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Preferential Homing of Tumor-specific and Functional CD8+ Stem Cell-like Memory T Cells to the Bone Marrow

Supplemental Digital Content is available in the text.

T memory stem cells (T SCM s), which possess the properties of self-renewal and multipotency, have been observed to play a role in the pathogenesis of various severe diseases such as graftversus-host disease, malignant melanoma, Chagas disease, as well as human immunodeficiency virus type 1 and simian immunodeficiency virus infections. [1][2][3][4][5][6][7][8][9][10][11][12][13] Mouse CD8 + T SCM s are a wellidentified subset of postmitotic CD44 low CD62L high T cells that express surface proteins, including stem cell antigen-1 (Sca-1), common interleukin (IL)-2, IL-15, receptor β-chain (CD122), as well as B-cell lymphoma protein-2 (Bcl-2) at high levels. It has been reported that T SCM s could be generated in vitro from naive T cells by activation of the Wnt, IL-7, or IL-15 signaling pathways. 1,2,14 Blood-derived T SCM s have also been tracked in vivo in patients after gene therapy for human severe combined immunodeficiency disease. 15 Similar to conventional memory T cells, CD8 + T SCM s can be detected in umbilical cord blood in humans. 3 Although this phenomenon indicates a tropism for lymphoid tissues, the anatomic characterization of T SCM -cell niches has not been performed to date.
The bone marrow (BM) functions as the major reservoir and site of recruitment for hematopoietic stem cells (HSCs) as well as memory B and T cells by providing appropriate cellular niches. [16][17][18][19][20][21][22][23][24][25][26][27][28][29] The common niche that supports HSCs or leukocytes in the BM is constituted by CXCL12 + stromal cells. 30 Under certain conditions, the BM supports the homeostasis of naive T cells and pro-B cells. [31][32][33] In addition, BM microvessels constitutively express prerequisite traffic molecules that support the recruitment of HSCs and conventional memory T cells, and potentially support the homing of other T-cell subsets. 34 More importantly, the function of BM-resident CD8 + T cells is distinct from that of those residing in other organs. For instance, BMresident CD8 + T cells from cancer patients elicit more efficient tumor-specific cytotoxicity than CD8 + T cells in peripheral blood (PB). Thus, T cells from the BM are regarded as potential resources for antitumor cellular therapy. 35 Nevertheless, it is not known whether T SCM s, which constitute a distinct T-cell subset with the highest antitumor activity reported to date, accumulate in the BM.
In the present study, we demonstrate that the BM acts as a hub to which most tumor-specific CD8 + T SCM s relocate. Importantly, BM-resident T SCM s showed higher inhibitory activity against tumor growth than spleen (SP)-resident T SCM s in a B16 murine model, implying potential applications for immunotherapy against melanoma.

CD8 + T Memory Stem Cells Preferentially Reside in the BM
Although T SCM s are categorized as memory cells, they display a naive-like phenotype; these cells include CD62L + CCR7 + CD45RO − CD45RA + in humans and CD44 low CD62L high in mice. [1][2][3]36 To determine whether T SCM s reside naturally in the BM, the expression levels of Sca-1 and CD122, which are typical markers used to distinguish T SCM s from naive T cells in mice, were first analyzed in CD44 low CD62L high CD8 + T cells (Fig. S1, Supplemental Digital Content 1, http://links.lww.com/JIT/A529). Significant elevation of the CD122 high Sca-1 high subset was observed in the BM-derived naive T-cell compartment (Fig. 1A, Fig. S2, Supplemental Digital Content 1, http://links.lww.com/JIT/A529) compared with those from other tissues, including SP, PB, and mesenteric lymph nodes (LN) (Fig. 1B). Thus, we hypothesized that CD122 high Sca-1 high T SCM s preferentially reside in the BM.
