Memory T Cells Response

Heterogeneity of the Memory CD4 T Cell Response: Persisting Effectors and Resting Memory T Cells1

Mojgan Ahmadzadeh, S. Farzana Hussain, and Donna L. Farber2

Defining the cellular composition of the memory T cell pool has been complicated by an inability to distinguish effector and memory T cells. We present here an activation profile assay, using anti-CD3 and antigenic stimuli, that clearly distinguishes effector and memory CD4 T cells and defines subsets of long-lived memory CD4 T cells based on CD62 ligand (CD62L) expression. The CD62Llow memory subset functionally resembles effector cells, exhibiting hyper-responsiveness to antigenic and anti-CD3 mediated stimuli, high proliferative capacity, and rapid activation kinetics. The CD62Lhigh memory subset functionally resembles resting memory cells, exhibiting hyporesponsiveness to anti-CD3 stimuli, lower proliferative capacity, and slower activation kinetics. Our results indicate that the memory CD4 T cell pool is heterogeneous, consisting of persisting effectors and resting memory T cells. The Journal of Immunology, 2001, 166: 926–935.

he memory immune response, characterized by increased ated with a naive phenotype, and loss of CD62L expression kinetics, higher levels of immune reactants, and effica- (CD62Llow) is a hallmark of effector/memory subsets (12), CD62L T cious clearance of Ag, is generally ascribed to the reac- heterogeneity among memory T cells remains unexplained at tivation of Ag-specific memory T lymphocytes. Although it is un- present. Whether CD62L expression delineates functional subsets known how memory T cells are generated, it has been shown that within the memory T cell pool is also not known. the majority of acutely activated effector T cells die in vivo (1), To characterize the cellular bases of heterogeneity within the while a smaller proportion of previously activated T cells persist as memory T cell pool, it is essential to establish parameters that memory T cells. Previous assumptions that these memory T cells reliably distinguish effector from resting memory T cells, as it has represent a long-lived, resting subset have been clouded by recent been difficult to assess the life span and identity of these subsets in findings of functional and/or phenotypic heterogeneity within the vivo due to similar functions and phenotypes. Functionally, effec- memory population. Functional heterogeneity based on differences tor and memory CD4 and CD8 T cells both mediate effector func- in activation kinetics has been identified among virus-specific, tions such as cytokine production and/or cytolysis, although effec- memory CD8 T cells in humans and mice (2, 3) and among subsets tor T cells generally perform these functions with more rapid of human memory CD4 and CD8 T cells that differ in expression kinetics (13). Progress has recently been made in defining new cell of the chemokine receptor CCR7 (4). These kinetic variations in surface phenotypes that distinguish effector and memory CD8 T by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd. both the CD4 and CD8 memory T cell pool result in similar func- cells (14); however, there is still no phenotypic marker that reliably tional outcomes, and subsets of memory T cells exhibiting defined distinguishes effector and memory CD4 T cells. We have been differences in activation or functional outcome have not been exploring alternate ways to distinguish effector and memory T identified. cells and have recently shown that effector and memory CD4 T Long-lived memory CD4 and CD8 T cells also exhibit pheno- cells differ biochemically, as assessed by the tyrosine phosphory- typic heterogeneity based on surface expression of the lymph lation profile (10). This biochemical analysis requires a relatively 3 node-homing receptor, CD62 ligand (CD62L). Memory CD8 T large number of purified cells; therefore, a functional assay em- cells that persist following viral or bacterial infection are hetero- ploying a small number of T cells to distinguish effector and mem- https://www.jimmunol.org geneous for CD62L expression (5, 6). Similarly, CD62L hetero- ory CD4 T cell subsets would be highly advantageous. geneity has been observed in memory CD8 T cells generated from In this study we demonstrate that effector and memory CD4 T in vivo activation of TCR-transgenic T cells in adoptive hosts cells can be clearly distinguished based on activation profile to (6–9) and in persisting memory CD4 T cells generated by transfer different types of TCR-mediated stimuli, and we use these assays of TCR-transgenic effector CD4 T cells into adoptive hosts (10, to analyze heterogeneity within the memory CD4 T cell pool. We high 11). Because CD62L expression (CD62L ) is generally associ- previously found that effector CD4 T cells could maintain effector

Downloaded from properties for several months in vivo based on biochemical analysis of the persisting population (10). However, we could not rule Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 out that a resting memory population was generated and masked Received for publication August 8, 2000. Accepted for publication October 24, 2000. by a dominant effector profile. Here, we demonstrate that both conventional resting memory T cells and persisting effector CD4 T The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cells comprise the memory T cell pool and can be isolated based with 18 U.S.C. Section 1734 solely to indicate this fact. on CD62L expression. These two memory subsets mediate recall 1 This work was supported by National Institutes of Health Grant R29AI42092 (to responses and produce effector cytokines, yet differ in overall pro- D.L.F.). liferative capacity, kinetics of activation, activation profile, and 2 Address correspondence and reprint requests to Dr. Donna L. Farber, Department of spontaneous proliferation to MHC class II. These findings suggest Surgery, University of Maryland, MSTF Building, Room 400, 685 West Baltimore Street, Baltimore, MD 21201. E-mail address: [email protected] that different subsets of memory CD4 T cells may play disparate 3 Abbreviations used in this paper: CD62L, CD62 ligand; HA, hemagglutinin; RAG2, roles in recall responses and may also have different requirements recombinase-activating gene 2. for maintenance in vivo.

