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• W2078656162 abstract "Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/α4β1 pathway. α4β1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Gαi proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood. Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/α4β1 pathway. α4β1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Gαi proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood. Although the importance of the BM in hematopoiesis is well known, its function in T cell-mediated immunity is only partly understood. Studies in mice have shown that the BM accumulates activated/memory T cells after exposure to environmental cognate antigens (Ags) (Price and Cerny, 1999Price P.W. Cerny J. Characterization of CD4+ T cells in mouse bone marrow. I. Increased activated/memory phenotype and altered TCR Vbeta repertoire.Eur. J. Immunol. 1999; 29: 1051-1056Crossref PubMed Google Scholar) and promotes the long-term persistence of antiviral memory cells (Kuroda et al., 2000Kuroda M.J. Schmitz J.E. Seth A. Veazey R.S. Nickerson C.E. Lifton M.A. Dailey P.J. Forman M.A. Racz P. Tenner-Racz K. Letvin N.L. Simian immunodeficiency virus-specific cytotoxic T lymphocytes and cell-associated viral RNA levels in distinct lymphoid compartments of SIVmac-infected rhesus monkeys.Blood. 2000; 96: 1474-1479PubMed Google Scholar, Marshall et al., 2001Marshall D.R. Turner S.J. Belz G.T. Wingo S. Andreansky S. Sangster M.Y. Riberdy J.M. Liu T. Tan M. Doherty P.C. Measuring the diaspora for virus-specific CD8+ T cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 6313-6318Crossref PubMed Scopus (246) Google Scholar, Slifka et al., 1997Slifka M. Whitmire J. Ahmed R. Bone marrow contains virus-specific cytotoxic T lymphocytes.Blood. 1997; 90: 2103-2108PubMed Google Scholar). In some circumstances, it can even support the priming of naive T cells (Feuerer et al., 2003Feuerer M. Beckhove P. Garbi N. Mahnke Y. Limmer A. Hommel M. Hammerling G.J. Kyewski B. Hamann A. Umansky V. Schirrmacher V. Bone marrow as a priming site for T-cell responses to blood-borne antigen.Nat. Med. 2003; 9: 151-157Crossref Google Scholar, Tripp et al., 1997Tripp R.A. Topham D.J. Watson S.R. Doherty P.C. Bone marrow can function as a lymphoid organ during a primary immune response under conditions of disrupted lymphocyte trafficking.J. Immunol. 1997; 158: 3716-3720PubMed Google Scholar). BM-resident T cells are also important for BM transplantation (BMT) because contaminating T cells in BM allografts can cause graft versus host disease (GvHD). On the other hand, T cell depletion from allogenic BM grafts compromises engraftment (Martinez et al., 1999Martinez C. Urbano-Ispizua A. Rozman C. Marin P. Rovira M. Sierra J. Montfort N. Carreras E. Montserrat E. Immune reconstitution following allogeneic peripheral blood progenitor cell transplantation: comparison of recipients of positive CD34+ selected grafts with recipients of unmanipulated grafts.Exp. Hematol. 1999; 27: 561-568Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) because CD8+ (but not CD4+) T cells and dendritic cells (DCs) facilitate engraftment (Adams et al., 2003Adams G.B. Chabner K.T. Foxall R.B. Weibrecht K.W. Rodrigues N.P. Dombkowski D. Fallon R. Poznansky M.C. Scadden D.T. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment.Blood. 2003; 101: 45-51Crossref PubMed Scopus (42) Google Scholar, Gandy et al., 1999Gandy K.L. Domen J. Aguila H. Weissman I.L. CD8+TCR+ and CD8+TCR− cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers.Immunity. 1999; 11: 579-590Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Zeng et al., 2002Zeng D. Hoffmann P. Lan F. Huie P. Higgins J. Strober S. Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation.Blood. 