Well-defined T SCM s express not only high levels of CD122 and Sca-1 but also high levels of Bcl-2 and CXCR3. 1,2 To validate whether these natural CD122 high Sca-1 high T SCM s in the BM are consistent with the previously well-defined T SCM s, the expression levels of Bcl-2 and CXCR3 were analyzed in CD122 high Sca-1 high T SCM s and CD122 low Sca-1 low naive T cells. As expected, the expression levels of both Bcl-2 and CXCR3 were higher in CD122 high Sca-1 high T SCM s than in naive CD8 + T cells (Figs. 1C, D, Fig. S3, Supplemental Digital Content 1, http://links.lww. com/JIT/A529). 1,2 Similar to BM naive T cells reported previously, BM CD8 + T SCM s were also positive for CD127 expression (Fig. 1E). 28 Despite the slightly lower expression of CD127 in BM CD8 + T SCM s, the difference was not statistically significant (Fig. S3, Supplemental Digital Content 1, http://links.lww.com/JIT/A529). It is interesting to note that, the expression of CD69 in BM-resident T SCM s was slightly higher than that of naive T cells (Fig. 1E,  Fig. S3, Supplemental Digital Content 1, http://links.lww. com/JIT/A529). Of note, the canonical T SCM s reported previously in peripheral immune organs were αβ T cells. 3 Therefore, to eliminate the potential interference of γδ T cells, which also reside in the BM and contain a fraction of cells expressing CD122 and Sca-1 molecules, 37-39 the expression of T-cell receptor (TCR) chains was evaluated by flow cytometry. We found that CD122 high Sca-1 high T SCM s expressed TCRβ chains other than TCRγδ chains (Fig. 1F,  Fig. S3, Supplemental Digital Content 1, http://links.lww. com/JIT/A529). In addition, to distinguish between T CM s, naive T cells and T SCM s in a postmitotic phenotype at the transcriptional level, we tested the expression of Ccr2, Ccr5, CD44, and Sca-1 (Ly6A/E) by quantitative real-time polymerase chain reaction (qRT-PCR) in accordance with previous reports 1 ; results showed that the messenger RNA (mRNA) expression of Ccr2 in T CM s was much higher than in naive T cells and T SCM s, whereas the mRNA expression of Ccr5 in naive T cells was much lower than in T SCM s and T CM s (Fig. 1G). It is notable that the mRNA expression of CD44 was lower in T SCM s than that in T CM s but the mRNA level of Sca-1 (Ly6A/E) was significantly higher than that in naive T cells, which was consistent with the previous report (Fig. 1G). 1 According to previous reports, cells with stem cell properties, including HSCs and T CM s, are most likely in the resting stage. 20,26,27 In order to verify the stem cell properties of T SCM s, we examined the cell cycle of naive T cells, T SCM s, and T CM s (Fig. 1H, Fig. S4, Supplemental Digital Content 1, http://links.lww.com/JIT/A529). Measurement of DNA content showed that BM CD8 + T SCM s were stalled in the G0/G1 cell interphase and resting state; these findings were almost consistent with those for BM memory T cells, as indicated by previous reports (Fig. 1H). 25,40 In addition, we examined other factors (Klf-7, Tcf-1, Lef-1, β-catenin) involved in the regulation of stemness of cells by qRT-PCR (Fig. 1G). The qRT-PCR data indicated that the mRNA expression of Tcf-1, Lef-1, and β-catenin in BM-resident T SCM s was notably higher than that in naive T cells (Fig. 1G). Collectively, these observations further support that BM-enriched CD122 high Sca-1 high naive-like CD8 + T lymphocytes can be identified as T SCM s that naturally inhabit the BM.

BM-resident CD8 + T SCM s Vigorously Respond to a Blood-borne Antigen
CD8 + T SCM s have been shown to elicit rapid immune responses upon antigen rechallenge. 3,41 To investigate the immune responses of CD8 + T SCM s in situ, the purified naive T cells (CD8 + CD44 low CD62L high CD122 low Sca-1 low ), T CM s (CD8 + CD44 high CD62L high ), and T SCM s (CD8 + CD44 low CD62L high CD122 high Sca-1 high ) from the BM of OT-I mice (CD45.2 + ) were adoptively transferred into congenic mice (CD45.1 + ), respectively, followed by antigen stimulation by ovalbumin (OVA) immunization ( Fig. 2A, Fig. S5, Supplemental Digital Content 1, http://links.lww.com/JIT/A529). Flow cytometric analysis showed that the BM T SCM s displayed significantly higher levels of cell proliferation and interferon-γ (IFN-γ) production than BM T CM s and BM naive T cells (Figs. 2B, C). In addition, to test the downstream differentiation potential of T SCM s upon antigen exposure, we compared the frequencies of BM CD44 + T cells in naive T-cell-transferred or T SCM s-transferred recipient mice. As expected, we detected larger numbers of CD45.2 + CD44 + CD8 + T cells in T SCM s-transferred recipient mice (Fig. 2D), which indicated that the transferred CD8 + T SCM s were capable of differentiation into conventional memory or effector T cells more rapidly. These results indicate that BM-enriched CD122 high Sca-1 high T SCM s response to a blood-borne antigen efficiently.

