CD28/B7 Costimulatory Pathway Control of Memory CD4 T Cell

Control of Memory CD4 T Cell Recall by the CD28/B7 Costimulatory Pathway Modesta P. Ndejembi, John R. Teijaro, Deepa S. Patke, Adam W. Bingaman, Meena R. Chandok, Agnes This information is current as Azimzadeh, Steven G. Nadler and Donna L. Farber of September 23, 2021. J Immunol 2006; 177:7698-7706; ; doi: 10.4049/jimmunol.177.11.7698 http://www.jimmunol.org/content/177/11/7698 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Control of Memory CD4 T Cell Recall by the CD28/B7 Costimulatory Pathway1

Modesta P. Ndejembi,*‡ John R. Teijaro,*‡ Deepa S. Patke,* Adam W. Bingaman,2* Meena R. Chandok,* Agnes Azimzadeh,† Steven G. Nadler,§ and Donna L. Farber3*

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of high low memory CD4 T cells, including central (CD62L ) and effector memory (TEM; CD62L ) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-␥ production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Downloaded from Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-␥ production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly TEM in control- treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on TEM responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical http://www.jimmunol.org/ implications for using CTLA4Ig to abrogate the pathologic consequences of TEM cells in autoimmunity and chronic disease. The Journal of Immunology, 2006, 177: 7698–7706.

emory T cells are known to exhibit enhanced activa- T cell subsets can differ in their protective responses to virus in- tion properties relative to naive T cell counterparts fection or tumors in vivo (14–17). These results suggest that dif- M manifested by robust responses to low doses of Ag (1), ferential modulation of memory subsets and/or migratory proper- rapid production of potent effector cytokines (2), and reduced ac- ties can have profound effects on controlling recall responses, tivation requirements (3–5). These features allow memory T cells

although strategies to achieve this level of regulation have not been by guest on September 23, 2021 to mediate protective immunity to pathogens; however, their ro- deﬁned. bust responses are detrimental when directed to self Ags in auto- Costimulatory pathways regulate T cell activation and differen- immunity and alloantigens in transplantation (6, 7). In addition, tiation, and serve as therapeutic targets for manipulation of T cell memory T cells are heterogeneous in expression of lymph node responses. Of particular importance is the CD28/B7 costimulatory homing receptors and tissue distribution delineating central mem- pathway, as interaction of the CD28 costimulatory receptor with its 4 high ϩ oryT(TCM; CD62L /CCR7 ) cells in lymphoid tissue and ligands B7-1/B7-2 (CD80/CD86) on APCs is required for activa- low Ϫ effector memory (TEM; CD62L /CCR7 ) cells in peripheral tis- tion of naive CD4 T cells. Blocking this pathway with the well- sues (8, 9). Memory CD4 T cell subsets exhibit functional diver- characterized CTLA4Ig fusion molecule that binds B7 ligands (18) sity in cytokine production and expansion (8, 10–12) and play inhibits naive CD4 T cell activation and proliferation (19), and can distinct roles in pathogenic processes (13), whereas memory CD8 prevent the development of autoimmunity and inhibit graft rejection in animal models (20, 21). By contrast, it is generally accepted

*Division of Transplantation, University of Maryland School of Medicine, Baltimore, that memory T cells do not require CD28/B7-derived costimulation MD 21201; †Division of Cardiac Surgery, Department of Surgery, University of for recall responses (22), based on previous studies showing that Maryland School of Medicine, Baltimore, MD 21201; ‡Department of Microbiology memory CD4 T cells are fully activated by B7-deficient APC in vitro and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; Ϫ/Ϫ and §Immunology and Inflammation Drug Discovery, Bristol-Myers Squibb Pharma- (5, 23), and that CD28 mice are not impaired in the generation ceutical Research Institute, Princeton, NJ 08543 or recall of memory CD8 T cell responses to lymphocytic chorio- Received for publication July 5, 2006. Accepted for publication September 19, 2006. meningitis virus infection (24). However, CTLA4Ig treatment can The costs of publication of this article were defrayed in part by the payment of page alleviate ongoing autoimmunity in animal models (25), and shows charges. This article must therefore be hereby marked advertisement in accordance clinical efficacy in treating rheumatoid arthritis (26) and psoriasis with 18 U.S.C. Section 1734 solely to indicate this fact. (27), diseases known to be driven by memory CD4 T cells (28). 1 This work was supported by a grant from Bristol-Myers Squibb, and National In- stitutes of Health Grants AI50632 and AI42092 (awarded to D.L.F.). These results suggest that the CD28/B7 pathway may regulate 2 Current address: Department of Surgery, University of Texas, San Antonio, TX memory CD4 T cell-mediated responses; however, the in vivo 78229. requirements for CD28/B7 costimulation and the CTLA4Ig sus- 3 Address correspondence and reprint requests to Dr. Donna L. Farber, University of ceptibility of Ag-specific memory CD4 T cells have not been Maryland, Baltimore, Department of Surgery, Medical School Teaching Facility, examined. Room 400, 685 West Baltimore Street, Baltimore, MD 21201. E-mail address: [email protected] In this study, we used CTLA4Ig to investigate the CD28/B7 co- 4 stimulatory requirements of heterogeneous memory CD4 T cells spe- Abbreviations used in this paper: TCM, central memory T; HA, hemagglutinin; TEM, effector memory T. cific for influenza hemagglutinin (HA) and chicken OVA peptides, as

