- Email: [email protected]
CD8 T-cell memory: the other half of the story
Microbes and Infection 5 (2003) 221–226 www.elsevier.com/locate/micinf
Forum in immunology
CD8 T-cell memory: the other half of the story David Masopust, Leo Lefrançois * Division of Immunology, Department of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, MC1319, Farmington, CT 06030-1319, USA
Abstract Historically, most immune response studies have been limited to analyses of lymphoid tissue. However, peripheral sites of infection are likely to represent important sites of cell-mediated immune surveillance and effector function. Recent debates have centered on the persistence, trafficking patterns, effector activity, and protective role of non-lymphoid memory T cells. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Infection; Memory; T cell; Migration; Non-lymphoid
1. Introduction Recent technological innovations such as the adoptive transfer system [1] and MHC tetramers [2], both of which allow tracking and identification of antigen-specific T cells, are driving major changes in our perceptions of cellular immunity. Consequently, our understanding of the dynamics of CD4 and CD8 T-cell responses is rapidly expanding. Nevertheless, many of the concepts related to the T-cell immune response are based on analyses of lymphocytes located in secondary lymphoid tissues. However, nonlymphoid peripheral organs may represent important T-cell effector sites in the context of infection, allograft rejection, and autoimmune disease. Lymphoid and non-lymphoid environments differ in many respects, including the density and quality of lymphocytes, antigen presenting cells, cytokines and innate immune system components, all of which are factors with important bearing on the evolution of adaptive immune responses. There are many critical questions which remain unresolved regarding non-lymphoid immunity including: 1. Can studies of lymphoid T-cell populations be extrapolated to non-lymphoid populations? Or, are these populations functionally distinct? 2. Are activated T cells only targeted to specific sites of inflammation/infection? 3. What is the trafficking pattern of memory T cells? Do they persist in or recirculate through non-lymphoid * Corresponding author. Tel. +1-860-679-3242; fax: +1-860-679-1868. E-mail address: [email protected] (L. Lefrançois). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 1 4 - 5
tissues and do they home preferentially to the original site of infection? 4. What are the relative roles of lymphoid vs. nonlymphoid effector and memory T cells in protection from infection and re-infection? Recently, several important findings have begun to shed light on some of these issues. It is clear that non-lymphoid T-cell populations hold many surprises. Subjecting these cells to further scrutiny will provide us with a more complete understanding of the dynamics of T-cell-mediated immunity.
2. Current paradigms for migration pathways of naive, activated and memory T cells Our current model of T-cell trafficking is based on a large body of elegant in vitro and in vivo experimentation and a complete treatment of this topic is beyond the scope of this article. Early studies demonstrated that thoracic duct lymph (TDL), which empties into the blood, contains many lymphocytes [3]. The source and fate of this population were of considerable controversy. In a landmark experiment, Gowans and Knight [4] transferred radiolabeled thoracic duct lymphocytes intravenously (i.v.) into naive syngeneic recipients and detected donor lymphocytes in their TDL. This provided a formal demonstration that lymphocytes recirculate continuously between blood and lymph. These investigators also performed autoradiographic analyses of high endothelial venules (HEVs) within lymph nodes (LNs) and demonstrated convincingly that small lymphocytes cross HEVs to enter LNs. Small lymphocytes (which would be
222
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
made up of primarily naive T cells but would contain some memory cells) were detected in LN, white pulp of spleen, and Peyer’s patches (PPs). The number of small lymphocytes in other tissues was minor in comparison providing an early suggestion that naive lymphocytes may not enter nonlymphoid tissues. With this and many other groundbreaking studies, a picture at the molecular level of the pathway of naive T-cell migration has emerged. Blood-borne naive T cells enter into LNs via interactions with receptor/ligand pairs expressed by the endothelium in the HEV and lymphocytes [5–7]. This is a highly orchestrated multistep process, which involves selectins to initiate lymphocyte rolling, chemokines to activate integrins, and activated integrins to trigger firm adhesion and eventual transendothelial migration into the LN. Lymphocytes can then exit the LNs via efferent lymphatics and eventually drain into the thoracic duct and the recirculatory process begins anew. In the case of the spleen, HEV are not present and so this pathway is not involved, but it is now clear that chemokines and no doubt other adhesion molecules are responsible for naive T- and B-cell migration into the appropriate areas of the splenic sub architecture [8]. Experiments have also demonstrated that naive lymphocytes rarely enter non-lymphoid tissue. This has been shown using short-term in vivo migration studies as well as adoptive transfer of trackable naive CD8 TCR transgenic T cells, which did not enter the intestinal lamina propria (LP) or epithelium unless activated [9]. Nevertheless, small populations of CD8 T cells with naive phenotypes can be found in the intestinal mucosa, liver and lung (our unpublished observations) suggesting that limited traffic of naive cells through non-lymphoid tissues may occur. Westermann and colleagues [10,11] have also argued that there is limited trafficking of naive T cells through skin and liver. More definitive analyses are required to prove this possibility. When T cells are activated following encounter with antigen in an LN, their homing program is dramatically altered at the lymphocyte level. In addition, the endothelium may also be activated in the face of local or systemic inflammation. The migration of activated CD8 T cells into the intestinal mucosa provides an excellent system for analysis of T-cell migration into tertiary lymphoid tissue. The intestinal immune system is composed of secondary lymphoid inductive sites (PPs and mesenteric LNs) and tertiary effector sites, the LP and the epithelium [12]. The vast majority of resident lymphocyte populations in the latter express activation and/or memory markers [13,14]. In the transfer studies of Gowans and Knight [4], large lymphocytes appeared to marginate primarily to gut (which included MLN and LP in this case) rather than PLN, although a few cells were detected in the liver and kidney using autoradiography, which allowed direct visualization of live cells within tissue. Later studies by Sprent [15] confirmed and extended this work regarding the intestine using a system of transfer of activated alloreactive lymphocytes. However, a detailed analysis of other tissues was not performed. In more recent studies in which the identity of transferred cells could be precisely determined,
only after activation via virus infection did TCR transgenic CD8 T cells migrate to the intestinal LP and epithelium in an a4b7-integrin-dependent pathway [16]. These results were corroborated in studies using vesicular stomatitis virus infection and detection of endogenous antigen-specific CD8 T cells with MHC class I tetramers [17]. Primary infection with a variety of viruses also leads to appearance of antigenspecific CD8 T cells in non-lymphoid organs. For example, after infection with either lymphocytic choriomeningitis virus (LCMV) [18] or influenza virus [19], antigen-specific CD8 T cells are found in bone marrow. Also, quantitation of the primary response to intranasal infections with influenza virus [20], Sendai virus [21] or gammaherpes virus [22] identified substantial numbers of antigen-specific CD8 T cells in the lung which in all likelihood migrated to that site after primary activation in draining LN. In one study, influenza-specific CD8 T cells were also found in the liver after intranasal infection [23]. This latter result is of interest considering that influenza virus only productively infects lung epithelial cells. Thus, the entry of activated CD8 T cells into the liver was not dependent on local virus production. Overall, these studies demonstrate that activated T cells migrate to sites of infection and may traffic to other, potentially uninfected tissues. Distinguishing between activated and memory T cells has historically been difficult. The use of adoptive transfer and MHC tetramers has greatly improved our ability to identify bona fide naive, effector and memory cells. To discriminate primary effector from memory cells, cell size appears to be a viable criterion and this along with phenotypic analysis of transferred or tetramer+ cells allows reasonable, if not absolute, identification of these subsets. Earlier studies by Mackay et al. [24] found that T lymphocytes in afferent lymph draining from tissues into the popliteal LN of sheep were largely CD44hi, a phenotype associated with activation or memory. While no definitive marker for memory vs. activated T cells existed at that time, cell size data indicated that at least some of this population represented true memory T cells. Further studies using sheep demonstrated that the afferent lymph of intestinal tissue also contained high percentages of memory phenotype T cells [25]. These results helped establish the current hypothesis that memory T cells circulate in the blood, extravasate into non-lymphoid tissues, drain into LNs via afferent lymph, and subsequently return to the blood stream via efferent lymphatics. Since most T cells in efferent lymph are of a naive phenotype (CD44lo) [24], memory/activated cells make up only a small percentage of cells migrating through LN from the tissues. 