA-DR, and enhanced recall responses to viral Ags. Our description of the phenotypic signature and function of this CD8+ TEM cell population within the human BM provides a requisite link to pave the way for clinical translation.

In our initial phenotypic analysis, we discovered that the memory T cells within the BM express low levels of CD62L, a marker for TEM. Memory T cells can be divided into two subsets, TEM and TCM, based on their expression of CCR7 and CD62L. TEM cells express high levels of activation and effector molecules, low levels of costimulatory receptors, and exert immediate effector function. In contrast, TCM cells express high levels of costimulatory receptors, low levels of effector molecules and possess high proliferative capacity (32). Based on the relatively large population of TEM within the human BM, we sought to determine whether a unique TEM population could account for the observed phenotypic and functional changes.

In comparison to correlate cells in the PB, BM-derived TEM express high levels of the CD27 and CD28 costimulatory receptors, which are postulated to serve as markers for antitumor and antiviral protection, and whose loss defines end-stage T cell differentiation (19, 33). For example, tumor-specific T cells bearing a CD27+CD28+ phenotype, similar to the BM-derived CD8+ T cells described here, have recently been associated with successful adoptive immunotherapy in patients with metastatic melanoma (34, 35). Similarly, in patients infected with HIV, CD27-positive CD8+ cells preferentially survive in vivo, proliferate following antigenic stimulation and are more resistant to apoptosis than CD27-negative cells (36). The high levels of CD27/CD28 on BM CD8+ TEM, suggest that this population of cells might be responsible for the potent effector function of BM memory T cells.

In addition to costimulatory receptors, BM-derived CD8+ TEM demonstrate up-regulation of the CD38, CD69, and HLA-DR activation markers. Specifically, expression of these activation markers is enhanced in comparison to both PB TEM and BM TCM. The high expression of CD38, CD69, and HLA-DR, correlates well with previous studies which demonstrate that BM T cells are in a heightened state of activation (37, 38) and are phenotypically distinct from memory cells in the PB (30). These data suggest that BM-derived memory TEM cells are phenotypically unique, bearing a signature associated with enhanced functional activity.

To correlate the phenotypic signature of BM TEM cells with effector function, we first analyzed their cytolytic potential and found lower levels of perforin and granzyme B in the resting state. However, upon TCR stimulation BM-derived CD8 cells evidence rapid intracellular accumulation of these molecules (stimulation studies were performed on the entire population of CD8 cells because of the potential changes in CD62L with activation). This profound accumulation of perforin and granzyme B is functionally significant as BM TEM evidence enhanced cytolytic capacity, exceeding that of the highly potent memory subset in PB.

Based on the potent cytotoxic potential of BM CD8+TEM cells, we sought to define their ability to respond to natural viral recall Ags, by analyzing CD107a expression in a series of short-term recall studies. These assays enabled us to monitor Ag-specific degranulation response in defined CD8+ T cell subsets with minimal manipulation in vitro (17). In comparison to CD8+ TEM cells in the PB, BM-derived CD8+ TEM exhibit increased expression of the degranulation marker CD107a in response to activating stimuli. This enhanced response to viral Ags is likely not dependent on differences in APC function in the BM and periphery as similar increases in CD107a and IFN-{gamma} production were observed in BM memory CD8+ T cells stimulated in a TCR-independent fashion. Furthermore, these findings do not appear to result from an increased percentage of Ag-specific T cells in the BM, as pentamer staining with an HLA-A2-restricted CMV peptide revealed similar percentages of Ag-specific cells in both the BM and PB.

To better understand why this unique population of CD8+ TEM cells is preferentially located in the BM, we analyzed specific patterns of chemokine and growth factor receptor expression. Our studies indicate that BM CD8+ TEM have increased expression of CXCR4 and CCR5 and decreased expression of CXCR1. High expression of CXCR4 on BM CD8+ TEM is likely physiologically relevant, as CXCL12, the ligand for CXCR4, is constitutively expressed by both BM stromal cells and the endothelium of BM microvessels (22, 23), and mediates the homing and localization of hemopoietic stem cells to the BM (39, 40). Similarly, CCL3 and CCL5, both ligands for CCR5, are produced by BM fibroblasts (24). In contrast, CXCL8, the ligand for CXCR1, induces mobilization of hemopoietic stem cells from BM. These data suggest that the high levels of CXCR4 and CCR5 stimulate TEM migration to the BM, while the low levels of CXCR1 found on BM CD8+ TEM might hinder migration (41).