Preferential Migration of CD8 + T SCM s to the BM
Owing to the limitation of low cell number of natural CD8 + T SCM s from wild-type (WT) mice, it was not practical to obtain the required cell numbers from WT mice on a large scale for investigation of the preferential migration of T SCM s to the BM. Although β-catenin signaling was not found to regulate the generation of memory T cells and inhibit T-cell proliferation, T SCM s could be generated efficiently from naive T cells in vitro with the glycogen synthase kinase-3β inhibitor TWS119 (a compound that induces T SCM cells by a mechanism of action that is currently debated) as previously reported. 42,43 The naive T cells and in vitro-generated T SCM s cells with TWS119 were isolated by flow cytometry (Fig. S6, Supplemental Digital Content 1, http://links.lww.com/JIT/A529). Subsequently, we labeled naive T cells and T SCM s with CMTPX and then mixed with FIGURE 1. CD8 + memory stem cells preferentially reside in BM. A, Expression of CD122 and Sca-1 in SP-resident and BM-resident naivelike T-cell compartment. Dot plots represent the frequencies of CD122 high Sca-1 high subset gated on CD3 + CD4 − CD8 + CD44 low CD62L high cells. Data are representative for 6 independent experiments (n = 8). B, T SCM s in BM, PB, SP, and LN organs. The frequencies of T SCM s accounting for CD44 low CD62L high CD8 + T cells and T SCM s cell counts in PB, LN, SP, and BM were shown as mean ± SD, 1-way ANOVA. The cell count of CD8 + T SCM s was calculated as the formulation: (the total number of cell count)×(CD3 + CD8 + )%×(CD44 − CD62L + )%×(CD122 + Sca-1 + )%. Data are representative for 4 independent experiments (n = 6). C, Expression of Bcl-2 in naive and T SCM subsets in BM-resident T cells. Overlaid histogram plots show the levels of Bcl-2 in BM-resident CD122 low Sca-1 low , CD122 low Sca-1 high , CD122 high Sca-1 low , and CD122 high Sca-1 high subsets gated on CD3 + CD4 − CD8 + CD44 low CD62L high cells. Data are representative for 5 independent experiments (n = 5). The MFI of Bcl-2 in CD122 low Sca-1 low , CD122 low Sca-1 high , CD122 high Sca-1 low , and CD122 high Sca-1 high subset was shown as mean ± SD, 1-way ANOVA. D-F, Flow cytometric analysis of SP-resident and BM-resident T SCM s overlaid with SP-resident and BM-resident naive T cells. Overlaid histogram plots show expression levels of a given molecule in different CD8 + T-cell subsets. CD8 + T-cell subsets were defined as follows: BM-resident T SCM s, CD3 + CD8 + CD4 − CD44 low CD62L high CD122 high Sca-1 high ; BMresident and SP-resident naive T cells, CD3 + CD8 + CD4 − CD44 low CD62L high CD122 low Sca-1 low . Data are representative for 5 independent experiments (n = 7). G, RT-PCR results show the expressions of Klf-7, Tcf-1, Lef-1, β-catenin, Ccr2, Ccr5, CD44, and Sca-1 in different CD8 + T-cell subsets. CD8 + T-cell subsets were defined as follows: BM-resident T SCM s, CD3 + CD8 + CD4 − CD44 low CD62L high CD122 high Sca-1 high ; BM-resident and SP-resident naive T cells, CD3 + CD8 + CD4 − CD44 low CD62L high CD122 low Sca-1 low ; BM-resident and SP-resident T CM s, CD3 + CD8 + CD4 − CD44 high CD62L high . Data were shown as mean ± SD, 1-way ANOVA. H, CD8 + naive T cells and T SCM s from BM, SP, of C57BL/6 mice and their DNA stained with PI. For the positive control of PI staining, the 2×10 6 SP cells from C57BL/6 mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) in the presence of IL-2 (10 ng/mL). Histograms show the percentage of DNA content in different CD8 + T-cell subsets. Data are representative for 3 independent experiments (n = 5). Data were shown as mean ± SD, 1-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). ANOVA indicates analysis of variance; BM, bone marrow; LN, lymph nodes; PB, peripheral blood; PI, propidium iodide; SP, spleen. equivalent carboxyfluorescein succinimidyl ester (CFSE)labeling SP cells, which served as a reference. The mixed cells were injected into recipient mice. After 6 hours, we detected the ratios of CMTPX-positive and CFSE-positive cells, and calculated the homing index (HI). The results showed that the HI of TWS119-induced T SCM s in the BM was ∼3-fold higher than that of those induced in the SP (Fig. 3A). To accurately compare the homing of each subset and avoid the off-target