3. Tissue-specific migration of activated and memory T cells The best examples of this phenomenon regard trafficking of T cells to the skin and intestinal mucosa. Picker et al. [26] demonstrated that following DTH reactions in human skin, about 50% of the T cells within the inflammatory site express
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
cutaneous associated lymphocyte antigen (CLA), which binds E-selectin expressed by endothelial cells [27]. As most T cells within blood do not express CLA, these data suggest that CLA is involved in migration into the skin. CLA expression could also be induced on in vitro activated T cells by adding certain cytokines to the culture. This process was more efficient using T cells derived from PLN rather than MLN. This result led to the notion that the particular lymphoid environment in which priming took place, imprinted upon activated T cells tropism for particular tissues. For example, priming in inguinal LNs would pre-dispose T cells to exhibit skin homing properties. Mediastinal LNs would generate lung specific responses, etc. Support for this hypothesis was also obtained in analyses of intestinal mucosal lymphoid tissue [25]. As noted above, the a4b7 integrin is involved in migration of activated T cells to the gut. However, it is important to note that activated T-cell migration to skin or gut is not absolutely dependent on expression of CLA [26] or a4b7, respectively [16,17]. Thus, approximately half of the T cells within inflamed skin lack CLA indicating that CLA is either down-regulated once within the tissue, or that there is some CLA independent migration into skin. In the case of the intestinal mucosa, most migration of activated antigen-specific CD8 T cells into the mucosa is a4b7dependent, but nevertheless a small population of cells traffic to the intestine in the absence of this integrin [16,17]. Memory cells in human blood have also been divided into tissue-specific subsets based on expression of CLA, a4b7 and chemokine receptors [28]. The expression of CLA and a4b7 by CD4+ CD45RA-small lymphocytes (presumably memory cells) is essentially mutually exclusive potentially defining two unique recirculating pools [28]. Furthermore, a large proportion of CLA+ memory cells express CCR4, while intestinal memory cells expressing a4b7 lack CLA and CCR4 [28,29]. The CCR4 ligand, TARC, is expressed in inflamed skin but is not expressed in intestinal tissue, further supporting the concept that memory cells may home in a tissue-specific manner. Whether this is reflective of the site of activation in the primary response remains to be determined. Also unknown is whether such memory cells continuously travel through non-lymphoid tissues or whether re-encounter with antigen and tissue inflammation is required for their movement out of the bloodstream. Interestingly, antigen-specific CD8 memory cells in the mouse intestinal mucosa express a distinct set of adhesion markers as compared to memory cells in spleen, lung and liver [30,31]. The integrin aEb7 is highly expressed by the majority of mucosal memory cells while very few memory cells outside of the mucosa express this molecule [30,31]. The carbohydrate determinant 1B11 present on CD43 and CD45 molecules [32] is present on nearly all mucosal memory CD8 cells while z1/2 of non-intestinal memory cells express this antigen [31]. While phenotypic evidence is circumstantial, the data suggest that intestinal memory T cells may represent a distinct subset of memory cells that does not intermix with the circulating pool of memory cells.
223
There are several mechanisms by which this situation could occur. Perhaps, after entry and subsequent regulation of cellsurface receptors, a portion of intestinal CD8 memory T cells remains in situ long-term and do not exit the tissue. Alternatively, lymphoid memory cells could enter the intestinal mucosa routinely and modify their cell-surface receptors upon entry. It is also possible that only very small numbers of intestinal memory T cells enter the circulation at any given time point such that recirculation of this population is routine but difficult to detect. Detailed in vivo migration studies are required to answer these questions. Tissue-specific migration of activated and memory T cells has important implications for immunity and vaccination. Such a system increases the efficiency of adaptive immune responses by limiting the volume of tissue that individual antigen-specific T cells are required to patrol. For example, restricting the traffic of naive T cells through secondary lymphoid tissue no doubt dramatically increases the speed with which rare antigen-specific naive precursors would encounter cognate antigen in the context of the appropriate anatomical and immunostimulatory environment. This theory suggests that activated T cells migrate preferentially to sites of inflammation, where they are most needed and that memory T cells migrate through tissues where they are most likely to re-encounter cognate antigen. Avoidance of other non-lymphoid organs may minimize the likelihood of crossreactive autoimmune responses to tissue-specific selfantigens. 4. Central vs. memory T cells: autonomous or tissue-specific regulation? Numerous recent studies have reported the presence of antigen-specific memory T cells within non-lymphoid tissues of mice. For example, transgenic memory CD8 T cells persist within the small intestinal mucosa following i.v. VSV infection [30]. While it has been suggested that the gut may be unique in its capacity to continuously harbor memory T cells, infection with LCMV leads to the appearance of virusspecific memory CD8 T cells within the bone marrow [18]. In addition, intranasal influenza [20] or Sendai virus [21] infections generate endogenous memory T cells within the lung. Oral Listeria monocytogenes infection leads to CD8 T-cell memory within the intestinal mucosa and liver [33]. Collectively, these data demonstrate that memory T cells can be found in multiple non-lymphoid organs. However, in most of these examples, the pathogen is thought to infect the organ in which memory cells are subsequently located. Thus, these studies do not demonstrate whether primary or memory T cells have the capacity to home to any non-lymphoid organ or solely to organs undergoing infection. Proving this point is difficult because although some viruses productively infect only certain tissues, it remains possible that disseminated non-productive infection of remote tissues may lead to some level of inflammation. In addition, robust infection at a local site could result in the release of sufficient inflammatory
224
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
Table 1 Absolute numbers of pathogen-specific CD8 T cells per tissuea Days post-infection 6 8 12 20 35
Spleen 2.94E6 970,833 321,833 109,667 133,833
Three tissues combined 1.903E6 701,167 434,167 132,667 86,000
Lung 761,500 503,667 238,333 41,667 49,833
LP 154,000 113,333 98,500 56,500 21,167
Liver 987,500 84,167 97,333 34,500 15,000
a
Lymphocytes were harvested from tissues at various time points following i.v. infection of C57BL/6 mice with 1 × 106 pfu vesicular stomatitis virus (VSV). Total numbers of antigen-specific CD8 T cells were enumerated by multiplying total lymphocytes isolated per tissue (as counted by light microscopy and trypan blue exclusion) by the percentage of lymphocytes staining positive with tetramerized H-2Kb/RGYVYQGL, representing an immunodominant VSV epitope. Staining with irrelevant tetramers was negligible.