well as polyclonal influenza virus-specific memory cells generated Intracellular cytokine staining from flu infection in vivo. In vitro activation of HA- and OVA-spe- Intracellular cytokine staining analysis of IFN-␥ and IL-2 was performed, cific memory CD4 T cells in the presence of CTLA4Ig induces se- as previously described (33, 35). Briefly, memory CD4 T cells were cul- lective inhibition of IL-2 and TNF-␣ production, without affecting tured with APC and 5 ␮g/ml HA or 1 ␮g/ml OVA peptide Ϯ 50 ␮g/ml early IFN-␥ production and up-regulation of activation markers. Like- murine CTLA4Ig or isotype control IgG2a for 18–36 h, and monensin wise, IL-2 production of influenza virus-specific memory CD4 T cells (Golgistop; BD Pharmingen) was added 6 h before harvesting. Specific ␥ timepoints for each experiment are indicated in the figure legends. Cells was profoundly inhibited by CTLA4Ig treatment, whereas the IFN- were stained with Abs for surface CD4, CD25, CD44, KJ1-26, or 6.5, fixed production was partially down-regulated. When administered in vivo, (Cytofix buffer; BD Pharmingen), permeabilized, and stained intracellu- CTLA4Ig did not affect very early activation of HA- and OVA-spe- larly with fluorochrome-conjugated anti-IFN-␥, anti-IL-2, or their respec- cific memory T cells, although it profoundly inhibited in vivo Ag- tive isotype controls in permeabilization buffer before washing and FACS analysis. Stained cells were analyzed using either FACSCalibur or LSRII driven proliferation, and triggered a loss of Ag-stimulated memory and CellQuest or FACSdiva software, respectively (BD Biosciences). CD4 T cells that was most striking in the effector memory (CD62Llow) subset. Importantly, the responding memory T cells in ELISPOT assay