effects of small-molecule inhibitors, T SCM s were generated by stimulation of Wnt3A protein in vitro, and the frequency of each subset in the BM was determined. Flow cytometric analysis showed that the proportion FIGURE 2. CD8 + TSCMs from BM can respond to blood-borne antigen in vivo. A, Schematic diagram of adoptive transfer. B and C, BMresident T SCM s possess the capacity of rapidly acquiring effector functions in vivo. The 5×10 5 each subset of T cells from BM of OT-Ⅰmice were adoptively transferred to CD45.1 mice, respectively. Recipients were immunized with 500 μg OVA in CFA and sacrificed after 3 days for further analysis. The T-cell subsets were determined by the following FACS isolations: CD45.2 + CD8 + CD44 low CD62L high CD122 low Sca-1 low for naive T cells; CD45.2 + CD8 + CD44 low CD62L high CD122 high Sca-1 high for T SCM s; CD45.2 + CD8 + CD44 high CD62L high for T CM s. B, Numbers in histograms represent the percentage of BrdU-positive cells in BM-resident naive, T CM and T SCM cells after OVA stimulation. C, Intracellular cytokine staining of naive, T CM and T SCM cells in BM. Numbers in histograms show the percentage of IFN-γ-expressing cells in BM after OVA stimulation. Data are representative for 3 independent experiments (n = 6). Frequencies of BrdU + (B) and IFN-γ + cells (C) were shown as mean ± SD, t test. D, In vitro-generated CD8 + T SCM s possess the capacity of rapidly acquiring effector functions in vivo. The 1×10 6 purified in vitro-generated CD8 + T SCM s with TWS119 or 1×10 6 purified natural naive T cells were adoptively transferred into CD45.1 mice and then were immunized with 500 μg OVA in CFA per mice by injection intraperitoneally. Three days after immunization, the expressions of CD44 molecule were detected. Histograms show the percentage of the CD44 + CD45.2 + CD8 + T cells (n = 3). Data were shown as mean ± SD, 1-way analysis of variance (*P < 0.05, **P < 0.01, ***P < 0.001). BM indicates bone marrow; CFA, complete Freund's adjuvant; IFN-γ, interferon-γ; OVA, ovalbumin. . Homing index was represented as mean ± SD, t test. B, Frequencies of transferred T SCM s in SP and BM. 1×10 6 Wnt3A-treated CD44 low CD62L high T cells were injected intravenously and analyzed for transferred T SCM s after 24 hours. Dot plots represent the expression of CD122 and Sca-1 on the surface of CD44 low CD62L high adoptive T cells. T-cell subsets were defined as in Figure 2A and Figure S4. Data are representative for 4 independent experiments (n = 6). Frequencies of CD122 high Sca-1 high subset were shown as mean ± SD, t test. C, Numbers of transferred T SCM s in SP and BM. Data were representative for 4 independent experiments (n = 6). The numbers of CD45.2 + CD8 + CD44 low CD62L high CD122 high Sca-1 high cells were shown as mean ± SD, t test. D-F, In vivo-generated CD8 + T SCM s preferentially homed to the BM. The 2×10 6 splenic CD44 low CD62L high CD8 + T cells sorted from OT-Ⅰ mice were adoptively transferred to CD45.1 mice. The recipients were injected intraperitoneally. 500 μg ovalbumin with complete Freund's adjuvant and 4 doses per day of TWS119 (20 mg/kg) or DMSO (as control) from day 0 to day 3. Mice were sacrificed and analyzed for the expressions of CD122, Sca-1, and Bcl-2 of CD44 low CD62L high transferred CD8 + T cells in SP and BM after 1 week. T-cell subsets were determined as Figure 2C and D. D, Dot plots represent the expression levels of CD122 and Sca-1 on the surface of CD44 low CD62L high transferred T cells. E, Number of CD8 + T SCM s relocated to SP and BM in vivo. F, Overlaid histograms represent the levels of Bcl-2 in CD44 low CD62L high transferred CD8 + T cells with treatment of TWS119 and freshly isolated naive T cells from SP (T N ). Data are representative for 4 independent experiments (n = 6). Frequencies of CD122 high Sca-1 high subset were shown as mean ± SD, 1-way ANOVA (D). Numbers of CD45.2 + CD8 + CD44 low CD62L high CD122 high Sca-1 high cells were shown as mean ± SD, 1-way ANOVA (E). MFI of Bcl-2 was shown as mean ± SD, 1-way ANOVA (F) (*P < 0.05, **P＜0.01, ***P < 0.001). ANOVA indicates analysis of variance; BM, bone marrow; CFA, complete Freund's adjuvant; CFSE, carboxyfluorescein succinimidyl ester; DMSO, dimethyl sulfoxide; IFN-γ, interferon-γ; OVA, ovalbumin; SP, spleen.