mediators into the bloodstream to activate endothelium resulting in extravasation of T cells and perhaps innate immune system components into uninfected tissues. The concept of two types of memory T cells delineated by homing molecule expression and functional differences has recently been put forth based on analysis of human peripheral blood. Sallusto et al. [34] demonstrated that memory phenotype CD4 T cells can be broken down into at least two subsets based on CCR7 expression. CCR7 mediates entry of naive and perhaps memory T cells into secondary lymphoid organs via interaction with the chemokines CCL19 and CCL21 [8,35]. Moreover, CCR7– memory-phenotype T cells rapidly produced c-IFN and IL-2 after in vitro stimulation, while CCR7+ memory-phenotype T cells produced IL-2 but little c-IFN. In the CD8 compartment, CCR7 expression also distinguished two putative memory subsets and the CCR7– cells expressed perforin while the CCR7+ cells did not. These intriguing results led to the theory that two subsets of memory cells exist, one geared toward migration through secondary lymphoid tissues with low immediate functional ability and a second geared toward migration through non-lymphoid tissues with immediate effector activity. One of the difficulties in these and other studies in which antigen-specific T cells were not analyzed, is the inability to definitively identify memory cells. Thus, one is left with using cell-surface markers to define memory cells which may or may not always be indicative of memory cells. This problem has recently been highlighted by the demonstration that T cells undergoing homeostatic proliferation acquire a memory phenotype but do not develop into true memory cells [36,37]. The degree of homeostatic proliferation occurring under normal circumstances is not known, but nevertheless, caution should be used in using phenotype to define memory T cells. To definitively identify memory cells, the adoptive transfer of antigen-specific TCR transgenic T cells or the use of MHC tetramers to track antigen-specific endogenous T-cell populations are the current methods of choice. The adoptive transfer approach was used by Reinhardt et al. [38] to track ovalbumin-specific TCR transgenic CD4+ T cells in naive and immunized recipients. Migration was evaluated in various contexts by analysis of the location of donor cells in whole mouse histological sections. Donor cells were enumerated by immunohistochemistry on a single section and the total number of ovalbumin-specific CD4 T cells per organ
was calculated by correcting for the total volume of the various tissues. Following transfer of naive CD4 T cells, the donor population was exclusively located within secondary lymphoid tissue of perfused recipients. However, upon injection of lipopolysacharride (LPS), a small number of naive lymphocytes were visualized within non-lymphoid tissue. These data imply that naive T cells may have the ability to traffic through non-lymphoid tissues after ‘bystander’ activation of lymphocytes or perhaps after activation of the endothelium via strong inflammatory signals. Further studies are needed to determine whether naive T-cell migration to nonlymphoid tissues is a common occurrence and whether such a pathway has functional significance for immunity or tolerance induction. When ovalbumin peptide and LPS were injected i.v. into adoptively transferred recipients, activated CD4 T cells migrated into almost all non-lymphoid tissues, including gut, liver, and kidney, but not brain. Memory T cells persisted within these tissues for at least 60 d post challenge. Surprisingly, the absolute number of memory CD4 T cells within non-lymphoid tissues exceeded those within secondary lymphoid tissue by ~10-fold, with the intestinal mucosa harboring the largest number of memory cells. The functional capabilities of memory T cells in the lung were similar to those reported by Sallusto et al. [34] for CD4 effector memory T cells in human blood, in that they produced more c-IFN than their splenic counterparts after short-term antigen activation in vivo. It will be important to perform similar in vivo analyses after local or systemic infections with a variety of pathogens to determine whether these findings are generally applicable to CD4 memory T cells in non-lymphoid organs. In the case of CD8 T cells, our studies also demonstrate a functional dichotomy between lymphoid and non-lymphoid memory T cells. Following i.v. infection with VSV (a lytic virus that is thought to transiently infect mice) or an oral infection with L. monocytogenes and using MHC class I tetramers to monitor the endogenous antigen-specific CD8 T-cell response, we detect primary effectors in many tissues [17,31,33] (Tables 1 and 2). Furthermore, tetramer-reactive memory cells are present in all tissues up to at least 296 d after primary VSV infection and 224 d after secondary infection ([31] and Table 2). Interestingly, the kinetics of the response are distinct in different compartments and are exemplified in most cases by a more gradual decline of antigen-
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226 Table 2 Non-lymphoid CD8 T cells maintain lytic activitya Tissue Spleen Lung Liver LP 51
D7 164 ± 30 434 ± 72 328 ± 62.3 820 ± 156
D224 recall 5.7b 519 219 336
a Lytic units per million lymphocytes were determined for 30% lysis in a Chromium release assay. b This value was extrapolated because 30% lysis was not achieved.
specific CD8 T cells from non-lymphoid vs. lymphoid tissue. Enumeration of the total number of memory cells indicated that at least half of the CD8 memory T cells are found in non-lymphoid tissue, and this value is likely to be an underestimate due to potentially inefficient cell isolation procedures. Functional analysis of non-lymphoid memory CD8 T cells resulted in a surprising finding. While splenic CD8 memory T cells exhibited low lytic activity, memory cells from lung, liver and intestinal LP mediated astonishingly high levels of lytic activity. The lytic activity of tertiary tissue memory cells was equal to that of primary effector cells from any tissue. The mechanisms by which memory T cells retain effector function are unknown. One possibility is that a small subset of recirculating effector memory cells preferentially enter non-lymphoid tissues. Thus far, such a subset has not been identified. For example, even several weeks after infection, nearly all splenic and tertiary tissue memory cells lack CD62L (the LN homing receptor) (unpublished data), which has been suggested to be expressed by central but not effector memory cells [39]. Yet, memory cells from spleen and tertiary tissues are functionally disparate. Perhaps when additional mAbs specific for mouse chemokine receptors become available, a distinction will become evident. It is also possible that the process of migration results in the modulation of the functional abilities of memory cells. If memory cells traverse endothelium to enter non-lymphoid tissues, then the multistep adhesion process required to do so may itself be stimulatory to the memory cell. Finally, the environmental milieu of lymphoid and non-lymphoid tissues may regulate memory T-cell function. While lymphoid tissue may be inhibitory toward certain memory cell functions such as cytotoxicity, non-lymphoid tissues may provide additional costimulatory ligands and/or cytokines which mediate upregulation of effector function. The finding that activated but non-lytic CD8 T cells acquire effector function following migration into the intestinal mucosa [40] supports the existence of one or all of these possible pathways. How do these data impact our current model of T-cell trafficking? The results suggest that the migration of activated T cells may not be strictly driven by localized inflammation and further suggest that resting endothelium is able to recruit activated as well as memory T cells. This possibility is supported by the finding that during primary oral rotavirus infection which is restricted to gut epithelium, antigenspecific CD8 T cells can be found in the lung and the liver
225
(unpublished results). These studies also question the idea that antigen-specific memory T cells specifically recirculate only through the originally infected tissue, although there is circumstantial evidence that this process may occur for intestinal tissue. However, some of these data are far from conclusive and are only as good as our detection systems. Within an antigen-specific T-cell population, multiple subsets of T cells may exist with unique migratory capabilities as defined by expression of adhesion molecules. In addition, particular antigenic challenges may elicit more specific recruitment to particular tissues than the models discussed above. In conclusion, the memory phase of the immune response is characterized by a more complex phenotype that has been previously appreciated. Functionally distinct subsets of memory T cells exist which can be distinguished by tissue localization. The current challenge is to determine how functional differences in memory T cells arise at the organismal, cellular, and eventually, molecular levels. At a more practical level, it is important to learn whether non-lymphoid memory cells provide heightened protection, and, if so, gear vaccination towards generation of such memory populations. Acknowledgements This work was supported by USPHS grants DK45260 and AI41576. DM was supported by USPHS Training Grant T32-AI07080. References [1]
E.R. Kearney, K.A. Pape, D.Y. Loh, M.K. Jenkins, Visualization of peptide-specific T-cell immunity and peripheral tolerance induction in vivo, Immunity 1 (1994) 327–339. [2] J.D. Altman, P.A.H. Moss, P.J.R. Goulder, D.H. Barouch, M.G. McHeyzer-Williams, J.I. Bell, A.J. McMichael, M.M. Davis, Phenotypic analysis of antigen-specific T lymphocytes, Science 274 (1996) 94–96. [3] J.M. Yoffey, Variation in lymphocyte production, J. Anat Lond. 70 (1936) 507–514. [4] J.L. Gowans, E.J. Knight, The route of re-circulation of lymphocytes in the rat, Proc. Roy. Soc. B. 159 (1964) 257–282. [5] T.A. Springer, Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm, Cell 76 (1994) 301–314. [6] E.C. Butcher, L.J. Picker, Lymphocyte homing and homeostasis, Science 272 (1996) 60–66. [7] U.H. von Andrian, C.R. Mackay, T-cell function and migration. Two sides of the same coin, N. Eng. J. Med. 343 (2000) 1020–1034. [8] R. Forster, A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp, CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs, Cell 99 (1999) 23–33. [9] S.K. Kim, D.S. Reed, W.R. Heath, F. Carbone, L. Lefrançois, Activation and migration of CD8 T cells in the intestinal mucosa, J. Immunol. 159 (1997) 4295–4306. [10] J. Westermann, R. Pabst, How organ-specific is the migration of ‘naive’ and ‘memory’ T cells? Immunol. Today 17 (1996) 278–282. [11] B. Luettig, L. Pape, U. Bode, E.B. Bell, S.M. Sparshott, S. Wagner, J. Westermann, Naive and memory T lymphocytes migrate in comparable numbers through normal rat liver: activated T cells accumulate in the periportal field, J. Immunol. 163 (1999) 4300–4307.