CTLA4Ig-treated mice exhibited a predominant TCM phenotype com- The relative frequencies of IFN-␥-, IL-2-, TNF-␣-, IL-4-, IL-5-, and IL- pared with a TEM phenotype in control-treated mice. These results 10-secreting memory CD4 T cells in response to stimulation with HA/ reveal a novel role for CD28 costimulation in regulating the expan- OVA peptide were determined using ELISPOT, as previously described ϫ 4 ϫ 5 ϫ sion and resultant homing capacities of Ag-recalled memory CD4 T (34, 35), using 5 10 -1 10 purified memory CD4 T cells, 1.5 105-3 ϫ 105 BALB/c APC, and HA or OVA peptide (5 or 1 ␮g/ml, re- cells, and have important clinical implications for the use of CTLA4Ig spectively) per well. Plates were incubated at 37°C for 12–18 h, washed, in controlling the balance of pathologic and protective immune re- and developed, as described (34, 35), and spots were counted using the Downloaded from sponses in autoimmunity and infectious diseases. Immunospot ELISPOT reader (CTL; BD Biosciences). Generation and recall of influenza-specific memory CD4 T cells Materials and Methods Polyclonal memory CD4 T cells specific for influenza were generated by infecting BALB/c mice intranasally with a sublethal dose (102 PFU in 20 Mice ␮l of PBS) of influenza A virus (A/PR/8/34, PR8) obtained from American BALB/c mice (8–16 wk of age) were obtained from the National Cancer Type Culture Collection and grown in the allantoic fluid of embryonated http://www.jimmunol.org/ Institute Biological Testing Branch. Influenza HA-TCR-transgenic mice chicken eggs in the laboratory of D. Perez (University of Maryland, Col- (29) and DO11.10 mice (30) bred as heterozygotes onto BALB/c (Thy1.2) lege Park, MD). Infection was monitored in mice by weight loss measured or BALB/c (Thy1.1) hosts, and RAG2Ϫ/Ϫ mice (31) on BALB/c back- daily for the first week, after which mice recovered. Splenic CD4 T cells grounds (Taconic Farms) were maintained under specific pathogen-free containing influenza virus-specific memory CD4 T cells were harvested conditions. BALB/c mice used for influenza infections were transferred from primed hosts 12–16 wk postinfection. The relative frequency of IL-2- ␥ into the biocontainment facility. All mice were maintained in the Animal and IFN- -secreting cells was determined using the ELISPOT assay, as ϫ 4 ϫ 5 Facility at the University of Maryland Medical Center, and the experimen- previously described (34, 35), using 5 10 -1 10 purified CD4 T cells ϫ 5 ϫ 5 tal protocols were approved by the Institutional Animal Care and Use and 1.5 10 -3 10 BALB/c APC preincubated with 1 multiplicity of ␮ Committee at the University of Maryland. infection of PR8 influenza for1hat37°C, and 50 g/ml CTLA4Ig or IgG2a was added to respective wells. Plates were incubated for 12–18 h, washed, and developed, as described (34, 35); spots were counted using the by guest on September 23, 2021 Reagents Immunospot ELISPOT reader (CTL; BD Biosciences). The following Abs were purified from bulk culture supernatants and pur- In vivo restimulation of memory CD4 T cells d chased from BioExpress: anti-CD8 (TIB 105), anti-CD4 (GK1.5), anti-I-A To monitor in vivo recall of Ag-specific memory CD4 T cells, HA- and (212.A1), and anti-Thy-1 (TIB 238). The 6.5 anti-clonotype Ab directed OVA-specific memory CD4 T cells were harvested from spleens of three against the HA-TCR (29) was purified and conjugated to biotin (Pierce). to six RAG2Ϫ/Ϫ adoptive hosts (see above) (33, 34), purified as described The clonotype-specific Ab KJ1-26 specific for the DO11.10 CD4 TCR was (34), labeled with 5 ␮M CFSE (Molecular Probes) (36), and injected into purchased conjugated to PE (BD Pharmingen) or allophycocyanin (Caltag ϫ 6 ␥ four groups of BALB/c (Thy 1.1) hosts (1.5–2.5 10 cells per mouse). Laboratories). Allophycocyanin- or PE-conjugated anti-IFN- , and -IL-2, These secondary hosts were treated with 250 ␮g of murine CTLA4Ig or -CD25, -CD44, -CD62L, and PerCP-conjugated anti-CD4 were purchased IgG2a on days 1, 3, and 5 with respect to memory transfer, and challenged from BD Pharmingen. Murine and human CTLA4Ig were obtained from with 100 ␮gofHAor25␮g of OVA peptide in PBS or PBS alone as a Bristol-Myers Squibb. Single-chain anti-mouse CD28-blocking Ab was negative control on day 5, or CD4 T cells were recovered from spleen 4 h provided by R. O’Hara (Wyeth Laboratories, Madison, NJ). after peptide administration, and from spleen and mesenteric lymph nodes HPLC-purified influenza HA peptide (110–120, SFERFEIFPKE) and 60 h after peptide administration (Fig. 4A). Cells were analyzed for ex- OVA peptide (323–339, ISQAVHAAHAEINEAGR) were synthesized by pression of CD4, CD62L, CD25, CD69, and 6.5TCR or KJ1-26 by flow the Biopolymer Laboratory at the University of Maryland School of cytometry, as described above. Analysis of proliferation was calculated Medicine. from the percentage divided at each cell division, as previously described (34, 37). Statistical comparison between treatment groups was done using Student’s t test. Generation of effector and memory CD4 T cells For generation of HA- and OVA-specific effector CD4 T cells, CD4 T cells Results were purified from spleens of HA-TCR and DO11.10 mice, as described CTLA4Ig differentially affects naive vs memory CD4 T cell (12), and cultured with 5.0 ␮g/ml HA peptide or 1.0 ␮g/ml OVA peptide, activation respectively, and mitomycin C-treated APCs were prepared from BALB/c splenocytes, as described (32), in complete Clicks medium (Irvine Scien- To study the role of CD28/B7 costimulation in memory CD4 T cell tific) (12) for 3 days at 37°C. The resultant effector cells were purified by function, we generated Ag-specific memory CD4 T cells using an Ficoll density gradient centrifugation (ICN/Cappel), resulting in 90–95% ϩ ϩ ϩ adoptive transfer system extensively characterized and validated CD4 6.5 or KJ1-26 T cells. Memory generation from these effector cells was achieved by i.v. transfer of 5 ϫ 106 purified effector cells into by our laboratory and others (5, 12, 23, 33–35, 38–40), and ana- RAG2Ϫ/Ϫ adoptive hosts, as done previously (12, 33–35). Persisting mem- lyzed their antigenic recall functions in vitro and in vivo in the ory CD4 T cells were harvested from spleen and mesenteric lymph node presence of CTLA4Ig to inhibit CD28/B7 costimulation. For mem- 2–5 mo posttransfer, as described (35), for subsequent in vitro and in vivo ory generation, we obtained CD4 T cells specific for influenza HA analysis (see below). In some cases, spleen-derived memory cells were further fractionated into CD62Lhigh and CD62Llow subsets with anti- from HA-TCR-transgenic mice (29), primed them in vitro with HA CD62L-coupled magnetic beads (Miltenyi Biotec) using the autoMACS peptide and APC, transferred the resultant effector cells into Ϫ Ϫ (Miltenyi Biotec), as described (35). RAG2 / or BALB/c (Thy1.1) hosts, and recovered persisting 7700 CTLA4Ig-MEDIATED INHIBITION OF MEMORY CD4 T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 1. Effect of CTLA4Ig on activation of HA-specific naive and heterogeneous memory CD4 T cells. HA-specific naive (or resting) CD4 T cells were isolated from the spleens of unmanipulated HA-TCR mice, and HA-specific memory CD4 T cells were isolated from spleen of RAG2Ϫ/Ϫ adoptive hosts Ͼ2 mo posttransfer of HA-specific effector cells, as described (12, 33, 34). A, Phenotype of HA-specific naive and memory CD4 T cells; the number indicates the percentage of CD25low, CD44high, and CD62Lhigh, respectively. B, HA-specific naive and memory CD4 T cells were cultured for 24 and 48 h with APCs and either 1 ␮g/ml HA peptide alone or Ϯ50 ␮g/ml CTLA4Ig or IgG2a. Harvested cells were stained with fluorescent-labeled Abs for CD4, 6.5, and CD25. C, HA-specific memory CD4 T cells were activated as in B for 24 h, then harvested and surface stained for CD4, CD25, and 6.5, before intracellular staining for IL-2 and IFN-␥. Stained cells were analyzed by flow cytometry; all events are gated on CD4ϩ/6.5ϩ live T cells. The numbers represent percentage of cells in the respective quadrant. D, HA-specific spleen memory CD4 T cells were fractionated into CD62Lhigh and CD62Llow subsets and activated with HA, as above, Ϯ human CTLA4Ig or human Ig as controls, and the frequency of IL-2 and IFN-␥ production was determined by 18-h by guest on September 23, 2021 ELISPOT; results are given as mean Ϯ SD of triplicates and are representative of three independent experiments.