The preferential accumulation of CD8+ TEM cells in the BM may also be due to microenvironmental considerations. BM contains T cell survival factors, such as IL-7 and IL-15, which are recognized to induce Ag-independent proliferation of memory T cells (25, 42, 43). Interestingly, we did not find differences in IL-7R{alpha} nor IL-15R{alpha} expression in BM vs PB TEM. The response of T cells to IL-7 relies on both the IL-7R expression on T cells and the concentration of available IL-7. Although, BM stromal tissue is one source of IL-7 production, the levels of soluble IL-7 are not increased in the BM microenvironment (44). Similarly, recent evidence suggests that homeostatic proliferation of memory T cells in response to IL-15 might be indirect (45, 46). BM cells, including stromal cells and dendritic cells, express both IL-15 and IL-15R{alpha}, which allows efficient transpresentation of IL-15 to memory CD8 T cells (47, 48). Therefore, the fact that BM cells do not evidence increased expression of the IL-7R or the IL-15R does not rule out the potential importance of these molecules on the survival of BM TEM.

Several limitations of our study are important to note. Specifically, OA is classically considered to result from mechanical degradation of the joint and synovial inflammation. However, new evidence suggests a potential role for autoimmune synovial attack (49, 50). Specifically, the infiltration of T cells and the presence of inflammatory cytokines have recently been demonstrated in the synovial membrane and synovial fluid of OA (51, 52). Furthermore, the persistence of CMV or EBV in the BM (53) might drive the local activation of viral-specific T cells. Finally, because OA commonly occurs in the elderly, we cannot eliminate the possibility that some of our observations are age related.

Three considerations are important when assessing the potential impact of synovial inflammation and patient age on our results. First, specimens were harvested directly from the medullary canal and demonstrated a heightened response to nonsynovial recall Ags. Second, we were unable to detect the site-specific expansion of CMV-specific CD8 T cells in the BM compared with PB. Furthermore, the expression of activation-associated markers on these CMV-specific CD8 T cells is similar to the total population of CD8 T cells in the BM (data not shown). Importantly, however, the potential impact of patient age on our results may be significant. For example, a recent study has shown that the human BM contains an approximately equal proportion of both CD8+ TCM and TEM cells (54). Therefore, the high percentage of TEM vs TCM in the BM of OA patients observed in our study maybe due to an age-associated change (55).

Taking these limitations into consideration, our data suggest that human BM is enriched with functionally enhanced population of CD8+ TEM cells, which bear a hybrid phenotype (CD27high, CD69high,CD38high, perforinlow), between classically defined TEM and TCM subsets. These BM TEM are characteristic of cells at intermediate stages of differentiation, which may have the potential for self-renewal and homeostasis in the absence of Ag. Although several elegant murine models have been described for studying T cell memory in BM (56, 57), to the best of our knowledge, this is the first report to define the unique phenotype of BM CD8+ TEM cells and characterize the viral recall response of CD8+ TEM cells in human BM from patients with no known history of cancer or well-defined immunologically mediated disease. The phenotypic and functional data in this report provide a solid foundation to study the use of BM-derived memory T cells for the treatment of infectious disease and cancer, and to promote their generation in vaccine design.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
S. E. Strome and X. Zhang have submitted a patent through the University of Maryland School of Medicine based on these findings.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 X.Z. effort was supported by a Postdoctoral Head and Neck Cancer Research Fellowship in honor of Richard H. Stoll. Back

2 Address correspondence and reprint requests to Dr. Scott E. Strome, Department of Otorhinolaryngology–Head and Neck Surgery, University of Maryland School of Medicine, 16 South Eutaw Street, Suite 500, Baltimore, MD 21201-1619. E-mail address: sstrome{at}smail.umaryland.edu Back

3 Abbreviations used in this paper: TEM, effector memory T; TCM, central memory T; BM, bone marrow; PB, peripheral blood; BMC, BM cell; CEF, CMV, Epstein-Barr virus and influenza virus; OA, osteoarthritis. Back

Received for publication April 12, 2006. Accepted for publication August 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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