Materials and Methods Adoptive transfers and cell purification Mice Effector CD4 T cells were purified through Ficoll (LSM, ICN, Costa Mesa, CA) after 3 days in culture, washed three or four times in PBS, and re- BALB/c mice were obtained from National Cancer Institute Biological 7 Testing Branch and were used between 8 and 12 wk of age. Hemagglutinin suspended in 10 cells/0.5 ml of PBS. Effector cells or equal numbers of purified resting HA-TCR CD4 T cells from naive mice were injected in 0.5 (HA)-TCR-transgenic mice (15) were bred as heterozygotes and main- ml into the tail vein of RAG2Ϫ/Ϫ tained in the Microbiology Animal Facility at the University of Maryland mice. Adoptive transfer recipient mice Ϫ Ϫ were sacrificed 8–13 wk post-transfer, and splenic CD4 T cells were iso- (College Park, MD). MHC class II / mice (16) and recombinase-activat- Ϫ Ϫ lated using MACS magnetic sorting. Briefly, splenocytes were first incu- ing gene 2 (RAG2) / mice (17) on a BALB/c genetic background were purchased as breeding pairs from Taconic Farms (Germantown, NY) and bated at 37°C for1htoremove adherent cells. Nonadherent cells were bred and maintained in the animal facility under specific pathogen-free collected and incubated with FcR blocking reagent (Miltenyi) for 15 min conditions. followed by anti-Thy 1.2 microbeads (Miltenyi) for 10 min on ice. Cells were washed, resuspended in PBS/1% FCS, and positively selected over a MACS magnetic column. Abs and reagents FACS sorting and analysis The following Abs were purified from culture supernatants from hybrid- omas maintained in the laboratory: C363.29B (anti-CD3⑀) (18), GK1.5 CD4 T cells from adoptive transfer recipient mice or HA-TCR mice were (anti-CD4) (19), anti-CD8 (Tib-105, American Type Culture Collection, enriched by immunomagnetic depletion. Briefly, splenocytes were incu- ␣ ␥ Manassas, VA), 212.A1 (anti-I-Ad), anti-Fc-␥R (clone 2.4G2), anti-Thy1.2 bated with an Ab cocktail containing anti-Mac-1 , anti-Fc R, anti-Ly6G, (TIB 238), and anti-Mac-1␣ (TIB 128). The 6.5 anti-clonotype Ab (15) and anti-MHC II followed by anti-rat IgG-, anti-mouse IgG-, and anti- directed against the HA-TCR (rat IgG) hybridoma was purified and FITC mouse IgM-coupled magnetic beads. The cells were subsequently stained conjugated as described previously (10). The following mAbs were pur- with FITC-conjugated anti-Thy 1.2 and PE-conjugated anti-CD62L before sorting on an EPICS Elite ESP flow cell sorter (Coulter, Miami, FL). The chased from PharMingen (San Diego, CA): PE- and biotin-conjugated anti- ϩ low ϩ high CD45RB (clone C363.16A) (20), PE-conjugated anti-CD62L (clone MEL- resultant Thy 1.2 CD62L and Thy 1.2 CD62L sorted populations 14), PE-conjugated anti-CD25 (clone 7D4), FITC-conjugated Thy-1.2 were 90–97% pure. For staining, cells were washed and resuspended in (clone 30-H12), purified anti-mouse Ly-6G (clone RB6-8C5), and PE-con- stain buffer (PBS, 5% FCS, and 0.05% sodium azide). Stained cells were jugated anti-CD4. Quantum Red-conjugated anti-CD4 (clone H129.19) analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA) with was purchased from Sigma (St. Louis, MO). The MACS anti-CD90 CellQuest software. (Thy1.2) microbeads were purchased from Miltenyi Biotec (Auburn, CA). The HA peptide 110–119 of the sequence, SFERFEIFPK, was synthesized Results by Biopolymer Laboratory, University of Maryland School of Medicine. Generation and functional analysis of HA-specific effector CD4 T cells Isolation of naive and memory HA-TCR subsets To characterize effector CD4 T cells relative to naive and memory The detailed procedure for isolation of mouse CD4 T cells and subsequent T cell counterparts, we first generated Ag-specific effector CD4 T separation into CD45RBlow (memory) and CD45RBhigh (naive) subsets cells from HA-TCR transgenic mice in which a large proportion of was detailed previously (21). Briefly, CD4 cells (Ͼ90% pure) were isolated peripheral CD4 T cells express the transgene-encoded TCR spe- from HA-TCR spleen using immunomagnetic depletion with anti-CD8 and cific for peptide 110–119 of influenza HA and I-Ed (15). We pre- anti-I-Ad mAbs followed by anti-rat IgG-, anti-mouse IgG-, and anti- mouse IgM-coupled magnetic beads (Perseptive Biosystems, Cambridge, viously showed that activation of naive HA-TCR CD4 T cells for MA). CD4 T cells were fractionated into naive and memory subsets by 5 days with HA peptide in the presence of splenic MHC class ϩ by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd. positive and negative selection by MACS separation using biotin-conju- II APC resulted in activated cells bearing the functional and phe- gated anti-CD45RB (C363.16A, PharMingen) and streptavidin MACS notypic attributes of differentiated effector CD4 T cells (10). We magnetic beads (Miltenyi Biotec). The resultant CD45RBlow (memory) and CD45RBhigh (naive) populations were Ͼ95% CD4. compared activation parameters of this effector CD4 T cell population relative to ex vivo naive (CD45RBhigh) HA-TCR CD4 T cell precursors by stimulating with different doses of HA peptide in the In vitro generation of effector cells presence of APC. Overall, we found that effector cells exhibit peak Ag-activated effector CD4 T cells were generated from HA-TCR CD4 T proliferative responses between 24–48 h, whereas naive CD4 T ϫ 6 ␮ ϫ 6 cells (1 10 cells/ml) incubated with 5 g/ml HA peptide and 3 10 cells exhibit maximal proliferation at 72 h (data not shown). As cells/ml T-depleted BALB/c splenocytes as APC in 24-well plates for 3–5 days at 37°C (10). For in vitro functional analyses, effector cells were shown in Fig. 1A, at 48 h effector CD4 T cells proliferate exten- https://www.jimmunol.org centrifuged through Ficoll after 5 days to remove dead cells and contam- sively at all Ag doses, whereas naive T cells proliferate at a lower inating accessory cells, washed in PBS, and resuspended in complete level than effectors only at higher Ag doses (see inset), consistent Ͼ Clicks medium (10). Effectors generated in this way were 95% pure and with similar findings by Iezzi (22). Effector CD4 T cells did not had no residual APC. proliferate in response to HA peptide alone, confirming the lack of contaminating APC (Fig. 1A). Restimulation of HA-TCR effectors Proliferation and cytokine assays with increasing doses of Ag also resulted in increasing levels of