memory CD4 T cells after 2–6 mo in vivo. We previously We have shown previously that CD25 up-regulation on Ag- showed that HA-specific memory CD4 T cells generated in ei- stimulated memory CD4 T cells does not necessarily reflect un- ther lymphocyte-deficient or intact mouse hosts exhibit the phe- derlying modulations in cytokine production that can occur by al- notypic and functional attributes of endogenous memory CD4 T tering recall parameters (33, 34). When additional memory cells (12, 32, 33, 35, 41). The phenotype of Ag-specific naive functions were assessed, we found that IL-2 production was sub- and memory CD4 T cells used in these experiments was con- stantially inhibited (average 65 Ϯ 14% inhibition; n ϭ 4) from sistent with our previous results (12, 32, 33, 42); HA-specific Ag-stimulated memory CD4 T cells in the presence of CTLA4Ig low low high naive CD4 T cells are CD25 , CD44 , and CD62L , compared with isotype controls (Fig. 1C, second row), whereas low high whereas memory CD4 T cells are CD25 , CD44 , and het- IFN-␥ production, a hallmark of memory T cell recall, was not erogeneous for CD62L expression (Fig. 1A). Functionally, HA- affected (third row). To determine whether this selective IL-2 in- specific memory CD4 T cells exhibit rapid production of IFN-␥ hibition by CTLA4Ig on unfractionated memory CD4 T cells was and IL-2 (12, 33–35). due to differential effects on a particular memory T cell subset, we We initially compared the ability of CTLA4Ig to inhibit activa- sorted splenic HA-specific memory CD4 T cells into CD62Llow tion of HA-specific naive and memory CD4 T cells in vitro. As (T ) and CD62Lhigh (T ) subsets and assessed Ag-driven cy- expected, in vitro activation of HA-specific naive CD4 T cells was EM CM significantly impaired by CTLA4Ig, as manifested by greatly re- tokine production in the presence of CTLA4Ig. We have deter- duced up-regulation of IL-2R␣ (CD25) (Fig. 1B), a marker of early mined previously that HA-specific TEM and TCM subsets both pro- ␥ activation. By contrast, activation of HA-specific memory CD4 T duced IL-2 as well as the effector cytokine IFN- (35), similar to cells in the presence of CTLA4Ig did not affect CD25 up-regula- findings that lymphocytic choriomeningitis virus-specific TCM and tion at any timepoint (Fig. 1B and data not shown). Thus, TEM CD8 T cell subsets both produced effector cytokines (17). We CTLA4Ig appeared to have selective inhibitory effects on naive found that CTLA4Ig selectively inhibited IL-2 production by 50– CD4 T cell activation, while not affecting memory T cell activa- 70% for both CD62Llow and CD62Lhigh memory CD4 T cell sub- tion, consistent with previous in vitro results comparing naive and sets without affecting IFN-␥ production, similar to results obtained memory CD4 T cell responses in the absence of CD28/B7 co- with unfractionated memory CD4 T cells (Fig. 1D). These results stimulation (5, 23). demonstrate a selective requirement for CD28/B7 costimulation The Journal of Immunology 7701 for optimal IL-2 production from heterogeneous subsets of memory T cells. Selective triggering of memory CD4 T cell IL-2 production through CD28/B7 costimulation Previous studies showed that memory CD4 T cells generated using similar adoptive transfer approaches from DO11.10 (OVA-specific) or AND (cytochrome c-specific) TCR-transgenic CD4 T cells produced high levels of IL-2 and effector cytokines when stimulated independent of CD28 costimulation with B7-deficient APC (5, 23). To establish that CTLA4Ig-mediated inhibition in our system was not peculiar to HA-TCR-derived memory T cells, we generated OVA-specific memory CD4 T cells by adoptive transfer of in vitro primed DO11.10 CD4 T cells, as previously described (3), and tested their requirement for CD28/B7 costimulation in vitro. Similar to HA-specific memory cells, IL-2 production by OVA-specific memory CD4 T cells was selectively inhibited (70%) in the presence of CTLA4Ig compared with controls (Fig.

2A, second row), whereas CD25 up-regulation and IFN-␥ produc- Downloaded from tion were not suppressed. These results clearly demonstrate that CTLA4Ig selectively inhibits IL-2 production by memory CD4 T cells in two different peptide Ag systems. To establish that CTLA4Ig-driven inhibition of IL-2 production was due to direct blockade of the CD28 pathway, we as-

sessed memory CD4 T cell responses in the presence of a block- http://www.jimmunol.org/ ing anti-CD28 Ab (see Materials and Methods). We found similar selective inhibition of IL-2, but not IFN-␥ production by OVA-stimulated memory CD4 T cells in the presence of anti- CD28-blocking Ab or CTLA4Ig (Fig. 2B). These results establish an intrinsic requirement for CD28/B7 costimulation for optimal IL-2 production by Ag-speciﬁc memory CD4 T cells, and that CTLA4Ig functions primarily to block positive CD28-mediated signals in memory CD4 T cells. We further assessed the role of CTLA4Ig-mediated blockade on the production of other by guest on September 23, 2021 cytokines, and observed an inhibition (50–70%) of early TNF-␣ in the presence of CTLA4Ig (Fig. 2C), whereas late TNF-␣ was unaffected and IL-4, IL-5, and IL-10 were produced at low levels and were unaffected by CTLA4Ig treatment (data not shown). Therefore, CTLA4Ig-mediated blockade selectively down-regulates IL-2 and early TNF-␣ production from memory CD4 T cells. CD28/B7 blockade inhibits cytokine production from in vivo generated memory CD4 T cells