2002; 99: 1449-1457Crossref PubMed Scopus (58) Google Scholar). Indeed, BM-resident T cells are functionally distinct from those in other compartments. Compared to their blood-derived cousins, BM-derived CD8+ T cells induce milder GvHD and tumor-specific cytotoxic lymphocytes (CTL) are more frequent and possess higher antitumor activity in BM than in blood of cancer patients (Feuerer et al., 2001aFeuerer M. Beckhove P. Bai L. Solomayer E.F. Bastert G. Diel I.J. Pedain C. Oberniedermayr M. Schirrmacher V. Umansky V. Therapy of human tumors in NOD/SCID mice with patient-derived reactivated memory T cells from bone marrow.Nat. Med. 2001; 7: 452-458Crossref PubMed Scopus (236) Google Scholar, Feuerer et al., 2001bFeuerer M. Rocha M. Bai L. Umansky V. Solomayer E.F. Bastert G. Diel I.J. Schirrmacher V. Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients.Int. J. Cancer. 2001; 92: 96-105Crossref PubMed Scopus (114) Google Scholar, Zeng et al., 2002Zeng D. Hoffmann P. Lan F. Huie P. Higgins J. Strober S. Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation.Blood. 2002; 99: 1449-1457Crossref PubMed Scopus (58) Google Scholar). Thus, the BM is being considered with growing interest as a source of T cells for cancer therapy (Schirrmacher et al., 2003Schirrmacher V. Feuerer M. Fournier P. Ahlert T. Umansky V. Beckhove P. T-cell priming in bone marrow: the potential for long-lasting protective anti-tumor immunity.Trends Mol. Med. 2003; 9: 526-534Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Here, we asked how T cells accumulate in the BM and how they differ from T cells elsewhere. BM stroma cells can support lymphoid precursor cell differentiation into mature T cells in vitro (Garcia-Ojeda et al., 1998Garcia-Ojeda M.E. Dejbakhsh-Jones S. Weissman I.L. Strober S. An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation.J. Exp. Med. 1998; 187: 1813-1823Crossref PubMed Scopus (47) Google Scholar) and in athymic mice in vivo (Tsark et al., 2001Tsark E.C. Dao M.A. Wang X. Weinberg K. Nolta J.A. IL-7 enhances the responsiveness of human T cells that develop in the bone marrow of athymic mice.J. Immunol. 2001; 166: 170-181PubMed Google Scholar). However, T cells in wild-type (wt) BM are probably immigrants from the blood because T cells are normally produced in the thymus. Indeed, the BM continuously recruits circulating hemopoietic stem cells (HSCs) (Wright et al., 2001Wright D.E. Wagers A.J. Gulati A.P. Johnson F.L. Weissman I.L. Physiological migration of hematopoietic stem and progenitor cells.Science. 2001; 294: 1933-1936Crossref PubMed Scopus (727) Google Scholar) because BM microvessels constitutively express prerequisite traffic molecules (Mazo and von Andrian, 1999Mazo I.B. von Andrian U.H. Adhesion and homing of blood-borne cells in bone marrow microvessels.J. Leukoc. Biol. 1999; 66: 25-32Crossref PubMed Scopus (94) Google Scholar), which might also support homing of T cell subsets. After exiting the thymus, naive T cells search for Ag by recirculating between blood and secondary lymphoid organs (SLOs). Upon Ag stimulation, T cells proliferate and become effector cells (TEffs); CD8+ TEffs produce cytokines, especially interferon (IFN)-γ, and become CTL. Upon Ag clearance most TEffs die, but a few give rise to memory cells providing long-term protection. Memory cells are subdivided into two subsets based on their migration pattern (Sallusto et al., 1999Sallusto F. Lenig D. Forster R. Lipp M. Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature. 