Downloaded from CD45RBhigh, CD45RBlow, and in vitro generated effector subsets (50,000 IFN-␥ production, whereas naive HA-TCR CD4 T cells did not cells/well) were incubated in flat-bottom 96-well plates with APC produce significant levels of IFN-␥ even at high peptide doses (150,000/well) in complete Clicks medium. Titrated amounts of either HA (Fig. 1B). These results demonstrate that the in vitro-generated peptide or anti-CD3⑀ Ab were added. Cells were incubated at 37°C, proliferation was assessed after 24 and 48 h by the addition of 1 ␮Ci [3H]thy- HA-TCR effector population is functionally hyper-responsive to midine (6.7 Ci/mmol)/well, and cells were harvested after 18 h using a antigenic stimulation compared with naive CD4 T cells, and that Tomtec 96-well plate harvester (Wallac, Gaithersburg, MD). Radioactivity effector CD4 T cell proliferation, when measured at early time was quantitated using a Microbeta Tri-luxe plate scintillation counter (Wal- points, correlates with cytokine secretion. lac), and proliferation was expressed as the average of triplicates in which errors were consistently Ͻ10%. Supernatants from duplicate cultures set up Activation profile of naive, memory, and effector CD4 T cells for proliferation were collected after 24 and 48 h. IFN-␥ in supernatants was measured by specific ELISA (Endogen, Cambridge, MA). Color re- Although naive and effector CD4 T cells can be clearly distin- actions were read at 450 absorbance in an ELISA reader (Bio-Rad), and guished by their response to antigenic stimulation (Fig. 1), func- units per milliliter of IFN-␥ were calculated by comparison to a known tional differences between memory and effector CD4 T cells are IFN-␥ standard. For analysis of CD4 T cells and CD62L subsets from adoptive transfer recipient mice, assays were set up as described above, not clearly defined. With the goal of defining functional parameters except that 37,500 T cells were added per well, and proliferation was as- that delineate effector and memory CD4 T cells, we considered the sessed after 24–72 h in culture. striking differences in activation requirements to anti-CD3-mediated 928 MEMORY CD4 T CELL HETEROGENEITY

Table I. Activation requirements of naive, effector, and memory CD4 T cells

Naivea Memoryb Activation Stimulus (CD45RBhigh) (CD45RBlow) Effectorc

Ag/MHC class IIϩ APC ϩϩ ϩϩ ϩϩϩϩd Anti-CD3 Ab/MHC ϩϩϩ ϪϪ ϩϩϩϩ class IIϩ APC Anti-CD3 Ab/MHC ϩϩϩ ϩϩϩ ϩϩϩϩ class IIϪ APC

aRefs. 21 and 22. bRefs. 21 and 29. cResults shown in this study. dRef. 22 and this study.

specific activation property, we asked whether effector CD4 T cells would likewise exhibit hyporesponsiveness to anti-CD3/IIϩAPC. For these experiments we isolated naturally occurring memory and naive CD4 T cells from HA-TCR mice as defined by CD45RB isoform expression (20). The majority (Ͼ90%) of splenic CD4 T cells in HA-TCR mice that express the transgene-encoded TCR (clonotype 6.5) exhibit a characteristic CD45RBhigh naive phenotype (10). However, a small proportion (between 5–8%) of 6.5ϩ CD4 T cells express the CD45RBlow memory phenotype (data not shown), presumably by priming via a second, endogenously expressed TCR as found in another TCR-transgenic mouse strain (24). We isolated the naturally occurring CD45RBlow (memory) and CD45RBhigh (naive) subsets from HA-TCR mice and analyzed these subsets phenotypically and functionally. The CD45RB expression profile of these purified subsets is shown in Fig. 2A.To ensure that these subsets functionally represented naive and memory CD4 T cells, we tested for the absence and the presence, respectively, of effector cytokine production in response to HA peptide Ag. As shown in Fig. 2B, CD45RBlow (memory) HA-TCR

by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd. CD4 T cells produce high levels of IFN-␥ in response to HA peptide, whereas equivalent numbers of 6.5ϩ CD45RBhigh (naive) HA-TCR CD4 T cells produce negligible levels of this effector cytokine. These results demonstrate that the CD45RBlow subset of HA-TCR CD4 T cells functionally represents memory T cells, thus FIGURE 1. Functional analysis of in vitro generated effector HA-TCR CD4 T cells. HA-TCR effector CD4 T cells generated from HA-TCR CD4 enabling a novel comparison of endogenous HA-specific memory T cells cultured with 5 ␮g/ml HA peptide and mitomycin C-treated to HA-specific effectors. BALB/c APC for 5 days were washed and recultured with different doses To establish whether effector CD4 T cells exhibit hyporespon- ϩ of HA peptide with or without splenic APC. Parallel cultures were set up siveness to anti-CD3/II APC similar to memory counterparts, we https://www.jimmunol.org with naive (CD45RBhigh) CD4 T cells freshly isolated from HA-TCR mice. cultured naive, effector, and memory HA-TCR CD4 T cells with A, Proliferation of naive (open symbols) and effector (closed symbols) anti-CD3/IIϩAPC and performed a dose and kinetic analysis of HA-TCR CD4 T cells assessed after 48 h in culture, normalized for equiv- their responses. As shown in Fig. 2C, at all doses and time points ϩ alent numbers of clonotype (6.5 ) CD4 T cells. The inset gives an enlarged effector CD4 T cells exhibit the highest level of proliferation to view of naive CD4 T cell proliferation to different peptide Ag doses. Re- anti-CD3/IIϩ APC, compared with ex vivo HA-TCR naive and sults are averages of triplicates with variances Ͻ10%. B, Production of ϩ memory subsets. Although effectors show maximal proliferation IFN-␥ from equivalent numbers of 6.5 naive and effector HA-TCR CD4 Downloaded from ␥ after 24 h of stimulation and hyper-responsiveness to anti-CD3/ T cells stimulated with different doses of HA peptide. IFN- was quanti- ϩ tated by specific ELISA from 24-h culture supernatants. Results are rep- II APC at the lowest doses of anti-CD3 stimulation (0.01–0.1 resentative of five different experiments. ␮g/ml), naive CD4 T cells show increased proliferation at 48 h only at higher doses of anti-CD3 (1–5 ␮g/ml; Fig. 2C). By contrast, at all time points and doses of anti-CD3 Ab, memory CD4 T cells fail to exhibit a significant proliferative response. These restimulation exhibited by ex vivo naive and memory CD4 T cells (see sults demonstrate that when stimulated with anti-CD3/IIϩAPC, ef- Table I). We and others had previously demonstrated that murine fector CD4 T cells are hyper-responsive, and memory CD4 T cells memory CD4 T cells are hyporesponsive to soluble anti-CD3 Ab in are hyporesponsive. the presence of MHC class IIϩ splenic APC (anti-CD3/IIϩAPC), Although effector and memory CD4 T cells can be clearly dis- whereas naive CD4 T cells are fully activated by this stimulus (21, 23) tinguished by their responses to anti-CD3/IIϩAPC, we wished to (see Table I). This memory CD4 T cell hyporesponsiveness is depen- examine multiple functional parameters to obtain an activation dent on the CD4-MHC class II interaction (21, 23), as memory CD4 profile that clearly defines naive, effector, and memory CD4 T cell T cells can be activated by anti-CD3 presented by MHC class IIϪ subsets. We thus analyzed proliferative responses of HA-TCR APC (anti-CD3/IIϪAPC) (21). Given this novel memory CD4 T cell- naive, effector, and memory CD4 T cells to Ag/APC, anti-CD3/ The Journal of Immunology 929