226
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
[12] A.M. Mowat, J.L. Viney, The anatomical basis of intestinal immunity, Immunol. Rev. 156 (1997) 145–166. [13] S.P. James, C. Fiocchi, A.S. Graeff, W. Strober, Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn’s disease and control patients, Gastroenterology 91 (1986) 1483–1489. [14] J.W. Huleatt, L. Lefrançois, Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8(+) T cells in vivo, J. Immunol. 154 (1995) 5684–5693. [15] J. Sprent, Fate of H2-activated T lymphocytes in syngeneic hosts. I. Fate in lymphoid tissues and intestines traced with 3H-thymidine, 125 -deoxyuridine and 51chromium, Cell Immunol. 21 (1976) 278–302. [16] L. Lefrançois, C.M. Parker, S. Olson, M.P. Schon, W. Muller, N. Wagner, L. Puddington, The role of b7 integrins in CD8 T cell trafficking during an antiviral immune response, J. Exp. Med. 189 (1999) 1631–1638. [17] D. Masopust, J. Jiang, H. Shen, L. Lefrancois, Direct analysis of the dynamics of the intestinal mucosa CD8 T cell response to systemic virus infection, J. Immunol. 166 (2001) 2348–2356. [18] M.K. Slifka, J.K. Whitmire, R. Ahmed, Bone marrow contains virusspecific cytotoxic T lymphocytes, Blood. 90 (1997) 2103–2108. [19] D.R. Marshall, S.J. Turner, G.T. Belz, S. Wingo, S. reansky, M.Y. Sangster, J.M. Riberdy, T. Liu, M. Tan, P.C. Doherty, Measuring the diaspora for virus-specific CD8+ T cells, Proc. Natl. Acad. Sci. USA (2001). [20] K.J. Flynn, G.T. Belz, J.D. Altman, R. Ahmed, D.L. Woodland, P.C. Doherty, Virus-specific CD8+ T cells in primary and secondary influenza pneumonia, Immunity 8 (1998) 683–691. [21] E.J. Usherwood, R.J. Hogan, G. Crowther, S.L. Surman, T.L. Hogg, J.D. Altman, D.L. Woodland, Functionally heterogeneous CD8(+) T-cell memory is induced by Sendai virus infection of mice, J. Virol. 73 (1999) 7278–7286. [22] R.J. Hogan, E.J. Usherwood, W. Zhong, A.A. Roberts, R.W. Dutton, A.G. Harmsen, D.L. Woodland, Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections, J. Immunol. 166 (2001) 1813–1822. [23] G.T. Belz, J.D. Altman, P.C. Doherty, Characteristics of virus-specific CD8(+) T cells in the liver during the control and resolution phases of influenza pneumonia, Proc. Natl. Acad. Sci. USA 95 (1998) 13812–13817. [24] C.R. Mackay, W.L. Marston, L. Dudler, Naive and memory T cells show distinct pathways of lymphocyte recirculation, J. Exp. Med. 171 (1990) 801–817. [25] C.R. Mackay, W.L. Marston, L. Dudler, O. Spertini, T.F. Tedder, W.R. Hein, Tissue-specific migration pathways by phenotypically distinct populations of memory T cells, Eur. J. Immunol. 22 (1992) 887–895. [26] L.J. Picker, J.R. Treer, B. Ferguson-Darnell, P.A. Collins, P.R. Bergstresser, L.W. Terstappen, Control of lymphocyte recirculation in man. II. Differential regulation of the cutaneous lymphocyteassociated antigen, a tissue-selective homing receptor for skinhoming T cells, J. Immunol. 150 (1993) 1122–1136.
[27] E.L. Berg, T. Yoshino, L.S. Rott, M.K. Robinson, R.A. Warnock, T.K. Kishimoto, L.J. Picker, E.C. Butcher, The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1, J. Exp. Med. 174 (1991) 1461–1466. [28] J.J. Campbell, G. Haraldsen, J. Pan, J. Rottman, S. Qin, P. Ponath, D.P. Andrew, R. Warnke, N. Ruffing, N. Kassam, L. Wu, E.C. Butcher, The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells, Nature 400 (1999) 776–780. [29] D.P. Andrew, N. Ruffing, C.H. Kim, W. Miao, H. Heath, Y. Li, K. Murphy, J.J. Campbell, E.C. Butcher, L. Wu, C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Th1 and Th2 potential, J. Immunol. 166 (2001) 103–111. [30] S.K. Kim, K.S. Schluns, L. Lefrançois, Induction and visualization of mucosal memory CD8 T cells following systemic virus infection, J. Immunol. 163 (1999) 4125–4132. [31] D. Masopust, V. Vezys, A.L. Marzo, L. Lefrançois, Preferential localization of effector memory cells in nonlymphoid tissue, Science 291 (2001) 2413–2417. [32] D.A. Carlow, B. Ardman, H.J. Ziltener, A novel CD8 T cell-restricted CD45RB epitope shared by CD43 is differentially affected by glycosylation, J. Immunol. 163 (1999) 1441–1448. [33] C. Pope, S.K. Kim, A. Marzo, D. Masopust, K. Williams, J. Jiang, H. Shen, L. Lefrançois, Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection, J. Immunol. 166 (2001) 3402–3409. [34] F. Sallusto, D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia, Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401 (1999) 708–712. [35] S. Mori, H. Nakano, K. Aritomi, C.R. Wang, M.D. Gunn, T. Kakiuchi, Mice lacking expression of the chemokines CCL21-ser and CCL19 (plt mice) demonstrate delayed but enhanced T cell immune responses, J. Exp. Med. 193 (2001) 207–218. [36] A.W. Goldrath, L.Y. Bogatzki, M.J. Bevan, Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation, J. Exp. Med. 192 (2000) 557–564. [37] K. Murali-Krishna, R. Ahmed, Cutting edge: naive T cells masquerading as memory cells, J. Immunol. 165 (2000) 1733–1737. [38] R.L. Reinhardt, A. Khoruts, R. Merica, T. Zell, M.K. Jenkins, Visualizing the generation of memory CD4 T cells in the whole body, Nature 410 (2001) 101–105. [39] S. Oehen, K. Brduscha-Riem, Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division, J. Immunol. 161 (1998) 5338–5346. [40] S.K. Kim, D.S. Reed, S. Olson, M.J. Schnell, J.K. Rose, P.A. Morton, L. Lefrançois, Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals, Proc. Natl. Acad. Sci. USA 95 (1998) 10814–10819.