of the CD122 high Sca-1 high subpopulation in the BM increased by ∼5-fold compared with that in the SP (Fig. 3B). Consistently, the number of T SCM s grew significantly when compared with that in the SP (Fig. 3C). As previously reported, CD8 + T SCM s could be generated by activating CD8 + T cells in vivo, when coupled with pharmacological activation of Wnt signaling. 2 To mimic physiological conditions, a relatively low dose of TWS119 was injected intraperitoneally into mice whose T cells were activated by OVA injection to facilitate the minimum generation of T SCM s in vivo (Fig. S7, Supplemental Digital Content 1, http://links.lww. com/JIT/A529). 2 The ratios and numbers of CD122 high Sca-1 high T SCM s significantly increased in both BM and SP upon TWS119 treatment (Fig. 3D). In particular, the number and ratio of T SCM s in the BM was 4-to 5-fold higher than that in the SP (Figs. 3D,  E, Fig. S2, Supplemental Digital Content 1, http://links.lww.com/ JIT/A529). In addition, the expression of Bcl-2 was upregulated in BM-resident T SCM s compared with that in their SP-derived counterparts or freshly isolated SP-derived naive T cells (Fig. 3F). Taken together, these results demonstrate that the CD122 high Sca-1 high T SCM s preferentially homed to the BM.

P-Selectin or E-Selectin/P-Selectin Glycoprotein 1 (PSGL-1) and Vascular Cell Adhesion Protein 1 (VCAM-1) Mediate the Homing of T SCM s to the BM
The translocation of CD8 + T CM s and HSCs to BM microvessels is dependent on the interactions between adhesion molecules such as VLA-4/VCAM-1 (CD106), P-selectin and E-selectin/PSGL-1, or CXCL-12/CXCR4. 18,25,44,45 Given the similar homing properties of T SCM s, T CM s, and HSCs, studies of the homing of T CM s and HSCs may help to elucidate the mechanism underlying the homing of T SCM s. To verify this hypothesis, the expression of candidate adhesion molecules in BM and SP was determined by flow cytometry. The results showed that adhesion molecules, including LFA-1, CXCR4, integrin α4β7, and PSGL-1, were involved in T-cell homing and did not exhibit significant differences in expression (Fig. 4A,  Fig. S8, Supplemental Digital Content 1, http://links.lww.com/ JIT/A529). 18,46,47 Of note, integrin-α2, a key mediator of the homing of CD4 + memory T cells to the BM, was detected at a low level on the surface of T SCM s (Fig. 4A, Fig. S8, Supplemental Digital Content 1, http://links.lww.com/JIT/A529). 20 P-selectin and E-selectin as well as VCAM-1 molecules are detected in the normal BM sinusoid. It is interesting to note that, the expression of VCAM-1 was moderately higher in BMresident T SCM s than in naive T cells (Fig. 4A, Fig. S8, Supplemental Digital Content 1, http://links.lww.com/JIT/A529). To determine whether these adhesion molecules mediate the homing of T SCM s to the BM, we generated T SCM s cells with TWS119 in vitro and isolated T SCM s by flow cytometry. CD8 + T SCM s were labeled with CMTPX and then mixed with CFSElabeling SP cells at ratio of 1:1. The mixed cells were injected into recipient mice, followed by the injection of corresponding antibodies intravenously 6 hours later. After 1 day, we examined the ratios of CMTPX-positive and CFSE-positive cells and calculated the HI. Compared with treatment with immunoglobulin G, flow cytometric analysis showed that the treatment with anti-VCAM-1, anti-P-selectin, anti-E-selectin, or anti-PSGL-1 antibodies significantly downregulated the homing of T SCM s into the BM (Figs. 4B, C). In addition, treatment with anti-CXCL-12 did not result in significant differences in the homing of T SCM s into all the organs (Figs. 4B, C). Together, these data indicate that P-selectin, E-selectin, PSGL-1, and VCAM-1 are the key mediators of the homing of T SCM s.