FIGURE 2. Activation profile of naive, memory, and effector HA-TCR CD4 T cells to antigenic vs anti-CD3 stimulation. A, CD45RB expression profile of naive (CD45RBhigh) and memory (CD45RBlow) CD4 T cells isolated from HA-TCR mice. Subsets were fractionated by MACS separation (see Materials and Methods) and stained with PE-labeled CD45RB. B, Cyto- kine production from equivalent numbers of clonotype-positive naive (CD45RBhigh) and memory (CD45RBlow) HA-TCR CD4 T cells stimulated with 5 ␮g/ml HA peptide and APC. C, Ki- netics of proliferation to activation by increasing doses of anti-CD3⑀ Ab in the presence of MHC class IIϩ APC (IIϩ APC). Ex vivo memory (CD45RBlow), naive (CD45RBhigh) and in vitro generated effector HA-TCR CD4 T cells (50,000 cells/well) were stimulated with 0.01–1 ␮g/ml anti- CD3 in the presence of 150,000 MHC class IIϩ APC. Proliferation was assessed by pulsing with [3H]thymidine after 24 and 48 h in culture. D, Activation profile of naive, effector, and memory HA-TCR CD4 cells. Naive, effector, and memory CD4 T cells were cultured either with 1 ␮g/ml HA peptide plus APC or with 1 ␮g/ml anti-CD3 in the presence of IIϩAPC or MHC class IIϪ APC (IIϪ APC). Negative controls include cells incubated with either IIϩ or IIϪ APC alone (stimulus- none) and cells incubated with either anti-CD3 Ab or HA peptide alone (APC type 0). Proliferation was assessed after 48 h of culture. Results are representative of four different experiments. by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd.

IIϩAPC and anti-CD3/IIϪAPC (memory CD4 T cell hyporespon- ory CD4 T cells. The hyper-responsiveness of effectors is inde- siveness to anti-CD3/IIϩ APC is overcome by anti-CD3/IIϪAPC pendent of stimulation by residual Ag and/or APC, because (21); see Table I), to obtain an activation profile. As shown in Fig. effectors stimulated with either APC alone or anti-CD3 or HA 2D, two themes emerge when examining individual activation pro- peptide alone exhibited negligible proliferation (Fig. 2 and data not files of naive, effector, and memory CD4 T cells. First, naive, shown). These results indicate that analysis of the activation profile effector, and memory CD4 T cells exhibit distinct activation pro- to cognate and noncognate stimuli and overall proliferative capac- files to the three types of TCR stimuli. (Each activation profile is ities can be used to unambiguously distinguish effector and mem- https://www.jimmunol.org depicted on a separate graph, so that relative responses to Ag-vs ory CD4 T cells (see Table I). anti-CD3 can be compared.) Naive CD4 T cells proliferate well in response to Ag/APC and exhibit higher proliferative responses to Analysis of memory generation in vivo anti-CD3-mediated stimuli in the presence of IIϩ or IIϪAPC Given our ability to distinguish effector and memory CD4 T cells (probably due to the activation of nonclonotype T cells). Effector based on activation profile, we next applied this assay to analyze CD4 T cells exhibit high proliferative responses to Ag/APC and the generation of long-lived memory T cells from effector CD4 T ϩ Downloaded from equally high proliferative responses to anti-CD3/II APC and anti- cells in vivo. We had previously demonstrated that transfer of HA- CD3/IIϪAPC (Fig. 2D, middle graph). Although ex vivo-derived TCR effector CD4 T cells into sublethally irradiated BALB/c memory (CD45RBlow) HA-TCR CD4 T cells proliferate in re- adoptive hosts resulted in the persistence of Ag-specific T cells for sponse to Ag/APC and anti-CD3/IIϪAPC, they do not exhibit sub- many months that mediated recall responses to Ag (10), consistent stantial proliferation in response to anti-CD3/IIϩAPC (Fig. 2D, with findings in other adoptive transfer systems (25, 26). Although right-most graph). Thus, while both naive and effector CD4 T cells these cells appeared smaller in size than effectors and were long- exhibit antigenic responses lower or equivalent to anti- lived, this persisting population exhibited an effector-specific bio- CD3/IIϩAPC responses, memory CD4 T cells exhibit greater re- chemical pattern of tyrosine phosphorylation. These results sug- sponses to Ag compared with anti-CD3/IIϩAPC. gested that a proportion of transferred effectors were maintained in The second theme that emerges from the activation assays in vivo as persisting effector CD4 T cells (10). Fig. 2D is that naive, effector, and memory CD4 T cells exhibit In this study, we asked whether these persisting cells likewise profound differences in proliferative capacity. Each subset re- exhibited an effector-specific or memory cell-specific activation sponded maximally to stimulation with anti-CD3/IIϪAPC, and profile. For these analyses, we transferred HA-TCR effectors into comparison of these responses reveals that effectors have the high- syngeneic RAG2Ϫ/Ϫ adoptive hosts to remove the complication of est proliferative capacity overall, followed by naive and then mem- endogenous T cells. We purified the persisting splenic CD4 T cells 930 MEMORY CD4 T CELL HETEROGENEITY