1999; 401: 708-712Crossref PubMed Scopus (4332) Google Scholar): effector memory cells (TEMs) migrate to peripheral tissues to eliminate pathogens, whereas TCMs express homing molecules, allowing them to migrate to SLOs, particularly lymph nodes (Weninger et al., 2001Weninger W. Crowley M.A. Manjunath N. von Andrian U.H. Migratory properties of naive, effector, and memory CD8(+) T cells.J. Exp. Med. 2001; 194: 953-966Crossref PubMed Scopus (406) Google Scholar). We show here that TCMs constitute the largest endogenous subset of CD8+ T cells in murine BM and are also prominent in human BM. Accordingly, adoptively transferred TCMs from immunized mice colonized recipient BM more effectively than TEMs and naive T cells. The mechanisms by which in vivo-generated memory cell subsets are recruited to tissues have been difficult to study, because such studies require unattainable numbers of purified cells. We were able to circumvent this obstacle by using a recently described in vitro method to generate Ag-specific TEffs and TCMs from TCR transgenic CD8+ T cells (Manjunath et al., 2001Manjunath N. Shankar P. Wan J. Weninger W. Crowley M.A. Hieshima K. Springer T.A. Fan X. Shen H. Lieberman J. von Andrian U.H. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.J. Clin. Invest. 2001; 108: 871-878Crossref PubMed Scopus (347) Google Scholar). After i.v. injection, naive T cells and TCMs were more efficiently recruited to the BM than TEffs. Adoptively transferred TCMs were much more efficiently retained in the BM than other subsets and mounted potent recall responses upon restimulation. By using IVM, we have dissected the multistep adhesion cascade for TCM recruitment to the BM and show that extravasated TCMs move actively within BM cavities. To investigate CD8+ T cell traffic in normal BM, we compared the composition of CD8+ T cells in BM, blood, and SLOs of adult mice. CD8+ T cells comprised 2.5% ± 0.1% of all mononuclear cells (MNCs) in the BM, which is a much smaller fraction than in blood (7.9% ± 1.8%) or spleen (11.8% ± 0.1%; Figure 1A ). Two-thirds of BM-resident T cells expressed surface markers indicative of Ag experience, i.e., they were CD44hi and CD122+, whereas most CD8+ T cells in spleen, peripheral lymph nodes (PLNs), and blood were naive (i.e., CD44lo/−CD122−; Figure 1B). Thus, the BM differs from classic SLOs in terms of its accumulation of memory cells. Memory T cells are subdivided based on their expression of CCR7 (Sallusto et al., 1999Sallusto F. Lenig D. Forster R. Lipp M. Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature. 1999; 401: 708-712Crossref PubMed Scopus (4332) Google Scholar). TCMs are CCR7+ and mostly coexpress L-selectin. They home efficiently to PLN and other SLOs, whereas CCR7− TEffs and the longer-lived TEMs are largely L-selectin– and accumulate in nonlymphoid tissues and to some degree in the spleen (Sallusto et al., 1999Sallusto F. Lenig D. Forster R. Lipp M. Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature. 1999; 401: 708-712Crossref PubMed Scopus (4332) Google Scholar, Weninger et al., 2001Weninger W. Crowley M.A. Manjunath N. von Andrian U.H. Migratory properties of naive, effector, and memory CD8(+) T cells.J. Exp. Med. 2001; 194: 953-966Crossref PubMed Scopus (406) Google Scholar). Thus, we examined CCR7 expression on CD44hi (memory) and CD44lo (naive) CD8+ T cells by using CCL19-Ig chimera as a probe (Manjunath et al., 2001Manjunath N. Shankar P. Wan J. Weninger W. Crowley M.A. Hieshima K. Springer T.A. Fan X. Shen H. Lieberman J. von Andrian U.H. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.J. Clin. Invest. 2001; 108: 871-878Crossref PubMed Scopus (347) Google Scholar). As expected, naive T cells in all organs were CCR7+ and L-selectin+, whereas CD44hi T cells expressed these markers variably (Figure 1C). CCR7− TEMs bound P-selectin (Table S1 available with this article online and data not shown) and represented approximately half of all memory cells in spleen and blood. By contrast, TEMs were more sparse among BM CD8 cells where the largest subset was comprised of CCR7+L-selectin+ TCMs that did not bind P-selectin. Consequently, although the spleen harbored many more naive CD8+ T cells and TEMs than the BM, the number of TCMs was similar in both organs. Thus, mouse BM is a major reservoir for Ag-experienced CD8+ T cells, most of which share a TCM phenotype. We also examined CD3+CD8+ T cells in BM from healthy human donors by using mAbs specific for CCR7 and CD45RA (Sallusto et al., 1999Sallusto F. Lenig D. Forster R. Lipp M. Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature. 1999; 401: 708-712Crossref PubMed Scopus (4332) Google Scholar). As shown in Figure 1D, naive T cells contributed only 21% of CD8+ T cells, whereas the largest subsets (∼30% each) were TCMs (CD45RA−CCR7+) and TEMs (CD45RA−CCR7−); a smaller fraction was comprised of TEffs (CD45RA+CCR7−). Similar results were obtained with two anti-CCR7 mAbs (clones 2H4 and 3D12), whereas a third anti-CCR7 mAb, 6B3 (Hasegawa et al., 2000Hasegawa H. Nomura T. Kohno M. Tateishi N. Suzuki Y. Maeda N. Fujisawa R. Yoshie O. Fujita S. Increased chemokine receptor CCR7/EBI1 expression enhances the infiltration of lymphoid organs by adult T-cell leukemia cells.Blood. 2000; 95: 30-38Crossref PubMed Google Scholar), stained nearly 70% of CD45RA− CD8+ T cells (not shown). Thus, mammalian BM is a rich source of TCMs not only in mice kept in a pathogen-free facility but also in people. The relatively larger proportion of CCR7− T cells in human versus murine BM might reflect the constant exposure to infectious Ags experienced in normal human life. To ask if this might also apply to mice, we transferred into wt recipients naive P14 CD8+ T cells expressing a TCR for LCMV gp33–41 in H2-Db. 1 day and again 3 weeks later, the mice received i.v. injections of mature peptide-pulsed DCs. After 5 more weeks, all CD8+ T cells detected by a P14 TCR-specific MHC tetramer were CD44high in both BM and spleen. At least as many TEMs as TCMs were found in recipients’ spleens, whereas TCMs in BM outnumbered TEMs. However, the TCM/TEM ratio in BM after Ag challenge (1.8:1) was markedly reduced compared to untreated pathogen-free mice (4.4:1), indicating that mouse BM can harbor substantial numbers of newly generated memory cells other than TCMs (Figure S1). Next, we asked whether the prevalence of TCMs in BM could be explained by their migratory properties. Thus, P14 memory cells were generated in vivo as above, and purified T cells (CD45.2+ containing 1.01% tetramer+ cells) were transfused into CD45.1 recipients. 2 hr later, tetramer+ memory cells were markedly enriched among CD45.2+ T cells in recipient spleens and, especially, BM compared to their input frequency (Figure 2A ). This effect was mostly due to selective accumulation of TCMs, which became 3.5-fold enriched in both tissues compared to TEMs. Nevertheless, the tetramer+ TEM concentration in BM was significantly higher than in the input, indicating TEMs have enhanced BM tropism, albeit less pronounced than TCMs. To identify the molecular mechanisms of memory CD8+ T cell homing to the BM, much larger numbers of purified memory subsets are needed than for simple homing experiments. Because it is not practical to obtain the required cell numbers from immunized mice, we resorted to in vitro production of TEffs and TCMs from naive T cells (Manjunath et al., 2001Manjunath N. Shankar P. Wan J. Weninger W. Crowley M.A. Hieshima K. Springer T.A. Fan X. Shen H. Lieberman J. von Andrian U.H. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.J. Clin. Invest. 2001; 108: 871-878Crossref PubMed Scopus (347) Google Scholar). We used T-GFP×P14 mice, in which naive T cells and TCMs express GFP, and effector CTLs become GFP− (Weninger et al., 2002Weninger W. Manjunath N. von Andrian U.H. Migration and differentiation of CD8+ T cells.Immunol. Rev. 2002; 185: 221-233Crossref Scopus (108) Google Scholar). Peptide-primed T-GFP×P14 T cells can be differentiated into GFP− TEffs or GFP+ TCM-like cells by several days exposure to IL-2 (CD8IL-2) or IL-15 (CD8IL-15), respectively (Manjunath et al., 2001Manjunath N. Shankar P. Wan J. Weninger W. Crowley M.A. Hieshima K. Springer T.A. Fan X. Shen H. Lieberman J. von Andrian U.H. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.J. Clin. Invest. 2001; 108: 871-878Crossref PubMed Scopus (347) Google Scholar). Their response to recall Ag and ability to migrate to SLOs and inflamed tissue were described previously (Goodarzi et al., 2003Goodarzi K. Goodarzi M. Tager A.M. Luster A.D. von Andrian U.H. Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to inflamed tissues.Nat. Immunol. 2003; 4: 965-973Crossref PubMed Scopus (276) Google Scholar, Manjunath et al., 2001Manjunath N. Shankar P. Wan J. Weninger W. Crowley M.A. Hieshima K. Springer T.A. Fan X. Shen H. Lieberman J. von Andrian U.H. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.J. Clin. Invest. 2001; 108: 871-878Crossref PubMed Scopus (347) Google Scholar, Weninger et al., 2001Weninger W. Crowley M.A. Manjunath N. von Andrian U.H. Migratory properties of naive, effector, and memory CD8(+) T cells.J. Exp. Med. 2001; 194: 953-966Crossref PubMed Scopus (406) Google Scholar). In vitro differentiated CD8IL-2 and CD8IL-15 cells are phenotypically similar to endogenous BM-resident TEMs and TCMs, respectively. This applies to the expression numerous traffic molecules, including CCR7; CXCR4; L-selectin; the integrins CD11a, CD11b, α4, α5, and α4β7; as well as PSGL-1 (Manjunath et al., 2001Manjunath N. Shankar P. Wan J. Weninger W. Crowley M.A. Hieshima K. Springer T.A. Fan X. Shen H. Lieberman J. von Andrian U.H. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.J. Clin. Invest. 2001; 108: 871-878Crossref PubMed Scopus (347) Google Scholar, Weninger et al., 2001Weninger W. Crowley M.A. Manjunath N. von Andrian U.H. Migratory properties of naive, effector, and memory CD8(+) T cells.J. Exp. Med. 2001; 194: 953-966Crossref PubMed Scopus (406) Google Scholar and data not shown). Thus, CD8IL-15 and CD8IL-2 cells represent faithful surrogates for bona fide TCMs and TEffs, respectively, and will be referred to under these names below. To determine whether in vitro-differentiated TCMs and TEffs migrate similarly to memory cells induced in situ, TCMs and TEffs were labeled red with TRITC and mixed with T-GFP×P14 splenocytes containing ∼30%–35% naive GFP+CD8+ T cells, which served as a reference. Cells were injected into naive mice and the homing index (HI; the ratio of TRITC+:GFP+ naive cells) was determined in various tissues 2 or 24 hr later. At 2 hr after injection, TCMs were most prominent in the blood, whereas naive T cells homed best to SLOs (Figure 2B). TEffs were recovered from all recipient tissues and the blood but at a lower concentration, probably because many of these relatively large blasts were sequestered in lung and liver (Weninger et al., 2001Weninger W. Crowley M.A. Manjunath N. von Andrian U.H. Migratory properties of naive, effector, and memory CD8(+) T cells.J. Exp. Med. 2001; 194: 953-966Crossref PubMed Scopus (406) Google Scholar). The BM was the only organ containing equivalent numbers of TCMs and naive T cells at 2 hr after injection. At 24 hr after transfer, the HI of TCMs and naive T cells was equivalent in PLN and blood and increased in the spleen, consistent with previous findings (Weninger et al., 2001Weninger W. Crowley M.A. Manjunath N. von Andrian U.H. Migratory properties of naive, effector, and memory CD8(+) T cells.J. Exp. Med. 2001; 194: 953-966Crossref PubMed Scopus (406) Google Scholar) (Figure 2C). Although naive T cells were most frequent in the blood, the HI in BM had increased significantly for TCMs and, to a lesser degree, also for TEffs. This shift in HI in the BM between 2 and 24 hr is explained by the fact that both naive T cells and TCMs were rapidly and efficiently recruited to the BM, but only TCMs were retained at this site, whereas naive T cells returned quickly to the circulation (Figure 2D). TEffs homed less well to the BM, but they too were efficiently retained. Thus, our homing experiments are in excellent agreement with our analysis of endogenous BM-resident T cells. Of note, there was a linear relationship between the number of homed and injected TCMs (Figure 2E), indicating that homing was not limited by competition with endogenous cells or the BM’s capacity to recruit TCMs. Having determined that TCMs possess BM tropism, we focused our further analysis on this subset. First, we employed multiphoton IVM to observe TCMs within BM cavities in skulls of anesthetized mice (Mazo et al., 1998Mazo I.B. Gutierrez-Ramos J.