The above results demonstrate that CTLA4Ig and anti-CD28 can FIGURE 2. Memory T cell activation in the presence of CTLA4Ig or inhibit IL-2 production from peptide-stimulated memory CD4 T anti-CD28. Spleen-derived OVA- and HA-specific memory CD4 T cells cells generated from TCR-transgenic mice. To establish that CD28 were harvested from RAG2Ϫ/Ϫ hosts 2–5 mo posttransfer of effector CD4 costimulation likewise plays a role in the recall function of mem- T cells and activated in culture for 12–24 h, and then phenotype and cy- ory CD4 T cells generated under physiological conditions in vivo, tokine production were determined as in Fig. 1. A, Dot plots showing the we used an influenza mouse infection system to generate poly- intracellular cytokine staining of IL-2 and IFN-␥, in OVA-specific memory clonal virus-specific memory CD4 T cells. For these experiments, CD4 T cells; percentage of cells in each quadrant indicated. B, In addition to above treatments, a single-chain anti-CD28-blocking Ab was added to we infected BALB/c hosts intranasally with a sublethal dose of activation cultures, and cytokine production of Ab- vs control-treated influenza A virus (A/PR/8/34), and 12–16 wk postinfection har- OVA-specific memory CD4 T cells is shown as the percentage of KJ1-26ϩ vested splenic CD4 T cells containing a polyclonal population of cytokineϩ cells expressed as mean Ϯ SD from three replicate experiments influenza-primed memory CD4 T cells. To quantitate the flu-spe- using intracellular cytokine staining. C, OVA- and HA-specific memory cific memory CD4 T cell response in the presence of CTLA4Ig or CD4 T cells activated in culture for 12 h; TNF-␣ production determined by control IgG2a, we stimulated total CD4 T cells from naive or in- ELISPOT; results are shown as mean Ϯ SD of triplicates, and are repre- fluenza-infected mice with splenic APC incubated with live influ- sentative of two experiments. enza virions and analyzed cytokine production by ELISPOT. As shown in Fig. 3, CD4 T cells harvested from flu-infected hosts produced significant levels of IL-2 and IFN-␥ within 12–18 h of inhibition of IL-2 and partial down-regulation (50%) of IFN-␥ pro- virus stimulation characteristic of a memory response, whereas duction from flu-specific memory CD4 T cells (Fig. 3). The greater CD4 T cells from uninfected controls lacked flu-specific responses. inhibition of IL-2 compared with IFN-␥ production by CTLA4Ig In the presence of CTLA4Ig, however, there was almost complete in polyclonal virus-specific memory CD4 T cells is similar to our 7702 CTLA4Ig-MEDIATED INHIBITION OF MEMORY CD4 T CELLS Downloaded from

FIGURE 3. CTLA4Ig selectively inhibits recall cytokine function of polyclonal inﬂuenza-speciﬁc memory CD4 T cells. Splenic CD4 T cells