Table II. Nomenclature used to designate T cell subsets persisting cells likewise exhibited hyporesponsiveness to anti- CD3/IIϩAPC. We thus assayed the ability of CD4ET cells 10 wk Cell Subset Designation Definition post-transfer to respond to different doses of anti-CD3 in the pres- ϩ NT ET ence of II APC compared with CD4 that persisted 10 wk post- CD4 CD4 T cells persisting following transfer of ET effector CD4 T cells into RAG2Ϫ/Ϫ hosts transfer. As shown in Fig. 3B, CD4 cells exhibit a high prolif- CD4NT CD4 T cells persisting following transfer of erative response to low doses of anti-CD3 Ab (0.1 and 1 ␮g/ml) naive CD4 T cells into RAG2Ϫ/Ϫ hosts compared with CD4NT cells, which respond substantially only at N N CD62Llo , CD62Lhi Subsets of CD4 T cells from naive HA- the highest dose tested (1 ␮g/ml). These results indicate that the TCR mice differing in CD62L expression ET ET ET ET CD4 cells that persist in vivo resemble effector CD4 T cells in CD62Llo , CD62Lhi CD4 cells fractionated into subsets ϩ differing in CD62L expression their hyper-responsiveness to anti-CD3/II APC, similar to in vitro generated effectors (Fig. 2C). To further analyze the activation properties of the CD4ET per- ET 8–13 wk post-transfer and designated these cells as CD4ET be- sisting population, we activated CD4 with Ag/APC, anti-CD3/ IIϩAPC, or anti-CD3/IIϪAPC to obtain an activation profile as in cause they derived from mice that received transferred effector ET cells (see Table II). For comparison, equal numbers of resting HA- Fig. 2D. The activation profile of CD4 cells in Fig. 3C demon- strates that CD4ET cells proliferate at high levels in response to TCR CD4 T cells were transferred in parallel, and the cells per- ϩ Ϫ sisting 8–13 wk in vivo were designated CD4NT (see Table II) Ag/APC, anti-CD3/II APC, and anti-CD3/II APC similar to in because they derived from mice that received resting HA-TCR vitro generated effector cells (Fig. 2D). Controls with APC, Ag, or CD4 T cells consisting predominantly of naive CD4 T cells. anti-CD3 Ab alone yielded negligible proliferative responses, con- ET firming the lack of contaminating Ag and APC in the purified In agreement with our previous results (10) the CD4 cells ET Ϫ/Ϫ CD4 population (Fig. 3B). From these results we conclude that persisting in RAG2 hosts were phenotypically different from ET the transferred effector CD4 T cells. In particular, the expression of at least a fraction of the persisting CD4 cells exhibit effector- CD25, while up-regulated in effector T cells relative to naive HA- specific activation profiles. This maintenance of effector-specific ET properties over time is consistent with our previous findings that TCR precursors (Fig. 3A) was down-regulated in the CD4 pop- ET ulation, consistent with analogous adoptive transfer systems (25, CD4 cells derived from BALB/c adoptive hosts exhibited effec- 26). In addition, while naive HA-TCR CD4 T cells were primarily tor-specific biochemical profiles (10). CD62Lhigh, both Ag-activated effectors and CD4ET persisting Isolation and functional analysis of heterogeneous 8–16 wk in adoptive hosts were heterogeneous for CD62L expres- ET sion, with two distinct populations of CD62Llo and CD62Lhi CD4 subsets clearly discernible in the CD4ET population (Fig. 3A). To deter- Although the persisting CD4ET cells exhibited a high proliferative mine whether CD4ET cells represented resting memory CD4 T capacity and hyper-responsiveness to anti-CD3/IIϩAPC similar to cells in terms of activation parameters, we asked whether these effector T cells, the presence of conventional resting memory T by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd.

FIGURE 3. Activation requirements of HA-TCR CD4 T cells persisting in adoptive hosts. In vitro-generated effector HA-TCR CD4 T cells or resting HA- TCR CD4 T cells were i.v. transferred into RAG2Ϫ/Ϫ hosts. Ten weeks post-transfer, the persisting splenic CD4 T cells were purified by positive selection with anti-Thy1.2 MACS magnetic beads. A, Expression of https://www.jimmunol.org CD62L and CD25 by naive, effector, and CD4ET.Pu- rified HA-TCR CD4 T cells, 5 day in vitro-generated HA-TCR effector CD4 T cells, and the persisting CD4ET population were triple stained for 6.5, CD4, and either CD62L or CD25, and the results shown are gated on CD4ϩ6.5ϩ T cells. The CD4ET phenotype was similar as measured 8–16 wk post-transfer. B, Downloaded from Dose response to anti-CD3/MHC class IIϩ APC (IIϩ APC) of the persisting CD4ET cells from hosts that received effector T cells compared with the persisting CD4NT cells from hosts that received predominantly naive HA-TCR CD4 T cells 10 wk previously. Prolif- eration was assessed after 3 days. C, Activation profile of persisting HA-TCR CD4 T cells (CD4ET). Purified CD4ET cells (50,000/well) were cultured with anti- CD3 (1 ␮g/ml) in the presence of 150,000 IIϩ APC or IIϪ APC or HA peptide (1 ␮g/ml) in the presence of IIϩ APC. Proliferation was assessed after 3 days. These results are representative of five transfer experiments. The Journal of Immunology 931 by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd.

FIGURE 4. Phenotypic and functional heterogeneity of the memory CD4 T cell pool. T cells were isolated from RAG2Ϫ/Ϫ hosts that received effector CD4 T cells 8–13 wk previously and were enriched by immunomagnetic depletion of macrophages and granulocytes. The enriched population was double stained with FITC-conjugated anti-Thy1.2 and PE-conjugated anti-CD62L before sorting into Thy1.2ϩ/CD62Llo and Thy1.2ϩ/CD62Lhi populations. A, CD62L expression of CD4ET cells before and after sorting into CD62LloET and CD62LhiET subsets. The histograms were gated on live Thy 1.2ϩ cells, and the resultant T cells were 93–97% pure. B, Functional analysis of CD62L sorted populations from CD4ET (CD62LloET and CD62LhiET) and fresh HA-TCR CD4 T cells (CD62LloN and CD62LhiN). Sorted T cells (37,500/well) were cultured with mitomycin C-treated APC (150,000/well) plus HA peptide Ag (0.05 ␮g/well) for 48 h. Sorted T cells from four to six adoptive hosts were pooled for the above analysis. C, Kinetics of cytokine production from CD62LET subsets isolated 13 wk post-transfer. CD62LloET and CD62LhiET subsets were cultured alone or with 0.1 or 1.0 ␮g/ml HA peptide in the https://www.jimmunol.org presence of APC. After 30 or 48 h in culture, supernatants were collected and analyzed for IFN-␥ content by ELISA. The above results are representative of four sorting experiments.