Forum in immunology
CD8 T-cell memory: the other half of the story David Masopust, Leo Lefrançois * Division of Immunology, Department of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, MC1319, Farmington, CT 06030-1319, USA
Abstract Historically, most immune response studies have been limited to analyses of lymphoid tissue. However, peripheral sites of infection are likely to represent important sites of cell-mediated immune surveillance and effector function. Recent debates have centered on the persistence, trafficking patterns, effector activity, and protective role of non-lymphoid memory T cells. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Infection; Memory; T cell; Migration; Non-lymphoid
1. Introduction Recent technological innovations such as the adoptive transfer system [1] and MHC tetramers [2], both of which allow tracking and identification of antigen-specific T cells, are driving major changes in our perceptions of cellular immunity. Consequently, our understanding of the dynamics of CD4 and CD8 T-cell responses is rapidly expanding. Nevertheless, many of the concepts related to the T-cell immune response are based on analyses of lymphocytes located in secondary lymphoid tissues. However, nonlymphoid peripheral organs may represent important T-cell effector sites in the context of infection, allograft rejection, and autoimmune disease. Lymphoid and non-lymphoid environments differ in many respects, including the density and quality of lymphocytes, antigen presenting cells, cytokines and innate immune system components, all of which are factors with important bearing on the evolution of adaptive immune responses. There are many critical questions which remain unresolved regarding non-lymphoid immunity including: 1. Can studies of lymphoid T-cell populations be extrapolated to non-lymphoid populations? Or, are these populations functionally distinct? 2. Are activated T cells only targeted to specific sites of inflammation/infection? 3. What is the trafficking pattern of memory T cells? Do they persist in or recirculate through non-lymphoid * Corresponding author. Tel. +1-860-679-3242; fax: +1-860-679-1868. E-mail address: [email protected] (L. Lefrançois). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 1 4 - 5
tissues and do they home preferentially to the original site of infection? 4. What are the relative roles of lymphoid vs. nonlymphoid effector and memory T cells in protection from infection and re-infection? Recently, several important findings have begun to shed light on some of these issues. It is clear that non-lymphoid T-cell populations hold many surprises. Subjecting these cells to further scrutiny will provide us with a more complete understanding of the dynamics of T-cell-mediated immunity.
2. Current paradigms for migration pathways of naive, activated and memory T cells Our current model of T-cell trafficking is based on a large body of elegant in vitro and in vivo experimentation and a complete treatment of this topic is beyond the scope of this article. Early studies demonstrated that thoracic duct lymph (TDL), which empties into the blood, contains many lymphocytes [3]. The source and fate of this population were of considerable controversy. In a landmark experiment, Gowans and Knight [4] transferred radiolabeled thoracic duct lymphocytes intravenously (i.v.) into naive syngeneic recipients and detected donor lymphocytes in their TDL. This provided a formal demonstration that lymphocytes recirculate continuously between blood and lymph. These investigators also performed autoradiographic analyses of high endothelial venules (HEVs) within lymph nodes (LNs) and demonstrated convincingly that small lymphocytes cross HEVs to enter LNs. Small lymphocytes (which would be
222
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
made up of primarily naive T cells but would contain some memory cells) were detected in LN, white pulp of spleen, and Peyer’s patches (PPs). The number of small lymphocytes in other tissues was minor in comparison providing an early suggestion that naive lymphocytes may not enter nonlymphoid tissues. With this and many other groundbreaking studies, a picture at the molecular level of the pathway of naive T-cell migration has emerged. Blood-borne naive T cells enter into LNs via interactions with receptor/ligand pairs expressed by the endothelium in the HEV and lymphocytes [5–7]. This is a highly orchestrated multistep process, which involves selectins to initiate lymphocyte rolling, chemokines to activate integrins, and activated integrins to trigger firm adhesion and eventual transendothelial migration into the LN. Lymphocytes can then exit the LNs via efferent lymphatics and eventually drain into the thoracic duct and the recirculatory process begins anew. In the case of the spleen, HEV are not present and so this pathway is not involved, but it is now clear that chemokines and no doubt other adhesion molecules are responsible for naive T- and B-cell migration into the appropriate areas of the splenic sub architecture [8]. Experiments have also demonstrated that naive lymphocytes rarely enter non-lymphoid tissue. This has been shown using short-term in vivo migration studies as well as adoptive transfer of trackable naive CD8 TCR transgenic T cells, which did not enter the intestinal lamina propria (LP) or epithelium unless activated [9]. Nevertheless, small populations of CD8 T cells with naive phenotypes can be found in the intestinal mucosa, liver and lung (our unpublished observations) suggesting that limited traffic of naive cells through non-lymphoid tissues may occur. Westermann and colleagues [10,11] have also argued that there is limited trafficking of naive T cells through skin and liver. More definitive analyses are required to prove this possibility. When T cells are activated following encounter with antigen in an LN, their homing program is dramatically altered at the lymphocyte level. In addition, the endothelium may also be activated in the face of local or systemic inflammation. The migration of activated CD8 T cells into the intestinal mucosa provides an excellent system for analysis of T-cell migration into tertiary lymphoid tissue. The intestinal immune system is composed of secondary lymphoid inductive sites (PPs and mesenteric LNs) and tertiary effector sites, the LP and the epithelium [12]. The vast majority of resident lymphocyte populations in the latter express activation and/or memory markers [13,14]. In the transfer studies of Gowans and Knight [4], large lymphocytes appeared to marginate primarily to gut (which included MLN and LP in this case) rather than PLN, although a few cells were detected in the liver and kidney using autoradiography, which allowed direct visualization of live cells within tissue. Later studies by Sprent [15] confirmed and extended this work regarding the intestine using a system of transfer of activated alloreactive lymphocytes. However, a detailed analysis of other tissues was not performed. In more recent studies in which the identity of transferred cells could be precisely determined,
only after activation via virus infection did TCR transgenic CD8 T cells migrate to the intestinal LP and epithelium in an a4b7-integrin-dependent pathway [16]. These results were corroborated in studies using vesicular stomatitis virus infection and detection of endogenous antigen-specific CD8 T cells with MHC class I tetramers [17]. Primary infection with a variety of viruses also leads to appearance of antigenspecific CD8 T cells in non-lymphoid organs. For example, after infection with either lymphocytic choriomeningitis virus (LCMV) [18] or influenza virus [19], antigen-specific CD8 T cells are found in bone marrow. Also, quantitation of the primary response to intranasal infections with influenza virus [20], Sendai virus [21] or gammaherpes virus [22] identified substantial numbers of antigen-specific CD8 T cells in the lung which in all likelihood migrated to that site after primary activation in draining LN. In one study, influenza-specific CD8 T cells were also found in the liver after intranasal infection [23]. This latter result is of interest considering that influenza virus only productively infects lung epithelial cells. Thus, the entry of activated CD8 T cells into the liver was not dependent on local virus production. Overall, these studies demonstrate that activated T cells migrate to sites of infection and may traffic to other, potentially uninfected tissues. Distinguishing between activated and memory T cells has historically been difficult. The use of adoptive transfer and MHC tetramers has greatly improved our ability to identify bona fide naive, effector and memory cells. To discriminate primary effector from memory cells, cell size appears to be a viable criterion and this along with phenotypic analysis of transferred or tetramer+ cells allows reasonable, if not absolute, identification of these subsets. Earlier studies by Mackay et al. [24] found that T lymphocytes in afferent lymph draining from tissues into the popliteal LN of sheep were largely CD44hi, a phenotype associated with activation or memory. While no definitive marker for memory vs. activated T cells existed at that time, cell size data indicated that at least some of this population represented true memory T cells. Further studies using sheep demonstrated that the afferent lymph of intestinal tissue also contained high percentages of memory phenotype T cells [25]. These results helped establish the current hypothesis that memory T cells circulate in the blood, extravasate into non-lymphoid tissues, drain into LNs via afferent lymph, and subsequently return to the blood stream via efferent lymphatics. Since most T cells in efferent lymph are of a naive phenotype (CD44lo) [24], memory/activated cells make up only a small percentage of cells migrating through LN from the tissues. 