were purified from mice that had been infected with PR8 influenza virus 16 http://www.jimmunol.org/ wk previously, and influenza-specific memory CD4 T cells were recalled in vitro with PR8-infected APCs. The frequency of IL-2- and IFN-␥-secreting FIGURE 4. CTLA4Ig treatment does not inhibit early activation of CD4 T cells from primed mice and unprimed controls treated with memory CD4 T cells in vivo. A, Protocol for assessing the effect of CTLA4Ig or isotype IgG2a was determined by ELISPOT. Control CD4 T CTLA4Ig on memory T cell recall in vivo. Ag-specific memory CD4 T cells incubated with uninfected APCs produced 23 and 7 spots for IL-2 and cells (HA and OVA specific) were CFSE labeled and transferred into four IFN-␥, respectively. The results are shown as mean Ϯ SD of triplicates and groups of BALB/c hosts; two groups were treated with 250 ␮gof are representative of three independent experiments. CTLA4Ig or IgG2a, respectively, on days 1, 3, and 5 with respect to memory transfer. On day 5, these hosts from each group were treated with HA results with TCR-transgenic peptide-specific memory CD4 T cells, or OVA peptide or PBS control; 4 h postpeptide administration or on day by guest on September 23, 2021 8 (2.5 days postpeptide administration) hosts were sacrificed; the spleens establishing that CTLA4Ig can exert its effects on memory responses and mesenteric lymph nodes were harvested; and transferred cells were to multiple Ags and in the presence of pathogen-associated signals. stained and subsequently analyzed by flow cytometry. B, Representative Interestingly, the extent of CTLA4Ig-mediated inhibition was higher plots of the CD25 and CD69 expression of OVA-specific spleen memory for recall of flu-specific compared with TCR-transgenic memory CD4 CD4 T cells harvested 4 h after peptide administration. C, Compilation of T cells, which is most likely due to the 20-fold difference in precursor CD5 and CD69 expression profiles of OVA-specific and HA-specific frequency in these two systems. These results further validate that the spleen memory CD4 T cells harvested 4 h postpeptide administration. Ag-specific memory cells generated in this study reflect the activation and functional and costimulatory requirements of memory cells gen- memory CD4 T cells boosted with antigenic peptide (34). Recalled erated under physiological conditions such as virus infection. memory CD4 T cells were recovered from spleen and lymph nodes at early times after boosting (4 h), and at a later timepoint (60 h) Effect of CTLA4Ig on Ag-driven recall of memory CD4 T cells (Fig. 4A), which we previously determined was the optimal time- in vivo point for measuring peak in vivo proliferation of Ag-stimulated It is well established that IL-2 is an important growth/survival memory CD4 T cells (34). Visualization of Ag-specific memory factor for T cells (43, 44), and recently it has been shown to be CD4 T cells in vivo was accomplished based on Thy-1 allelic important for the expansion of memory CD8 T cells during sec- differences for HA-specific memory CD4 T cells, which likewise ondary responses (45). Based on our in vitro results showing a represented 6.5ϩ T cells, and based on KJ1-26 clonotype expres- decrease in memory CD4 T cell IL-2 production, we hypothesized sion for OVA-specific memory CD4 T cells (data not shown). that the CD28/B7 pathway may be important for recall expansion For immediate recall in vivo, we found that OVA-specific mem- in vivo. To examine the effect of CTLA4Ig-mediated costimula- ory CD4 T cells exhibited rapid up-regulation of the early activation blockade on a specific population of Ag-specific memory CD4 tion markers CD25 and CD69 4 h after peptide boosting that was T cells in vivo, we used a secondary transfer system previously similar in IgG2a- and CTLA4Ig-treated mice (Fig. 4B). CTLA4Ig optimized in the laboratory to follow the fate of Ag-recalled mem- treatment likewise did not affect rapid CD25 and CD69 up-regu- ory CD4 T cells in an intact mouse host (34). We transferred lation on HA-specific memory CD4 T cells, although a lower pro- CFSE-labeled HA- or OVA-specific memory CD4 T cells into portion of HA-specific cells was CD25ϩ or CD69ϩ at this time- BALB/c (Thy1.2 or Thy1.1) hosts that were administered 250 ␮g point (Fig. 4C). These results establish that the CD28 pathway is of CTLA4Ig (weight equivalent for 10 mg/kg dose used in patients not required for rapid activation of memory CD4 T cells in vivo, (26)) or murine IgG2a isotype control for three successive treat- and that the majority of memory T cells have encountered the Ag. ments (Fig. 4A). Each group of treated mice was subsequently Despite the lack of CTLA4Ig-mediated effects at this early time- boosted with HA or OVA peptide Ag (or PBS as a control), as we point, we found that in vivo proliferation and expansion of Ag- have shown previously robust in vivo secondary responses by specific memory CD4 T cells were significantly inhibited by The Journal of Immunology 7703 Downloaded from FIGURE 5. CTLA4Ig limits in vivo Ag-driven proliferation of HA- and FIGURE 6. CTLA4Ig diminishes Ag-driven expansion of memory CD4 OVA-specific memory CD4 T cells. A, In vivo proliferation of spleen- T cells. Quantification of the yield of HA- and OVA-specific memory CD4 derived HA- and OVA-specific memory T cells. The percentage of undi- T cells harvested on day 8 from the spleens of BALB/c hosts that were vided memory cells indicated in the plot; box outlines cells undergoing two treated with CTLA4Ig or IgG2a and boosted with respective peptide or or more division cycles. B, Average percentage of transferred memory T control PBS, as in Fig. 4A. A, Absolute yield was quantified by microscopic cell precursors undergoing 0–4 divisions calculated from four independent cell count multiplied by the proportion of clonotype-positive cells deter-

experiments (six mice/group) for HA-specific memory CD4 T cells, and mined by flow cytometry. Scatter plots showing the absolute yield of Ag- http://www.jimmunol.org/ from two experiments (four mice/group) for OVA-specific memory CD4 T specific memory CD4 T cells from CTLA4Ig-treated hosts as percentage of cells. Significant differences between IgG2a- and CTLA4Ig-treated hosts yield from IgG2a-treated hosts of HA-specific (left) and OVA-specific for p Ͻ 0.01. (right) memory CD4 T cells with the mean percentage shown. B, Bar ءء for p Ͻ 0.05, and ء indicated as graphs showing the average absolute yield of Ag-specific memory CD4 T cells recovered from CTLA4Ig- and isotype IgG2a-treated hosts, HA spe- CTLA4Ig treatment that was apparent at later times (60 h). In cific (left) and OVA specific (right). Error bars represent the SD; data are control-treated mice, Ag-specific memory CD4 T cells underwent representative of two to four experiments with at least three mice per Indicates significant difference in yield between CTLA4Ig- and ,ء .significant proliferation in response to peptide boosting compared group with PBS boosting (Fig. 5A, first row), with OVA-specific memory IgG2a-treated hosts for HA- and OVA-specific memory CD4 T cells CD4 T cells proliferating more extensively than HA-specific mem- (p Ͻ 0.05). by guest on September 23, 2021 ory CD4 T cells. In the presence of CTLA4Ig, however, recall proliferation of HA- and OVA-specific memory CD4 T cells was Attrition of Ag-activated memory CD4 T cells by CTLA4Ig dramatically reduced compared with isotype controls (Fig. 5A, second row), manifested by a significant increase ( p Ͻ 0.05) in the To determine whether CTLA4Ig-mediated inhibition of memory percentage of undivided (CFSEhigh) HA- and OVA-specific mem- CD4 T cell proliferation affected the overall yield of boosted memory CD4 T cells (right quadrant), and substantially fewer number ory CD4 T cells, we calculated the absolute numbers of Ag-spe- of cells undergoing Ͼ2 divisions (area outlined by box). These cific memory CD4 T cells present in CTLA4Ig vs control-treated results show striking decreases in recall proliferation of memory mice. There was a striking loss of Ag-recalled HA- and OVA- CD4 T cells in CTLA4Ig-treated hosts in two distinct Ag systems specific memory CD4 T cells in the hosts that received CTLA4Ig with different magnitudes of proliferation. compared with IgG2a isotype control expressed either as a per- To obtain a quantitative assessment of CTLA4Ig-mediated in- centage of isotype control (Fig. 6A) or as absolute numbers of hibition of memory CD4 T cell proliferation in vivo, we calculated Ag-specific memory T cells (Fig. 6B). For OVA-specific memory the percentage of undivided precursors at each division cycle from CD4 T cells, a dramatic expansion of OVA-specific memory T multiple experiments, according to Lyons (37). There was a sig- cells was observed in spleen and lymph nodes after boosting with nificant ( p Ͻ 0.05, p Ͻ 0.01) decrease in HA- and OVA-specific OVA, and this expansion was dramatically inhibited by CTLA4Ig memory CD4 T cells, respectively, undergoing more than two di- treatment (Fig. 6B, right). For HA-specific memory T cells, there vision cycles in the hosts treated with CTLA4Ig in comparison was no net expansion upon recall consistent with our previous with IgG2a (Fig. 5B). From this analysis, one can clearly discern findings (34), probably due to the lower proliferative capacity of the higher magnitude of the OVA-driven proliferative response this system. However, there was a significant loss of HA-boosted compared with HA-specific memory CD4 T cells, with an in- memory CD4 T cells in CTLA4Ig-treated mice (Fig. 6B, left). creased proportion of OVA-specific cells undergoing three and These results demonstrate that CTLA4Ig treatment results in a dra- four division cycles (Fig. 5B). CTLA4Ig-mediated effects can be matic decrease in the yield of Ag-activated memory CD4 T cells, observed as a significant increase in nondividing cells for both consistent in multiple mice studied and in two diverse antigenic HA- and OVA-specific T cells (Fig. 5B), and striking decreases in systems. the proportion of cells undergoing two or more divisions for HA- high specific memory CD4 T cells, and three and four divisions for CTLA4Ig induces a predominant CD62L phenotype of the OVA-specific memory CD4 T cells (Fig. 5B). These results dem- responding memory CD4 T cells onstrate substantial inhibition of the progression of Ag-driven We asked whether the loss of Ag-specific memory CD4 T cells memory CD4 T cell proliferation by CTLA4Ig independent of the seen in the presence of CTLA4Ig was specific to a particular mem- magnitude of the recall response. ory subset, by analyzing CD62L expression of in vivo boosted 7704 CTLA4Ig-MEDIATED INHIBITION OF MEMORY CD4 T CELLS