cells that exhibit lower proliferative capacities and hyporespon- times greater than the CD62LhiET subset, when individual mice siveness to anti-CD3/IIϩAPC would be masked by dominant ef- were examined (data not shown). ET Downloaded from fector functions. The phenotypic heterogeneity in CD62L expres- To determine whether both CD62L subsets mediated recall sion found in the CD4ET-persisting population (see Fig. 3A) responses to specific Ag, we measured IFN-␥ secretion by suggested that the long-lived memory T cell pool may consist of CD62LET and CD62LN subsets in response to Ag. As demon- more than one subset of CD4 T cells. To determine whether dif- strated in Fig. 4B, both CD62LET subsets secrete IFN-␥ in references in CD62L expression delineated subsets of long-lived sponse to antigenic stimulation compared with the negligible level memory T cells that differed in function and/or activation profile, of IFN-␥ secreted by CD62LN subsets in response to antigenic we sorted the persisting CD4ET cells into CD62LloET and stimulation. Interestingly, the level of IFN-␥ in culture superna- CD62LhiET populations (histogram plots in Fig. 4A) for subse- tants from CD62LloET cells is 2- to 3-fold higher than that in quent functional analysis. For comparison, we also sorted CD62L supernatants from CD62LhiET cells, although both express com- T cell subsets directly from purified fresh HA-TCR CD4 T cells parable numbers (ϳ85%) of 6.5ϩ cells (data not shown). To fur- that were predominantly naive (CD62LloN and CD62LhiN, refer to ther examine the cytokine properties of the CD62LET subsets, we Table II for nomenclature). Although the CD62LhiN subset pre- performed a dose and kinetic analysis of Ag-specific cytokine pro- dominated in CD4 T cells derived from naive HA-TCR mice as duction (Fig. 4C). As illustrated in Fig. 4C, CD62LloET produces previously reported (10), the proportion of the CD62LloET subset a higher level of IFN-␥ with faster kinetics than the CD62LhiET in CD4ET cells ranged from equivalent (see Fig. 4A)toupto3 subset. After 30 h in culture with Ag/APC, CD62LloET produces 932 MEMORY CD4 T CELL HETEROGENEITY

FIGURE 6. Spontaneous proliferation of CD62L subsets to MHC class IIϩ and MHC class IIϪ APC. CD62L populations isolated and sorted from effector transfer recipients (CD62LET) 10 wk post-transfer and fresh transgenic HA-TCR CD4 T cells (CD62LN) were incubated with MHC class IIϩ APC (IIϩ APC) or IIϪ APC, and proliferation was assessed after 72 h. These results are representative of ﬁve sorting experiments.

response to either anti-CD3/IIϩAPC or anti-CD3/IIϪAPC in- creases ϳ2-fold between 48 and 72 h. By contrast, CD62LhiET proliferation in response to anti-CD3/IIϩAPC is negligible at 48 h and remains low after 72 h in culture, while proliferation in response to anti-CD3/IIϪAPC at 48 h is comparable to that in the ET FIGURE 5. Activation profile of CD62LET subsets in the memory CD4 CD62Llo subset and doubles at 72 h (Fig. 5B, right). Taken T cell pool. A, Proliferation of CD62LloET and CD62LhiET subsets isolated together, the results in Fig. 5 demonstrate that the sorted 11 wk post-transfer to HA peptide/MHC class IIϩ APC (IIϩ APC) and CD62LloET and CD62LhiET subsets exhibit distinct activation pro- anti-CD3 in the presence of IIϩ APC or IIϪ APC was assessed after 3 days. files. The CD62LloET subset has a high proliferative capacity and B, Proliferation kinetics of CD62LET subsets in response to stimulation is hyper-responsive to stimulation by Ag/APC, anti-CD3/IIϩ APC, ϩ Ϫ with 1 ␮g/ml anti-CD3 Ab in the presence of II APC or II APC. Pro- and anti-CD3/IIϪ APC similar to effector CD4 T cells, whereas the liferation was assessed after 48 and 72 h in culture. These results are rep- CD62LhiET subset has a lower proliferative capacity overall and is resentative of five sorting experiments. hyporesponsive to stimulation by anti-CD3/IIϩ APC, similar to resting memory CD4 T cells. by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd. significant levels of IFN-␥, while the CD62LhiET subset does not During the course of functional analyses of the sorted CD62L ␥ subsets, we also found that spontaneous proliferation in the pres- produce appreciable IFN- levels. After 48 h in culture, however, ϩ ET ET ␥ ence of II APC differs between CD62L subsets. As shown in the CD62Lhi subset produces substantial levels of IFN- ,al- ET ET Fig. 6, only the CD62Llo population exhibited significant back- though this level is still one-third of that produced by CD62Llo . ϩ ET ground proliferation in response to II APC alone, whereas the These results establish that both CD62L subsets mediate recall ET N responses, yet differ in the level and kinetics of effector cytokine CD62Lhi subset, and both CD62L subsets sorted from fresh ET naive HA-TCR CD4 T cells exhibited negligible background pro- production, with the CD62Llo subset exhibiting rapid kinetics ϩ ET ␥ liferation to II APC. This CD62Llo -specific proliferation to and higher levels of IFN- production compared with the ϩ

https://www.jimmunol.org ET II APC was consistently present in all the sorting experiments CD62Lhi subset. ET N ET performed. Interestingly, neither the CD62L nor the CD62L Because the CD62L subsets that persisted in vivo differ in Ϫ kinetics of activation similar to previous distinctions between ef- subset exhibited background proliferation to II APC, indicating that the proliferation in response to IIϩAPC most likely derives fector and resting memory T cells (27), we asked whether these ϩ CD62LET subsets could likewise be distinguished based on acti- from interaction with MHC class II and endogenous peptide. We vation profile, demonstrated here to delineate effector and memory also found that in vitro-generated effectors, but not ex vivo-derived memory CD4 T cells defined by CD45RBlow expression, prolifer- Downloaded from T cells (see Fig. 2). We thus measured the proliferative response of ϩ Ϫ sorted CD62LET subsets to Ag or anti-CD3 Ab in the presence of ate in response to MHC class II but not MHC class II APC (data IIϩ or IIϪ APC. As shown in Fig. 5A, the CD62LloET and not shown), suggesting that MHC class II-dependent turnover may CD62LhiET subsets exhibit disparate activation profiles to anti- be an effector-specific property. genic and anti-CD3-mediated stimuli. The CD62LloET subset proliferates vigorously in response to Ag/APC or anti-CD3 in the Discussion presence of IIϩ or IIϪ APC. By contrast, the CD62LhiET subset is Recent findings of heterogeneity in the memory T cell pool in hyporesponsive to anti-CD3/IIϩ APC, while it proliferates well in humans and mice suggest that more than one type of previously response to Ag/APC and anti-CD3/IIϪ APC, similar to the acti- activated T cell may mediate recall responses. In this study, we vation profile observed with ex vivo isolated resting memory CD4 analyzed the cellular composition of the memory CD4 T cell T cells (see Fig. 2C). pool based on novel activation criteria shown here to distin- To further analyze responses of the CD62LET subsets to non- guish effector and memory CD4 T cells. These activation cri- cognate stimuli, we performed a kinetic analysis of proliferation in teria were found to delineate subpopulations of memory CD4 T response to anti-CD3/IIϩ APC and anti-CD3/IIϪ APC after 48 and cells that differed in CD62L expression, with the CD62Llo 72 h in culture. As shown in Fig. 5B, CD62LloET proliferation in memory population resembling effector T cells and the The Journal of Immunology 933

FIGURE 7. Possible schemes for the generation of heterogeneous subsets of memory T cells.