3. Tissue-specific migration of activated and memory T cells The best examples of this phenomenon regard trafficking of T cells to the skin and intestinal mucosa. Picker et al. [26] demonstrated that following DTH reactions in human skin, about 50% of the T cells within the inflammatory site express
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
cutaneous associated lymphocyte antigen (CLA), which binds E-selectin expressed by endothelial cells [27]. As most T cells within blood do not express CLA, these data suggest that CLA is involved in migration into the skin. CLA expression could also be induced on in vitro activated T cells by adding certain cytokines to the culture. This process was more efficient using T cells derived from PLN rather than MLN. This result led to the notion that the particular lymphoid environment in which priming took place, imprinted upon activated T cells tropism for particular tissues. For example, priming in inguinal LNs would pre-dispose T cells to exhibit skin homing properties. Mediastinal LNs would generate lung specific responses, etc. Support for this hypothesis was also obtained in analyses of intestinal mucosal lymphoid tissue [25]. As noted above, the a4b7 integrin is involved in migration of activated T cells to the gut. However, it is important to note that activated T-cell migration to skin or gut is not absolutely dependent on expression of CLA [26] or a4b7, respectively [16,17]. Thus, approximately half of the T cells within inflamed skin lack CLA indicating that CLA is either down-regulated once within the tissue, or that there is some CLA independent migration into skin. In the case of the intestinal mucosa, most migration of activated antigen-specific CD8 T cells into the mucosa is a4b7dependent, but nevertheless a small population of cells traffic to the intestine in the absence of this integrin [16,17]. Memory cells in human blood have also been divided into tissue-specific subsets based on expression of CLA, a4b7 and chemokine receptors [28]. The expression of CLA and a4b7 by CD4+ CD45RA-small lymphocytes (presumably memory cells) is essentially mutually exclusive potentially defining two unique recirculating pools [28]. Furthermore, a large proportion of CLA+ memory cells express CCR4, while intestinal memory cells expressing a4b7 lack CLA and CCR4 [28,29]. The CCR4 ligand, TARC, is expressed in inflamed skin but is not expressed in intestinal tissue, further supporting the concept that memory cells may home in a tissue-specific manner. Whether this is reflective of the site of activation in the primary response remains to be determined. Also unknown is whether such memory cells continuously travel through non-lymphoid tissues or whether re-encounter with antigen and tissue inflammation is required for their movement out of the bloodstream. Interestingly, antigen-specific CD8 memory cells in the mouse intestinal mucosa express a distinct set of adhesion markers as compared to memory cells in spleen, lung and liver [30,31]. The integrin aEb7 is highly expressed by the majority of mucosal memory cells while very few memory cells outside of the mucosa express this molecule [30,31]. The carbohydrate determinant 1B11 present on CD43 and CD45 molecules [32] is present on nearly all mucosal memory CD8 cells while z1/2 of non-intestinal memory cells express this antigen [31]. While phenotypic evidence is circumstantial, the data suggest that intestinal memory T cells may represent a distinct subset of memory cells that does not intermix with the circulating pool of memory cells.
223
There are several mechanisms by which this situation could occur. Perhaps, after entry and subsequent regulation of cellsurface receptors, a portion of intestinal CD8 memory T cells remains in situ long-term and do not exit the tissue. Alternatively, lymphoid memory cells could enter the intestinal mucosa routinely and modify their cell-surface receptors upon entry. It is also possible that only very small numbers of intestinal memory T cells enter the circulation at any given time point such that recirculation of this population is routine but difficult to detect. Detailed in vivo migration studies are required to answer these questions. Tissue-specific migration of activated and memory T cells has important implications for immunity and vaccination. Such a system increases the efficiency of adaptive immune responses by limiting the volume of tissue that individual antigen-specific T cells are required to patrol. For example, restricting the traffic of naive T cells through secondary lymphoid tissue no doubt dramatically increases the speed with which rare antigen-specific naive precursors would encounter cognate antigen in the context of the appropriate anatomical and immunostimulatory environment. This theory suggests that activated T cells migrate preferentially to sites of inflammation, where they are most needed and that memory T cells migrate through tissues where they are most likely to re-encounter cognate antigen. Avoidance of other non-lymphoid organs may minimize the likelihood of crossreactive autoimmune responses to tissue-specific selfantigens. 4. Central vs. memory T cells: autonomous or tissue-specific regulation? Numerous recent studies have reported the presence of antigen-specific memory T cells within non-lymphoid tissues of mice. For example, transgenic memory CD8 T cells persist within the small intestinal mucosa following i.v. VSV infection [30]. While it has been suggested that the gut may be unique in its capacity to continuously harbor memory T cells, infection with LCMV leads to the appearance of virusspecific memory CD8 T cells within the bone marrow [18]. In addition, intranasal influenza [20] or Sendai virus [21] infections generate endogenous memory T cells within the lung. Oral Listeria monocytogenes infection leads to CD8 T-cell memory within the intestinal mucosa and liver [33]. Collectively, these data demonstrate that memory T cells can be found in multiple non-lymphoid organs. However, in most of these examples, the pathogen is thought to infect the organ in which memory cells are subsequently located. Thus, these studies do not demonstrate whether primary or memory T cells have the capacity to home to any non-lymphoid organ or solely to organs undergoing infection. Proving this point is difficult because although some viruses productively infect only certain tissues, it remains possible that disseminated non-productive infection of remote tissues may lead to some level of inflammation. In addition, robust infection at a local site could result in the release of sufficient inflammatory
224
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
Table 1 Absolute numbers of pathogen-specific CD8 T cells per tissuea Days post-infection 6 8 12 20 35
Spleen 2.94E6 970,833 321,833 109,667 133,833
Three tissues combined 1.903E6 701,167 434,167 132,667 86,000
Lung 761,500 503,667 238,333 41,667 49,833
LP 154,000 113,333 98,500 56,500 21,167
Liver 987,500 84,167 97,333 34,500 15,000
a
Lymphocytes were harvested from tissues at various time points following i.v. infection of C57BL/6 mice with 1 × 106 pfu vesicular stomatitis virus (VSV). Total numbers of antigen-specific CD8 T cells were enumerated by multiplying total lymphocytes isolated per tissue (as counted by light microscopy and trypan blue exclusion) by the percentage of lymphocytes staining positive with tetramerized H-2Kb/RGYVYQGL, representing an immunodominant VSV epitope. Staining with irrelevant tetramers was negligible.