BM-resident T SCM s Have Enhanced Antitumor Activity Relative to SP-resident T SCM s in Response to Tumor-Antigen Challenge
Tumor-specific cytotoxic T cells or chimeric antigen receptor T cells are currently applied clinically for targeted cancer therapy. The B16 murine melanoma model is useful for the study of malignant melanoma in humans. 31 In order to perform OVAspecific OT-I mice for studying T SCM specificity against tumor antigens, we constructed the B16-OVA cell line as previously described. We hypothesized that the microenvironment of the BM enables the generation of T SCM s with higher antitumor activity; accordingly, we generated OVA-specific CD8 + T SCM s in vivo via injection of TWS119 and tested the antitumor activities. As expected, the BM-derived T SCM s possessed higher antitumor reactivity than the SP-derived T SCM s in the adoptive transfer experiment (Fig. 5A). Furthermore, the transfer of BMderived T SCM s significantly extended the survival of mice compared with that achieved following the transfer of SP-derived T SCM s (Fig. 5B). Thus, the results suggest that the adoptively transferred BM-derived T SCM s exhibited enhanced antitumor activity and higher therapeutic efficacy than conventional SPderived T SCM s in mice.

DISCUSSION
In this study, CD8 + T SCM s that highly expressed CD122, Sca-1, Bcl-2, and CXCR3 were identified within the BM-resident naive-like T-cell compartments. Although a small number of natural T SCM s were detected in the peripheral lymphoid organs, including in the SP, PB, or LN, the ratios of natural T SCM s in these organs were much lower than those in the BM. Notably, similar to SP-derived T SCM s, the BM-resident T SCM s were capable of acquiring effector functions more rapidly upon bloodborne antigen exposure than naive T cells. These data also suggest that T SCM s significantly accumulated in the BM, rather than being simply confined to peripheral inflammatory sites. Nevertheless, the differences in species (mouse and nonhuman primates) resulted in differences in the distribution of CD8 + T-cell subsets in lymphoid organs, which was manifested in the distribution of CD8 + T CM s (larger numbers of mouse CD8 + T CM s in BM, in contrast with larger numbers of rhesus CD8 + T CM s in the LNs). 9 Further, the distribution of CD8 + T SCM s in mice was different from that in nonhuman primates. Therefore, the natural distribution of CD8 + T SCM s in the human body requires further in-depth study. In addition, our data provided novel insights into the cytotoxic activity of T SCM s in the BM, whose function has thus far been ambiguous, and suggested a possible mechanism for the enhanced antitumor activity of BM T cells.
Despite the concomitant expression of numerous markers of naive T cells on the surface of T SCM s, both bioinformatics analysis of microarray data and antigenic stimulation experiments have suggested that T SCM s are most closely related to T CM s. 3 However, it remains unknown whether the other characteristics of T SCM s, especially the trafficking properties, are similar to those of T CM s. The BM plays an important role in controlling immune responses by influencing the generation of lymphocytes and the maintenance of immunologic memory. 16 Through investigations of the homing and retention of T SCM s in the BM, we found that both in vivo-generated and in vitro-generated T SCM s preferentially relocated to the BM (Fig. 3). Unfortunately, the results of the homing assay with CD8 + T SCM s generated by Wnt3A protein in vitro were not absolutely consistent with the results of in vivo studies. We speculated that introduction in vitro may be more efficient. Therefore, the homing efficacy of in vivo-generated T SCM s was higher than that of in vitro-generated T SCM s. Although T SCM s could be induced from naive T cells in vivo and were detected in the BM, the anatomic sites of transition from the naive state have not yet been determined.
The process of the accumulation of leukocytes in tissue depends on a series of adhesive interactions with vessels that involve tethering, rolling, or sticking. Although each adhesion procedure is mediated by several specific receptorligand pairs, each leukocyte possesses its own indispensable,  6). B, Effect of monoclonal antibodies on T SCM s homing to BM vessels. The CMTPX-labeled (10 μM) 1×10 6 purified in vitro-generated CD8 + T SCM s with TWS119 and then mixed with CFSE-labeled (10 μM) total SP cells (as a reference) at ratio of 1:1. The mixed cells were injected into recipient mice. After 6 hours, the mice were injected with 100 μg of neutralizing antibodies and IgG control intravenously per mice and analyzed for homing index in SP and BM after 24 hours. C, Data are representative for 3 independent experiments (n = 4). Data were shown as mean ± SD, 1-way analysis of variance (*P < 0.05, **P < 0.01, ***P < 0.001). BM indicates bone marrow; CFSE, carboxyfluorescein succinimidyl ester; IgG, immunoglobulin G; PSGL-1, P-selectin glycoprotein 1; SP, spleen; VCAM-1, vascular cell adhesion protein 1.