specific memory T cells was not significantly altered in CTLA4Ig- treated mice, and that the CTLA4Ig-induced loss of CD62Llow phenotype memory CD4 T cells was only observed among the Ag-stimulated memory CD4 T cells (Fig. 7B). Furthermore, the biased CD62Lhigh profile of Ag-stimulated memory CD4 T cells in CTLA4Ig-treated mice occurred in both spleen and lymph node (Fig. 7C) with a substantial fraction of CD62Llow phenotype cells in spleen and a predominance of CD62Llow memory CD4 T cells in lymph node of control-treated mice, compared with a prepon- derance of CD62Lhigh phenotype memory CD4 T cells in both spleen and lymph node of CTLA4Ig-treated mice. These results low indicate a pronounced reduction of CD62L TEM phenotype cells during antigenic recall in the presence of CTLA4Ig.

Discussion We demonstrate in this study that inhibition of the CD28/B7 pathway using CTLA4Ig profoundly alters the recall function of memory CD4 T cells generated by priming with Ag or during infection with inﬂu-

enza virus. In vitro, we observed a biased inhibition of IL-2 produc- Downloaded from tion by CTLA4Ig from TCR-transgenic or polyclonal inﬂuenza- speciﬁc memory CD4 T cells. In vivo, although CTLA4Ig does not inhibit early activation events by Ag-stimulated memory CD4 T cells, as shown in two Ag systems, there are striking defects in proliferative expansion in vivo coincident with a biased loss of the low CD62L TEM subset in the responding population. Our results http://www.jimmunol.org/ identify a novel role for the CD28/B7 pathway as a regulator of