-C. Frenette P.S. Hynes R.O. Wagner D.D. von Andrian U.H. Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and VCAM-1.J. Exp. Med. 1998; 188: 465-474Crossref PubMed Scopus (353) Google Scholar). Optical serial sections were rendered as 3D images of BM cavities in which blood vessels were delineated by FITC-dextran (green) and hemopoietic tissue was stained with rhodamine 6G (red). Extravascular TCMs carrying blue fluorescence were found within 3 hr after injection (Figure 2F, Movie 1). 3D time-lapse movies showed that the homed TCMs were highly motile and migrated at 6.5 ± 0.2 μm/min (mean ± SEM, n = 189 cells), often over long distances (Movie 2). TCM movement was confined to the rhodamine 6G-filled BM cavities and did not occur in adjacent bone but otherwise was random in direction. Next, we performed epifluorescence-based video IVM in skull BM (Figure 3). Although this technique does not permit 3D imaging, the fast acquisition rate (30 frames/s) is useful to dissect the molecular mechanisms of rapid adhesion events (Mazo et al., 1998Mazo I.B. Gutierrez-Ramos J.-C. Frenette P.S. Hynes R.O. Wagner D.D. von Andrian U.H. Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and VCAM-1.J. Exp. Med. 1998; 188: 465-474Crossref PubMed Scopus (353) Google Scholar). Fluorescently labeled naive CD8+ T cells, TCMs or TEffs were injected into anesthetized mice and their passage through BM microvessels was recorded (Movie 3). TCMs rolled ∼1.6 times more frequently in BM vessels than TEffs, and the frequency at which rolling cells arrested (sticking fraction) was ∼2.2-fold higher (Figure 3A). Consequently, the frequency at which cells entering a microvessel completed the entire multistep adhesion cascade (sticking efficiency) was 3.8-fold higher for TCMs than TEffs. These findings agree well with our homing experiments and analysis of BM resident memory subsets. Naive T cells underwent rolling and sticking interactions that were similar in frequency to those of TCMs (data not shown) and in line with short-term homing experiments (Figure 2B). However, the low frequency of long-term resident naive T cells suggests that additional, as yet unidentified, factors influence the magnitude of steady-state CD8+ T cell subsets in BM. Normal BM sinusoids express P- and E-selectin as well as VCAM-1 (Mazo et al., 2002Mazo I.B. Quackenbush E.J. Lowe J.B. von Andrian U.H. Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow.Blood. 2002; 99: 4182-4191Crossref PubMed Scopus (74) Google Scholar). The latter is a ligand for α4β1 (VLA-4), which mediates stem cell rolling in BM microvessels (Mazo et al., 1998Mazo I.B. Gutierrez-Ramos J.-C. Frenette P.S. Hynes R.O. Wagner D.D. von Andrian U.H. Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and VCAM-1.J. Exp. Med. 1998; 188: 465-474Crossref PubMed Scopus (353) Google Scholar). However, TCM rolling was normal in BM of wt mice treated with anti-VCAM-1 (Figure 3B) and in mice with a conditional deficiency in endothelial VCAM-1 (Figure 3E). Moreover, TCM treatment with anti-β7 had no effect, indicating that α4β7 is also not involved. By contrast, anti-L-selectin reduced rolling by 64%, whereas mAbs to P- and E-selectin attenuated it by 49% and 46%, respectively (Figure 3B). TCM rolling was also reduced by 48% in mice deficient in PSGL-1, a major selectin ligand (Figure 3C, right). A combination of mAbs to P- and E-selectin blocked additively (by 78%), but there was no further inhibition when anti-L-selectin was added. This suggests that L-selectin mediated secondary tethering whereby endogenous leukocytes adhere to P- and/or E-selectin in BM microvessels and present PSGL-1 as an L-selectin ligand to circulating TCMs (Bargatze et al., 1994Bargatze R.F. Kurk S. Butcher E.C. Jutila M.A. Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells.J. Exp. Med. 1994; 180: 1785-1792Crossref PubMed Scopus (238) Google Scholar, Fuhlbrigge et al., 1996Fuhlbrigge R.C. Alon R. Puri K.D. Lowe J.B. Springer T.A. Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions.J. Cell Biol. 1996; 135: 837-848Crossref PubMed Scopus (88) Google Scholar, Walcheck et al., 1996Walcheck B. Moore K.L. McEver R.P. Kishimoto T.K. Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.J. Clin. Invest. 1996; 98: 1081-1087Crossref PubMed Scopus (292) Google Scholar). Indeed, anti-L-selectin had no effect on TCM rolling in BM of PSGL-1-deficient mice (Figure 3C, left). Thus, the endothelial selectins in BM microvessels interact with PSGL-1 on circulating TCMs, whereas L-selectin on TCMs likely contributes indirectly by allowing TCMs to tether to other adherent leukocytes. In most in vivo settings, rolling leukocytes can only arrest by using activation-dependent integrins (von Andrian and Mackay, 2000von Andrian U.H. Mackay C.R. T-cell function and migration. Two sides of the same coin.N. Engl. J. Med. 2000; 343: 1020-1034Crossref PubMed Scopus (1175) Google Scholar). Because both the β2 integrin LFA-1 and the α4 integrin VLA-4 are highly expressed on TCMs (not shown), we examined these pathways. However, although TCMs use LFA-1 to arrest in PLN HEV (Weninger et al., 2001Weninger W. Crowley M.A. Manjunath N. von Andrian U.H. Migratory properties of naive, effector, and memory CD8(+) T cells.J. Exp. Med. 2001; 194: 953-966Crossref PubMed Scopus (406) Google Scholar), anti-LFA-1 had no effect on TCM sticking in BM venules or sinusoids (data not shown). By contrast, anti-VCAM-1, the major vascular ligand for VLA-4, reduced TCM sticking by 77% (Figure 3D) and TCM sticking was also 72% lower in conditional VCAM-1 knockout mice than in wt mice (Figure 3E). The importance of VCAM-1 was confirmed in homing experiments; the BM of conditional VCAM-1 knockout mice recruited significantly fewer TCMs than wt BM (Figure 3F). However, it is possible that this result reflects not only decreased TCM sticking, because VCAM-1 might also participate in subsequent diapedesis. TCMs also express α4β7 (data not shown), which can bind to VCAM-1, but inhibition of this integrin did not affect sticking (Figure 3D). Therefore, VCAM-1–VLA-4 is the principal pathway for TCM arrest in BM microvessels. However, because sticking was not completely abolished without VCAM-1, there is probably at least one additional mechanism for TCM sticking. Integrins support leukocyte sticking only in their high-affinity conformation, which requires activation signal(s) from chemoattractant receptor(s). G protein-coupled receptors (GPCRs), e.g., those for chemokines, can rapidly transmit integrin activation signals. Because most chemoattractant receptors signal through Gαi, they are inhibitable by PTX. Therefore, we performed competitive homing assays comparing differentially labeled, PTX-treated TCMs with sham-treated control cells. As expected, 2 hr after injection," @default.
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• W2078656162 date "2005-02-01" @default.
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• W2078656162 title "Bone Marrow Is a Major Reservoir and Site of Recruitment for Central Memory CD8+ T Cells" @default.
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• W2078656162 doi "https://doi.org/10.1016/j.immuni.2005.01.008" @default.
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