CD62Lhi memory population resembling resting memory T systems (25, 26, 32). By applying our activation profile assay, we cells. Based on these observations, we conclude that the mem- found that the persisting memory population (designated CD4ET) ory CD4 T cell pool consists of persisting effectors and resting exhibited an effector-specific activation profile in both its high pro- memory T cells. liferative capacity and its hyper-responsiveness to anti- It has been difficult to follow the differentiative fate of effector CD3/IIϩAPC. This finding was consistent with our previous re- cells in vivo and characterize memory T cell heterogeneity due to sults demonstrating that CD4ET exhibited an effector-specific the lack of phenotypic markers and functional assays that reliably biochemical profile (10). When taken together, our data suggest distinguish effector and memory T cells. Cell surface phenotypes that effector CD4 T cells may persist for long periods of time in have been shown to delineate effector and memory CD8 T cells in vivo, longer than previous estimates of effector T cell life span mice and humans (14, 28); however, clear-cut phenotypic or func- (33), yet consistent with recent findings that effector B cells or tional differences between effector and memory CD4 T cells have plasma cells are likewise long-lived in vivo (34). not yet been reported. To characterize the cells that comprise the We and others have found that the memory T cell population memory CD4 T cell pool, we first established novel functional and persisting after adoptive transfer of effectors was heterogeneous activation differences between effector and memory CD4 T cells. for the expression of CD62L (7, 10, 25). CD62L heterogeneity has As our standard for resting memory CD4 T cells generated in vivo, likewise been observed in memory T cells that persist following low

by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd. we used the CD45RB subset of splenic CD4 T cells, because viral (6, 35) or bacterial (5) infection. We found that the CD62Llo this subset bears the phenotypic and functional properties of mem- and CD62Lhi subsets differed strikingly in activation profile, pro- ory CD4 T cells (29). As presented here, ex vivo-derived memory liferative capacity, and activation kinetics, similar to distinctions CD4 T cells can be clearly distinguished from effector CD4 T cells between effector and resting memory CD4 T cells, respectively. based on activation profile to antigenic and anti-CD3-mediated The association of effector-like functions with CD62Llo phenotype stimuli. Specifically, effector CD4 T cells exhibit a greater prolif- has been suggested in studies in which splenic LCMV-specific mem- erative capacity than memory CD4 T cells and are hyper-respon- ory CD8 T cells heterogeneous for CD62L expression exhibited more sive to anti-CD3/IIϩAPC and Ag, whereas memory CD4 T cells rapid effector function than lymph node memory CD8 T cells that are hyporesponsive to anti-CD3/IIϩAPC relative to Ag. were primarily CD62Lhi (35). Similarly, the CD62Llo fraction of https://www.jimmunol.org The differences in activation parameters identified here suggest long-lived Sendai virus-specific memory CD8 T cells was found to that effector and memory CD4 T cells signal differently through exhibit a higher proliferative capacity in vitro than the CD62Lhi frac- the TCR/CD3 complex. We have previously demonstrated striking tion, as assessed by CFSE staining (6). Although loss of CD62L ex- differences in the tyrosine phosphorylation profile of naive, effec- pression is typically associated with effector/memory function, tor, and memory CD4 T cells (10) and in proximal kinase phos- CD62Lhi memory CD4 T cells have been found in unmanipulated phorylation in ex vivo derived naive and memory subsets (30). mice (36) and were found to predominate in aged mice (37). These lck Downloaded from Differences in the expression of CD8-associated p56 kinase have results suggest that resting memory T cells may reacquire CD62L also been identified in naive vs effector/memory CD8 T cells (31). expression over time in vivo. These functional and biochemical disparities suggest that alter- Subsets of human memory CD4 T cells have recently been iso- ations in the TCR-coupled signaling machinery control the distinct lated by Lanzavecchia and colleagues based on expression of the functional properties of naive, effector, and memory T cells. We chemokine receptor CCR7 (4). The CCR7Ϫ memory subset was

are currently examining both proximal and distal TCR-mediated designated as effector-memory (TEM) due to rapid activation ki- signal intermediates in these three subsets to address this netics, whereas the CCR7ϩ subset was designated central memory

hypothesis. (TCM) due to slower activation kinetics. Although the TCM subset The identiﬁcation of a functional assay to distinguish between is predominantly CD62Lhi (4) and may correspond to the mouse

effector and memory CD4 T cells enabled analysis of the differ- CD62Lhi resting memory subset identified here, the TEM subset is entiative fate of effector CD4 T cells in vivo and the cellular com- heterogeneous for CD62L expression (4). Taken together, our re- position of the memory T cell pool. The memory T cell pool is sults and those reported by Lanzavecchia suggest that both the defined here as the Ag-specific HA-TCR CD4 T cells that persist CD62L homing receptor and the CCR7 chemokine receptor may 8–13 wk following transfer of HA-TCR effectors into RAG2Ϫ/Ϫ be differentially expressed on memory T cell subsets. Once the adoptive hosts, analogous to other CD4 and CD8 adoptive transfer murine CCR7-specific reagents are available, we can assess the 934 MEMORY CD4 T CELL HETEROGENEITY