mediators into the bloodstream to activate endothelium resulting in extravasation of T cells and perhaps innate immune system components into uninfected tissues. The concept of two types of memory T cells delineated by homing molecule expression and functional differences has recently been put forth based on analysis of human peripheral blood. Sallusto et al. [34] demonstrated that memory phenotype CD4 T cells can be broken down into at least two subsets based on CCR7 expression. CCR7 mediates entry of naive and perhaps memory T cells into secondary lymphoid organs via interaction with the chemokines CCL19 and CCL21 [8,35]. Moreover, CCR7– memory-phenotype T cells rapidly produced c-IFN and IL-2 after in vitro stimulation, while CCR7+ memory-phenotype T cells produced IL-2 but little c-IFN. In the CD8 compartment, CCR7 expression also distinguished two putative memory subsets and the CCR7– cells expressed perforin while the CCR7+ cells did not. These intriguing results led to the theory that two subsets of memory cells exist, one geared toward migration through secondary lymphoid tissues with low immediate functional ability and a second geared toward migration through non-lymphoid tissues with immediate effector activity. One of the difficulties in these and other studies in which antigen-specific T cells were not analyzed, is the inability to definitively identify memory cells. Thus, one is left with using cell-surface markers to define memory cells which may or may not always be indicative of memory cells. This problem has recently been highlighted by the demonstration that T cells undergoing homeostatic proliferation acquire a memory phenotype but do not develop into true memory cells [36,37]. The degree of homeostatic proliferation occurring under normal circumstances is not known, but nevertheless, caution should be used in using phenotype to define memory T cells. To definitively identify memory cells, the adoptive transfer of antigen-specific TCR transgenic T cells or the use of MHC tetramers to track antigen-specific endogenous T-cell populations are the current methods of choice. The adoptive transfer approach was used by Reinhardt et al. [38] to track ovalbumin-specific TCR transgenic CD4+ T cells in naive and immunized recipients. Migration was evaluated in various contexts by analysis of the location of donor cells in whole mouse histological sections. Donor cells were enumerated by immunohistochemistry on a single section and the total number of ovalbumin-specific CD4 T cells per organ
was calculated by correcting for the total volume of the various tissues. Following transfer of naive CD4 T cells, the donor population was exclusively located within secondary lymphoid tissue of perfused recipients. However, upon injection of lipopolysacharride (LPS), a small number of naive lymphocytes were visualized within non-lymphoid tissue. These data imply that naive T cells may have the ability to traffic through non-lymphoid tissues after ‘bystander’ activation of lymphocytes or perhaps after activation of the endothelium via strong inflammatory signals. Further studies are needed to determine whether naive T-cell migration to nonlymphoid tissues is a common occurrence and whether such a pathway has functional significance for immunity or tolerance induction. When ovalbumin peptide and LPS were injected i.v. into adoptively transferred recipients, activated CD4 T cells migrated into almost all non-lymphoid tissues, including gut, liver, and kidney, but not brain. Memory T cells persisted within these tissues for at least 60 d post challenge. Surprisingly, the absolute number of memory CD4 T cells within non-lymphoid tissues exceeded those within secondary lymphoid tissue by ~10-fold, with the intestinal mucosa harboring the largest number of memory cells. The functional capabilities of memory T cells in the lung were similar to those reported by Sallusto et al. [34] for CD4 effector memory T cells in human blood, in that they produced more c-IFN than their splenic counterparts after short-term antigen activation in vivo. It will be important to perform similar in vivo analyses after local or systemic infections with a variety of pathogens to determine whether these findings are generally applicable to CD4 memory T cells in non-lymphoid organs. In the case of CD8 T cells, our studies also demonstrate a functional dichotomy between lymphoid and non-lymphoid memory T cells. Following i.v. infection with VSV (a lytic virus that is thought to transiently infect mice) or an oral infection with L. monocytogenes and using MHC class I tetramers to monitor the endogenous antigen-specific CD8 T-cell response, we detect primary effectors in many tissues [17,31,33] (Tables 1 and 2). Furthermore, tetramer-reactive memory cells are present in all tissues up to at least 296 d after primary VSV infection and 224 d after secondary infection ([31] and Table 2). Interestingly, the kinetics of the response are distinct in different compartments and are exemplified in most cases by a more gradual decline of antigen-
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226 Table 2 Non-lymphoid CD8 T cells maintain lytic activitya Tissue Spleen Lung Liver LP 51
D7 164 ± 30 434 ± 72 328 ± 62.3 820 ± 156
D224 recall 5.7b 519 219 336
a Lytic units per million lymphocytes were determined for 30% lysis in a Chromium release assay. b This value was extrapolated because 30% lysis was not achieved.
specific CD8 T cells from non-lymphoid vs. lymphoid tissue. Enumeration of the total number of memory cells indicated that at least half of the CD8 memory T cells are found in non-lymphoid tissue, and this value is likely to be an underestimate due to potentially inefficient cell isolation procedures. Functional analysis of non-lymphoid memory CD8 T cells resulted in a surprising finding. While splenic CD8 memory T cells exhibited low lytic activity, memory cells from lung, liver and intestinal LP mediated astonishingly high levels of lytic activity. The lytic activity of tertiary tissue memory cells was equal to that of primary effector cells from any tissue. The mechanisms by which memory T cells retain effector function are unknown. One possibility is that a small subset of recirculating effector memory cells preferentially enter non-lymphoid tissues. Thus far, such a subset has not been identified. For example, even several weeks after infection, nearly all splenic and tertiary tissue memory cells lack CD62L (the LN homing receptor) (unpublished data), which has been suggested to be expressed by central but not effector memory cells [39]. Yet, memory cells from spleen and tertiary tissues are functionally disparate. Perhaps when additional mAbs specific for mouse chemokine receptors become available, a distinction will become evident. It is also possible that the process of migration results in the modulation of the functional abilities of memory cells. If memory cells traverse endothelium to enter non-lymphoid tissues, then the multistep adhesion process required to do so may itself be stimulatory to the memory cell. Finally, the environmental milieu of lymphoid and non-lymphoid tissues may regulate memory T-cell function. While lymphoid tissue may be inhibitory toward certain memory cell functions such as cytotoxicity, non-lymphoid tissues may provide additional costimulatory ligands and/or cytokines which mediate upregulation of effector function. The finding that activated but non-lytic CD8 T cells acquire effector function following migration into the intestinal mucosa [40] supports the existence of one or all of these possible pathways. How do these data impact our current model of T-cell trafficking? The results suggest that the migration of activated T cells may not be strictly driven by localized inflammation and further suggest that resting endothelium is able to recruit activated as well as memory T cells. This possibility is supported by the finding that during primary oral rotavirus infection which is restricted to gut epithelium, antigenspecific CD8 T cells can be found in the lung and the liver
225
(unpublished results). These studies also question the idea that antigen-specific memory T cells specifically recirculate only through the originally infected tissue, although there is circumstantial evidence that this process may occur for intestinal tissue. However, some of these data are far from conclusive and are only as good as our detection systems. Within an antigen-specific T-cell population, multiple subsets of T cells may exist with unique migratory capabilities as defined by expression of adhesion molecules. In addition, particular antigenic challenges may elicit more specific recruitment to particular tissues than the models discussed above. In conclusion, the memory phase of the immune response is characterized by a more complex phenotype that has been previously appreciated. Functionally distinct subsets of memory T cells exist which can be distinguished by tissue localization. The current challenge is to determine how functional differences in memory T cells arise at the organismal, cellular, and eventually, molecular levels. At a more practical level, it is important to learn whether non-lymphoid memory cells provide heightened protection, and, if so, gear vaccination towards generation of such memory populations. Acknowledgements This work was supported by USPHS grants DK45260 and AI41576. DM was supported by USPHS Training Grant T32-AI07080. References [1]
E.R. Kearney, K.A. Pape, D.Y. Loh, M.K. Jenkins, Visualization of peptide-specific T-cell immunity and peripheral tolerance induction in vivo, Immunity 1 (1994) 327–339. [2] J.D. Altman, P.A.H. Moss, P.J.R. Goulder, D.H. Barouch, M.G. McHeyzer-Williams, J.I. Bell, A.J. McMichael, M.M. Davis, Phenotypic analysis of antigen-specific T lymphocytes, Science 274 (1996) 94–96. [3] J.M. Yoffey, Variation in lymphocyte production, J. Anat Lond. 70 (1936) 507–514. [4] J.L. Gowans, E.J. Knight, The route of re-circulation of lymphocytes in the rat, Proc. Roy. Soc. B. 159 (1964) 257–282. [5] T.A. Springer, Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm, Cell 76 (1994) 301–314. [6] E.C. Butcher, L.J. Picker, Lymphocyte homing and homeostasis, Science 272 (1996) 60–66. [7] U.H. von Andrian, C.R. Mackay, T-cell function and migration. Two sides of the same coin, N. Eng. J. Med. 343 (2000) 1020–1034. [8] R. Forster, A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp, CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs, Cell 99 (1999) 23–33. [9] S.K. Kim, D.S. Reed, W.R. Heath, F. Carbone, L. Lefrançois, Activation and migration of CD8 T cells in the intestinal mucosa, J. Immunol. 159 (1997) 4295–4306. [10] J. Westermann, R. Pabst, How organ-specific is the migration of ‘naive’ and ‘memory’ T cells? Immunol. Today 17 (1996) 278–282. [11] B. Luettig, L. Pape, U. Bode, E.B. Bell, S.M. Sparshott, S. Wagner, J. Westermann, Naive and memory T lymphocytes migrate in comparable numbers through normal rat liver: activated T cells accumulate in the periportal field, J. Immunol. 163 (1999) 4300–4307.