subset-specific set of traffic molecules. We have demonstrated that the homing of T SCM s to the BM is dependent on adhesive interactions between T SCM s and BM vessels. Furthermore, the blocking experiment with certain antibodies resulted in a dramatic decline of BM-resident T SCM s in vivo, whereas the CD8 + T SCM s were arrested in the SP. CD8 + T SCM s, as a long-term subset, would be relocated to the immune organs in favor of their own self-renewal. SP, as the largest peripheral lymphoid organ, is one of the main locations at which the CD8 + T SCM s are arrested. Therefore, the migration of CD8 + T SCM s to the SP is indispensable when their migration to the BM is blocked. This phenomenon suggests that all 4 adhesion molecules analyzed in our study, namely P-selectin, E-selectin, PSGL-1, and VCAM-1, act as mediators for the homing of T SCM s into the BM. A significant decline of T SCM s in the SP was observed after injection of anti-PSGL-1. Of note, P-selectin and E-selectin as well as VCAM-1 are enriched in BM microvessels, whereas PSGL-1, the ligand of P-selectin and E-selectin, is expressed on most immune cells and facilitates the homing of resting T cells into lymphoid organs. 48 Therefore, the mechanism underlying the presence of T SCM s in the BM involves adhesive interactions between PSGL-1 and P-selectin or E-selectin, whereas T SCM s appear to home to the SP in a PSGL-1-independent manner. In particular, VCAM-1, the adhesion protein expressed in T SCM s and microvessels, was found to be crucial for the homing of T SCM s into the BM rather than into the SP; however, we have not yet identified the specific tissue-resident molecules that predominate in this process. The administration of various neutralizing antibodies altered the BM microenvironment surrounding the BM-resident CD8 + T SCM s and blocked the homing of CD8 + T SCM s into the BM. Under such conditions, the adoptively transferred CD8 + T SCM s were retained in the PB. Our data elucidate the specific migratory routes of T SCM s to the BM. Nevertheless, there may be additional unidentified factors involved in this process. The induction of adhesion-related molecules appears to be a critical step in the development of a stable T SCM compartment in the BM; however, the underlying mechanism merits further investigation. In contrast to previous reports describing the relocation of CD8 + T CM s in BM, the homing of CD8 + T SCM s did not seem to be dependent on CXCL-12. 18 In a previous study, the dependence of memory T-cell homing to the BM on CXCL12 was strongly implicated, with reduced integrin activation after anti-CXCL12 treatment. 18 We attempted to confirm the changes of integrin activation in CD8 + T SCM s; however, no differences were observed, at least in integrin-α4β7, integrin-α2, and integrin-β1 protein. It was speculated that the CD8 + T SCM subset is different from CD8 + T CM subset at this point, as they are 2 different subsets.
An appropriate microenvironment for BM-resident T SCM s requires conditions that facilitate the homeostasis of T SCM s in specific areas. In particular, the microenvironment in the BM provides not only a homeostatic proliferation signal but also a survival signal by upregulating the expression of Bcl-2 for the maintenance of T SCM s. The cellular DNA content, which was determined by detection using propidium iodide (PI), indicated that BM-resident T SCM s were in a resting status and represented a certain degree of stem cell characteristics; however, the expression of CD69 in BM-resident T SCM s was slightly higher than that in naive T cells. Simultaneously, BM-resident T SCM s could be reactivated rapidly upon exogenous antigen invasion, which suggests that active T SCM s are more likely to reside in areas close to APCs. We speculate that antigen-specific responses may be accompanied by the confluence of T SCM s into large aggregates with several APCs, leading to the activation of T SCM s in the BM. Given that a minority of T SCM s was found in the peripheral immune organs and a large number of T SCM s accumulated in inflammatory sites rapidly, we proposed that these cells utilized the niches in the BM as a refuge, and could be temporarily hidden from antigenic exposure before executing immunologic surveillance.
Our data indicate that the BM-resident T SCM s exert much stronger antitumor activity, which may be instructive for development of tumor immunotherapy. In melanoma patients, high frequencies of tumor-specific T cells were detected. However, most of these cells were anergic or nonresponsive. 49,50 Compared with other subsets, T SCM s show higher antitumor activity. Although the expression of IFN-γ and cell proliferation of natural SP-derived T SCM s was almost equal to those of natural BM-derived T SCM s, we exploited the fact that the microenvironment in the BM could generate more functional and tumor-specific T SCM s in vivo. In addition, we demonstrated that the in vivogenerated T SCM s derived from the BM could significantly extend the survival of mice compared with SP-derived T SCM s. Therefore, the present work showed that the BM microenvironment was more conductive to the induction of functional CD8 + T SCM s; however, the underlying mechanisms remain unknown and need further study. Of note, in the present work, approximately half of the mice with BM T SCM s died, although the tumors were still small. We speculate that tumor metastasis occurs in the B16 model, despite subcutaneous injection as previously described. 51 Therefore, tumor migration may lead to the death of mice in the B16 model, even when the tumor size is not very large. 2 Our data suggest that the selection of an appropriate microenvironment for tumor-specific T SCM s represents a novel strategy to improve the efficacy of anti-tumor immunotherapies.