pattern of CCR7 expression in the mouse CD62L memory subsets differ in phenotype and multiple functional parameters. Elucida- and determine whether additional memory subsets can be defined tion of the generation and maintenance of these subsets in vivo and by coordinate expression of these two markers. their roles in protective immunity can lead to improved vaccine Current models for the generation of memory T cells do not take strategies to generate the appropriate type of anamnestic response. into account heterogeneity of the memory T cell pool (38). The linear model predicts the generation of resting memory T cells Acknowledgments directly from effectors that have reverted to the resting state. By We thank Karen M. Wolcott (Biomedical Instrumentation Center, Uni- contrast, a divergent model predicts distinct generation of effectors formed Services University of the Health Sciences, Bethesda, MD) for and memory T cells directly from a naive T cell precursor. We excellent assistance with cell sorting, and Dr. Gregg Hadley (Department present in Fig. 7 a modified model for the generation of memory of Surgery, University of Maryland Medical School, Baltimore, MD) and in which the two memory subsets diverge either from an effector Dr. Sandeep Krishnan (University of Maryland, College Park, MD) for intermediate (scheme 1) or from a pre-effector intermediate critical reading of this manuscript. (scheme 2). Two other possibilities that cannot be ruled out are that resting memory T cells derive from persisting effectors (Fig. 7, References 1. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, scheme 3) and, conversely, persisting effectors may derive from a J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T memory intermediate as has been suggested by Lanzavecchia (4). cells: a reevaluation of bystander activation during viral infection. Immunity Following the development of each memory subset in vivo will 8:177. 2. Selin, L. K., and R. M. Welsh. 1997. Cytolytically active memory CTL present enable identification of the cellular precursor for each memory T in lymphocytic choriomeningitis virus-immune mice after clearance of virus in- cell subset. fection. J. Immunol. 158:5366. 3. Lalvani, A., R. Brookes, S. Hambleton, W. J. Britton, A. V. S. Hill, and Generation of these two memory subsets may be differentially ϩ A. J. McMichael. 1997. Rapid effector function in CD8 memory T cells. J. Exp. regulated. Zinkernagel and colleagues have shown that the same Med. 186:859. Ag delivered by a viral or bacterial pathogen elicited memory CD8 4. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector T cells differing in protective capacity, longevity, and activation functions. Nature 401:708. kinetics (39), suggesting that the type and duration of the anamnestic 5. Busch, D. H., K. M. Kerksiek, and E. G. Pamer. 2000. Differing roles of inflam- response are affected by the activating stimulus. Experiments are cur- mation and antigen in T cell proliferation and memory generation. J. Immunol. 164:4063. rently underway to determine whether the initial activation condition 6. Usherwood, E. J., R. J. Hogan, G. Crowther, S. L. Surman, T. L. Hogg, affects the proportion of CD62Llo and CD62Lhi subsets in the mem- J. D. Altman, and D. L. Woodland. 1999. Functionally heterogeneous CD8ϩ ory CD4 T cell pool, an important consideration for vaccine design T-cell memory is induced by Sendai virus infection of mice. J. Virol. 73:7278. 7. Kedl, R. M., and M. F. Mescher. 1998. Qualitative differences between naive and where a certain type of memory response may have protective memory T cells make a major contribution to the more rapid and efficient memory advantage. CD8ϩ T cell response. J. Immunol. 161:674. 8. Cho, B. K., C. Wang, S. Sugawa, H. Eisen, and J. Chen. 1999. Functional dif- What is the purpose of these two subsets of memory T cells in ferences between memory and naive CD8 T cells. Proc. Natl. Acad. Sci. USA anamnestic responses? It is feasible that each memory subset pro- 96:2976. vides a unique role in memory responses in both the type of pro- 9. Zimmerman, C., K. Brduscha-Riem, C. Blaser, R. M. Zinkernagel, and H. Pircher. 1996. Visualization, characterization, and turnover of CD8ϩ memory tection provided and the type of APC they encounter or activate. T cells in virus-infected hosts. J. Exp. Med. 183:1367.

by guest on October 1, 2021. Copyright 2001 Pageant Media Ltd. The persisting effector population with its rapid recall response and 10. Ahmadzadeh, M., S. F. Hussain, and D. L. Farber. 1999. Effector CD4 T cells are effector function may form the first line of protection in peripheral biochemically distinct from the memory subset: evidence for long-term persistence of effectors in vivo. J. Immunol. 163:3053. tissues. It has been shown that the CD62Llo effector subset rapidly 11. London, C. A., M. P. Lodge, and A. K. Abbas. 2000. Functional responses and migrates to inflammatory sites such as bronchial tissue to eradicate costimulator dependence of memory CD4ϩ T cells. J. Immunol. 164:265. 12. Bradley, L. M., D. D. Duncan, S. L. Tonkonogy, and S. L. Swain. 1991. Char- influenza infection (40) or to tumor sites to combat fibrosarcoma acterization of antigen-specific CD4ϩ effector T cells in vivo: immunization re- (41). In the tissues the CD62Llo subset may also recruit and prime sults in a transient population of MEL14-, CD45RB-helper cells that secretes tissue macrophages and immature dendritic cells. By contrast, the IL-2, IL-3, and IL-4. J. Exp. Med. 174:547. 13. Bachmann, M. F., M. Barner, A. Viola, and M. Kopf. 1999. Distinct kinetics of CD62Lhi resting memory subset migrates to secondary lymph cytokine production and cytolysis in effector and memory T cells after viral nodes to be reprimed by resident APC (40), enabling them to pro- infection. Eur. J. Immunol. 29:291. https://www.jimmunol.org vide help to B cells. We are currently examining the role of each 14. Harrington, L. E., M. Galvan, L. G. Baum, J. D. Altman, and R. Ahmed. 2000. Differentiating between memory and effector CD8 T cells by altered expression CD62L memory subset in recall responses and their potential pro- of cell surface O-glycans. J. Exp. Med. 191:1241. tective capacity in vivo. 15. Kirberg, J., A. Baron, S. Jakob, A. Rolink, K. Karjalainen, and H. von Boehmer. 1994. Thymic selection of CD8ϩ single positive cells with a class II major his- In addition to playing distinct roles in recall responses, these two tocompatibility complex-restricted receptor. J. Exp. Med. 180:25. memory subsets may have different life spans and/or requirements 16. Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, and for long term maintenance in vivo. Although it has recently been D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051. 17. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, Downloaded from demonstrated that CD4 T cell memory can persist in the absence of J. Charron, M. Datta, F. Young, A. M. Stall, et al. 1992. RAG-2-deficient mice MHC class II (42), it is unclear whether this phenomenon applies lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell to both subsets of memory T cells. Our results that only the 68:855. 18. 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Although many studies murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to cor- have focussed on the question of memory persistence in the ab- relate primarily with class II MHC antigen-reactivity. Immunol. Rev. 74:29. sence of Ag (43–45), in vivo Ag may indeed persist for an ex- 20. Bottomly, K., M. Luqman, L. Greenbaum, S. Carding, J. West, T. Pasqualini, and tended period of time depending on the nature of the pathogen or D. B. Murphy. 1989. A monoclonal antibody to murine CD45R distinguishes CD4 T cell populations that produce different cytokines. Eur. J. Immunol. 19:617. Ag (46). Whether the longevity of persisting effectors is dependent 21. Farber, D. L., M. Luqman, O. Acuto, and K. Bottomly. 1995. Control of memory on the presence or the absence of Ag remains to be determined. 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