low memory T cell responses and as a potential target for modula- FIGURE 7. CTLA4Ig treatment results in lack of CD62L TEM cells among the Ag-responsive memory CD4 T cells in vivo. HA- or OVA- tion of memory CD4 T cell expansion and homing capacities. specific memory CD4 T cells were transferred into BALB/c hosts that were Our findings challenge the generally accepted view that memory T treated with CTLA4Ig and HA or OVA peptide, as described in Fig. 4A. A, cells are minimally dependent on CD28/B7 costimulation for function CD62L vs CFSE expression of OVA (left)- and HA (right)-specific mem- based on in vitro recall studies and in vivo studies in CD28-deficient ory CD4 T cells recalled in vivo with antigenic peptide in control (IgG2a)- mouse models (22, 28). In vitro studies established a CD28-indepen- or CTLA4Ig-treated mice, with numbers in quadrants indicating percentage dent mechanism for memory T cell activation by showing that B7- of CD62Lhigh (upper left) or CD62Llow (lower left) dividing memory CD4 deficient APCs could activate memory, but not naive CD4 T cells, by guest on September 23, 2021 low T cells. B, Average percentage of CD62L phenotype cells observed in using memory CD4 T cells generated by adoptive transfer, as accom- PBS-boosted vs Ag-boosted OVA- and HA-specific memory CD4 T cells plished in this study (5, 23). However, B7 deficiency on APC elim- in IgG2a- vs CTLA4Ig-treated mice. Significant differences in yield be- inates both positive signaling through CD28 and negative regulation (when p Ͻ 0.05) ء tween CTLA4Ig- and IgG2a-treated hosts denoted as when p Ͻ 0.01). All plots gated on transgene-positive CD4 T cells. through CTLA4, which is known to be constitutively up-regulated in) ءء and C, Representative histogram plot showing the CD62L expression profile of memory T cells (46). In our system, we observed a consistent and OVA-specific spleen memory CD4 T cells from CTLA4Ig- or IgG2a- selective inhibition of IL-2 production in the presence of CTLA4Ig or treated hosts boosted with OVA peptide, as in Fig. 4A. Data from at least an anti-CD28 Ab, consistent with previous findings showing that three mice per treatment; experiment done at least twice. CTLA4Ig-mediated inhibition preferentially blocks the lower avidity CD28/B7 interaction, leaving the higher avidity CTLA4-B7 interaction intact (47). memory CD4 T cells as a function of CFSE dilution in CTLA4Ig- Results showing unimpaired recall responses by memory CD4 and control-treated mice. We found that in control-treated mice, all and CD8 T cells in CD28-deficient (CD28Ϫ/Ϫ) mice (24, 48) like- of the OVA-stimulated memory T cells underwent division, and wise supported a negligible role for CD28 costimulation and mem- these OVA-stimulated memory CD4 T cells exhibited heteroge- ory. However, CD28Ϫ/Ϫ mice exhibit generalized immune abnor- neous CD62L expression with 30–40% exhibiting a CD62Llow malities, including a lack of regulatory T cells (49), and naive phenotype (Fig. 7A, upper left). By contrast, in CTLA4Ig-treated CD28Ϫ/Ϫ CD4 T cells can also be activated in vitro and in vivo mice, although the majority of OVA-stimulated cells underwent (despite the potent ability of CTLA4Ig to down-regulate naive T division (although not as extensively as controls; see Fig. 5), these cell activation (21)), suggesting that additional pathways are op- responding OVA-specific CD4 T cells exhibited a predominant erable in CD28Ϫ/Ϫ mice (50) that may or may not be operable in CD62Lhigh (80–90%) phenotype, with a very low proportion of wild-type hosts in vivo. The tracking and analysis of an Ag-spe- CD62Llow phenotype cells even among cells undergoing extensive cific population of memory CD4 T cells in vivo in the presence of division (Fig. 7A, lower left). We obtained similar results with CD28/B7 inhibition, as shown in this study, have not been previ- HA-specific memory CD4 T cells in that the responding (dividing) ously reported. memory CD4 T cells in control-treated mice exhibited a substan- Our results indicate that whereas specific recall functions of tially higher proportion of CD62Llow phenotype cells, compared memory CD4 T cells, such as up-regulation of activation marker with memory CD4 T cells activated in the presence of CTLA4Ig expression, are independent of CD28 costimulation, optimal IL-2 (Fig. 7A, right). production and proliferative expansion of memory CD4 T cells The CD62L profile of Ag-specific memory CD4 T cells from require CD28 costimulation. It is known that CD28/B7 costimu- multiple PBS- and Ag-boosted mice is shown in Fig. 7B. These lation promotes naive T cell activation by enhancing IL-2 produc- results show that the CD62L profile of unactivated HA- and OVA- tion (51–54), and that CTLA4Ig-mediated inhibition of the The Journal of Immunology 7705

and greatly reduced migration of activated memory CD4 T cells to nonlymphoid compartments, inhibiting peripheral responses (Fig. 8). Our results showing that influenza-primed memory CD4 T cells are susceptible to CTLA4Ig-mediated inhibition suggest that protective response to influenza challenge in vivo may likewise be affected by CTLA4Ig, a possibility that we are currently investigating. This susceptibility of memory CD4 T cells to CTLA4Ig-mediated inhibition of CD28 costimulation has important clinical implications. Currently, use of human CTLA4Ig (Abatacept) is effi- cacious in treating ongoing rheumatoid arthritis (26), which is an autoimmune disease likely driven by memory T cells (28). Al- though CTLA4Ig is considered exclusively to inhibit naive T cell activation (22, 28), our findings together with clinical results strongly imply that CTLA4Ig can be highly effective in inhibiting immune responses driven by memory T cells, by repressing the

propagation of pathogenic memory T cells. The prevalent TCM phenotype of Ag-recalled memory CD4 T cells in the presence of

CTLA4Ig suggests a mechanism whereby CTLA4Ig may inhibit Downloaded from FIGURE 8. Dynamics of memory CD4 T cell recall in the presence or the propagation of pathogenic T cells that could potentially me- absence of CD28 costimulation. The memory T cell pool consisting of T EM CM diate pathology at peripheral tissue sites. Our results therefore and TEM subsets is shown, with a biased production of TEM phenotype cells in the presence of ample CD28 costimulation. Inhibition of CD28 costimu- identify a novel role for the CD28/B7 pathway as a key regulator lation results in a loss of memory T cell expansion and a predominance of of memory T cell responses and as a new target for modulation of

TCM phenotype cells, with consequent effects on memory T cell protective pathogenic and productive memory CD4 T cell responses. and pathological responses. http://www.jimmunol.org/ Acknowledgments We extend gratitude to Wendy Lai and Elizabeth Kadavil for mouse colony CD28/B7 pathway in naive T cells blocks progression, but not the maintenance; to Laureanne Lorenzo for help with the influenza ELISPOT; induction of proliferation in vitro (19). Our findings, that CD28 to Dr. Daniel Perez for growing influenza virus; and to Dr. Anita Tang for costimulation is likewise required for optimal IL-2 production experimental support. (Figs. 1 and 3) and proliferation (Fig. 5) of memory CD4 T cells, suggest similar control of IL-2 production and cell cycle progres- Disclosures sion by CD28 in naive and memory CD4 T cells in vivo. 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