Overall, our study has demonstrated that T SCM s, a distinct memory cell subset, exist naturally in WT mice and principally accumulate in the BM. In addition, the present findings show that PSGL-1 interacted with P-selectin and E-selectin to mediate the homing of T SCM s to the BM. In addition, VCAM-1 appears to be involved in this process. Moreover, these findings should contribute to the development of effective antitumor immunotherapy strategies by potentially enabling the production of tumor-antigenspecific T SCM s for patients with cancer.

Mice
OT-I, C57BL/6J, and CD45.1 (B6.SJL-Ptprc a Pep3 b /BoyJ) mice were purchased from Jackson Laboratories and bred under specific pathogen-free conditions at Sun Yat-Sen University. All experiments performed on mice were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University.

Flow Cytometry and Sorting
Single-cell suspensions were prepared from SP, mesenteric LN, blood, or BM of individual mice. For cell staining, cells were preincubated in 0.1% bovine serum albumin/phosphate-buffered saline solution of 10 μg/mL anti-FcgRII/III (2.4G2) (BD Pharmingen, San Jose, CA) for 10 minutes at 4°C. The cells were then stained for 20 minutes at 4°C with primary antibodies. For cell sorting, BD FACS AriaII cell sorter (BD Biosciences, San Jose, CA) was used. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (100 ng/mL; Sigma-Aldrich, St. Louis, MO) and ionomycin (1 μg/mL; Sigma-Aldrich) in the presence of 5 μg/mL brefeldin A (Sigma-Aldrich) for 4 hours. Cells were washed twice in phosphate-buffered saline, and then fixed and permeabilized with BD Cytofix/Cytoperm Fixation/Permeabilization Kit. Stained samples were analyzed in BD LSR II Fortessa (BD Biosciences). Flow cytometric data were analyzed with FlowJo (Tree Star) software.

qRT-PCR
Total RNA was isolated with TRIzol reagent (Life Technologies) and then subjected to complementary DNA (cDNA) synthesis with PrimeScript reverse transcription (RT) reagent kit (TaKaRa, Shiga, Japan). All primers were annealed at 37°C and RT was performed at 42°C. Quantitative PCR was performed with SYBR premix Ex Taq II kit (TaKaRa) following the manufacturer's instructions. Sequences of primers are listed in Supplemental

BrdU Labeling
For in vitro labeling with BrdU, cells were stimulated and then incubated with BrdU at a final concentration of 10 µM in cell culture medium before being harvested at 16 hours. For the in vivo proliferation assay, BrdU was diluted at a concentration of 10 mg/mL as a stock solution; then, 200 µL of the stock solution was injected into each mouse. some modifications. 53 Briefly, 2×10 6 CMTPX-labeled (10 μM) TWS119-induced CD8 + T SCM s or vehicle-treated control cells (or SP naive T cells) were mixed with the same number of newly isolated and CFSE-labeled (10 μM) SP cells and then injected intravenously into the CD45.1 recipients. The recipients were sacrificed after 24 hours, and cells from SP and BM were obtained as described to measure CMTPX + /CFSE + ratios by flow cytometry.

B16 Murine Model
B16 cells were kindly provided by Professor Jie Zhou and B16-OVA cells were established as described. 31,54 Briefly, we extracted OVA mRNA from hen oviduct and obtained cDNA by RT. OVA-expressing plasmid was constructed by inserting the OVA cDNA into retroviral vector plasmid pMSCV-puro (Clontech Laboratories Inc.) to generate plasmid pMSCV-OVA. pMSCV-OVA was cotransfected with the pIK packaging plasmid into 293T cells using the calcium phosphate transfection method. Forty-eight hours after transfection, supernatants were collected and incubated with B16 cells. The transfected cells were incubated for 24 hours in the presence of polybrene (2.5 μg/mL; Sigma-Aldrich). Puromycin (4 μg/mL; Sigma-Aldrich) was then used to select the stably transfected cells over a 12-day period. Next, C57BL/6J mice were injected intradermally with 1.5×10 6 B16-OVA cells. After 10 days, 5×10 4 OVA-specific T SCM s, which were isolated from SP or BM of CD45.1 recipient mice after injection of OVA (0.5 mg per mouse) with CFA and TWS119 (40 mg/kg), were adoptively transferred into the tumor-bearing WT mice. The tumor burden was measured by testing tumor size and survival rates.