226
D. Masopust, L. Lefrançois / Microbes and Infection 5 (2003) 221–226
[12] A.M. Mowat, J.L. Viney, The anatomical basis of intestinal immunity, Immunol. Rev. 156 (1997) 145–166. [13] S.P. James, C. Fiocchi, A.S. Graeff, W. Strober, Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn’s disease and control patients, Gastroenterology 91 (1986) 1483–1489. [14] J.W. Huleatt, L. Lefrançois, Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8(+) T cells in vivo, J. Immunol. 154 (1995) 5684–5693. [15] J. Sprent, Fate of H2-activated T lymphocytes in syngeneic hosts. I. Fate in lymphoid tissues and intestines traced with 3H-thymidine, 125 -deoxyuridine and 51chromium, Cell Immunol. 21 (1976) 278–302. [16] L. Lefrançois, C.M. Parker, S. Olson, M.P. Schon, W. Muller, N. Wagner, L. Puddington, The role of b7 integrins in CD8 T cell trafficking during an antiviral immune response, J. Exp. Med. 189 (1999) 1631–1638. [17] D. Masopust, J. Jiang, H. Shen, L. Lefrancois, Direct analysis of the dynamics of the intestinal mucosa CD8 T cell response to systemic virus infection, J. Immunol. 166 (2001) 2348–2356. [18] M.K. Slifka, J.K. Whitmire, R. Ahmed, Bone marrow contains virusspecific cytotoxic T lymphocytes, Blood. 90 (1997) 2103–2108. [19] D.R. Marshall, S.J. Turner, G.T. Belz, S. Wingo, S. reansky, M.Y. Sangster, J.M. Riberdy, T. Liu, M. Tan, P.C. Doherty, Measuring the diaspora for virus-specific CD8+ T cells, Proc. Natl. Acad. Sci. USA (2001). [20] K.J. Flynn, G.T. Belz, J.D. Altman, R. Ahmed, D.L. Woodland, P.C. Doherty, Virus-specific CD8+ T cells in primary and secondary influenza pneumonia, Immunity 8 (1998) 683–691. [21] E.J. Usherwood, R.J. Hogan, G. Crowther, S.L. Surman, T.L. Hogg, J.D. Altman, D.L. Woodland, Functionally heterogeneous CD8(+) T-cell memory is induced by Sendai virus infection of mice, J. Virol. 73 (1999) 7278–7286. [22] R.J. Hogan, E.J. Usherwood, W. Zhong, A.A. Roberts, R.W. Dutton, A.G. Harmsen, D.L. Woodland, Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections, J. Immunol. 166 (2001) 1813–1822. [23] G.T. Belz, J.D. Altman, P.C. Doherty, Characteristics of virus-specific CD8(+) T cells in the liver during the control and resolution phases of influenza pneumonia, Proc. Natl. Acad. Sci. USA 95 (1998) 13812–13817. [24] C.R. Mackay, W.L. Marston, L. Dudler, Naive and memory T cells show distinct pathways of lymphocyte recirculation, J. Exp. Med. 171 (1990) 801–817. [25] C.R. Mackay, W.L. Marston, L. Dudler, O. Spertini, T.F. Tedder, W.R. Hein, Tissue-specific migration pathways by phenotypically distinct populations of memory T cells, Eur. J. Immunol. 22 (1992) 887–895. [26] L.J. Picker, J.R. Treer, B. Ferguson-Darnell, P.A. Collins, P.R. Bergstresser, L.W. Terstappen, Control of lymphocyte recirculation in man. II. Differential regulation of the cutaneous lymphocyteassociated antigen, a tissue-selective homing receptor for skinhoming T cells, J. Immunol. 150 (1993) 1122–1136.
[27] E.L. Berg, T. Yoshino, L.S. Rott, M.K. Robinson, R.A. Warnock, T.K. Kishimoto, L.J. Picker, E.C. Butcher, The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1, J. Exp. Med. 174 (1991) 1461–1466. [28] J.J. Campbell, G. Haraldsen, J. Pan, J. Rottman, S. Qin, P. Ponath, D.P. Andrew, R. Warnke, N. Ruffing, N. Kassam, L. Wu, E.C. Butcher, The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells, Nature 400 (1999) 776–780. [29] D.P. Andrew, N. Ruffing, C.H. Kim, W. Miao, H. Heath, Y. Li, K. Murphy, J.J. Campbell, E.C. Butcher, L. Wu, C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Th1 and Th2 potential, J. Immunol. 166 (2001) 103–111. [30] S.K. Kim, K.S. Schluns, L. Lefrançois, Induction and visualization of mucosal memory CD8 T cells following systemic virus infection, J. Immunol. 163 (1999) 4125–4132. [31] D. Masopust, V. Vezys, A.L. Marzo, L. Lefrançois, Preferential localization of effector memory cells in nonlymphoid tissue, Science 291 (2001) 2413–2417. [32] D.A. Carlow, B. Ardman, H.J. Ziltener, A novel CD8 T cell-restricted CD45RB epitope shared by CD43 is differentially affected by glycosylation, J. Immunol. 163 (1999) 1441–1448. [33] C. Pope, S.K. Kim, A. Marzo, D. Masopust, K. Williams, J. Jiang, H. Shen, L. Lefrançois, Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection, J. Immunol. 166 (2001) 3402–3409. [34] F. Sallusto, D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia, Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401 (1999) 708–712. [35] S. Mori, H. Nakano, K. Aritomi, C.R. Wang, M.D. Gunn, T. Kakiuchi, Mice lacking expression of the chemokines CCL21-ser and CCL19 (plt mice) demonstrate delayed but enhanced T cell immune responses, J. Exp. Med. 193 (2001) 207–218. [36] A.W. Goldrath, L.Y. Bogatzki, M.J. Bevan, Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation, J. Exp. Med. 192 (2000) 557–564. [37] K. Murali-Krishna, R. Ahmed, Cutting edge: naive T cells masquerading as memory cells, J. Immunol. 165 (2000) 1733–1737. [38] R.L. Reinhardt, A. Khoruts, R. Merica, T. Zell, M.K. Jenkins, Visualizing the generation of memory CD4 T cells in the whole body, Nature 410 (2001) 101–105. [39] S. Oehen, K. Brduscha-Riem, Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division, J. Immunol. 161 (1998) 5338–5346. [40] S.K. Kim, D.S. Reed, S. Olson, M.J. Schnell, J.K. Rose, P.A. Morton, L. Lefrançois, Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals, Proc. Natl. Acad. Sci. USA 95 (1998) 10814–10819.