Immune Response to an Intracellular Pathogen Role of CD80 (B7.1) And

Role of CD80 (B7.1) and CD86 (B7.2) in the Immune Response to an Intracellular Pathogen1

Carlos S. Subauste,2*† Rene de Waal Malefyt,‡ and Franklin Fuh*

The costimulatory ligands CD80 and CD86 play a crucial role in the initiation and maintenance of an immune response. We demonstrate that whereas infection of human monocytes with viable tachyzoites of Toxoplasma gondii resulted in rapid induction of expression of CD80 and up-regulation of expression of CD86, incubation with killed organisms failed to alter the levels of expression of these costimulatory ligands. The T. gondii-mediated changes in levels of expression of these molecules are critical to the T cell response to the parasite. Proliferation of resting T cells in response to parasite-infected cells was dependent on both CD80 and CD86. More importantly, early production of IFN-␥ in response to T. gondii by T cells from T. gondii-seronegative individuals occurred only after stimulation with monocytes that exhibited increased expression of CD80 and CD86 (monocytes infected with viable parasites) and was almost completely ablated by the combination of anti-CD80 plus anti-CD86 mAb. Moreover, prolifer- .ation and IFN-␥ production by CD4؉ CD45RA؉ T cells from unexposed individuals were dependent on both CD80 and CD86 These data indicate that pathogen-monocyte interaction inﬂuences the ensuing T cell response. The Journal of Immunology, 1998, 160: 1831–1840.

he generation of a protective immune response against It has been reported that the expression of CD80 and CD86 on invading infectious organisms requires that the immune APC can be regulated. Incubation with IFN-␥ induces the ex- T system discriminate microbial Ags from self Ags. It has pression of CD80 and up-regulates the expression of CD86 on been proposed that one way the immune system accomplishes this monocytes (10, 11). In addition, activation of B cells results in task is by requiring the presence of a microbially induced second induction of the expression of CD80 and up-regulation of the signal during T cell activation (1). While recognition by the TCR expression of CD86 (9, 12). Regulation of the expression of of peptide-MHC complexes initiates Ag-specific T cell responses, CD80 and CD86 on APC may be an important feature of the a second signal (costimulatory signal) provided by the APC is biology of these molecules with potential implications in self/ necessary for T cell clonal expansion and optimal cytokine pro- nonself discrimination. duction (2). The relevance of costimulatory signals is emphasized The outcome of infections with certain organisms has been by the induction of T cell nonresponsiveness or anergy when T shown to correlate with the type of cytokines produced by T cells by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. cells encounter Ags in the absence of costimulation (2–4). (13). In this regard, there is ample evidence of the pivotal role that Data accumulated to date suggest that the interaction between IFN-␥ plays in the induction of protective immunity against intra- CD28 expressed on T cells and its counter-receptors CD80 (B7-1) cellular micro-organisms (14). Thus, the identification of factors and CD86 (B7-2) expressed on specialized APC provides the most that influence cytokine production triggered by infectious organ- important costimulatory signal (5, 6). Both CD80 and CD86 can isms are crucial to our understanding of the mechanisms that de- provide costimulation to T cells for proliferation and IL-2 produc- termine whether a protective immune response is elicited. Toxo- tion (7, 8). However, these molecules differ in their expression on plasma gondii, an obligate intracellular protozoan that infects all APC. Whereas dendritic cells express both costimulatory ligands, nucleated cells, provides an example of a micro-organism against

http://classic.jimmunol.org ␥ monocytes/macrophages constitutively express only CD86 (9). which cell-mediated immunity with resulting IFN- production Resting B cells have low levels of expression of CD86 and no plays a critical role in controlling infection (15, 16). Indeed, this signiﬁcant expression of CD80 (9). parasite has become a major opportunistic pathogen in immuno- compromised individuals (17). The demonstration that presumably unprimed human T cells respond in vitro to this parasite (18) made T. gondii well suited for study of the roles of CD80 and CD86 in

Downloaded from *Research Institute, Palo Alto Medical Foundation, Palo Alto, CA 94301; †Division the early events of the T cell response against an intracellular or- of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH ganism. Our results indicate that human monocytes discriminate 45267; and ‡DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA 94304 between noxious and innocuous preparations of T. gondii, which Received for publication July 14, 1997. Accepted for publication October 28, 1997. translates into the induction of the expression of CD80 and the The costs of publication of this article were defrayed in part by the payment of page up-regulation of the expression of CD86 only when these cells charges. This article must therefore be hereby marked advertisement in accordance encounter viable (noxious) organisms. Our results reveal that, in with 18 U.S.C. Section 1734 solely to indicate this fact. turn, T cell proliferation is dependent on CD80 and CD86, and that 1 This work was supported by National Institutes of Health Grant AI37936–01A1 (to the induction/up-regulation of expression of these molecules is as- C.S.S.), an AmFAR grant made in memory of Walter J. Smith (to C.S.S.), a grant from the University of California Universitywide AIDS Research Program (to C.S.S.) sociated with the generation of an IFN-␥ response by T cells from and in part by National Institutes of Health Grants AI04717 and AI30230. unexposed individuals. These data provide evidence of the impor- 2 Address correspondence and reprint requests to Dr. Carlos S. Subauste, Division of tance of pathogen-monocyte interaction, especially through patho- Infectious Diseases, Department of Medicine, University of Cincinnati College of Medicine, P.O. Box 670560, Cincinnati, OH 45267–0560. E-mail address: gen-mediated induction/up-regulation of costimulatory ligands, in [email protected] shaping the ensuing T cell response.

Materials and Methods hyde in PBS (18). To obtain T. gondii lysate Ags (TLA), tachyzoites were harvested from infected human foreskin fibroblasts and lysed in H Oby Abs and cytokines 2 three cycles of freezing and thawing followed by reconstitution with 10ϫ The following mAbs were used for cell purifications: anti-CD2, anti-CD3, PBS. Antigenic preparations were devoid of detectable levels of endotoxin anti-CD8, anti-CD14, and anti-CD56 (all from Becton Dickinson, San (Ͻ10 pg/ml) using a Limulus amebocyte lysate assay (Sigma). Jose, CA); anti-CD16 (Medarex, Inc., Annandale, NJ); anti-CD19 (Coulter, Unless stated otherwise, PBMC were incubated with tachyzoites of T. Hialeah, FL); anti-CD45RA, anti-CD45RO, and anti-CD66b (all from Im- gondii at a ratio of 1:1 for 24 h, and purified monocytes were incubated munotech, Westbrook, MA); and anti-glycophorin A (10F7 MN, American with four tachyzoites per cell for 48 h before cytofluorometric analysis. Type Culture Collection, Rockville, MD). Cells were cultured in Teflon vessels in CM. The percentage of cells with Anti-CD14 (RM052, Immunotech), anti-CD80 (L307, Becton Dickin- intracellular parasites was determined by light microscopy (22). In some son), anti-CD86 (Fun-1, PharMingen, San Diego, CA), CTLA-4-Ig (gift experiments, Transwell inserts (Corning Costar Corp., Cambridge, MA) from Dr. Peter Linsley) (19), and neutralizing mAb against the cytokines were used to separate monocytes plus T. gondii tachyzoites from mono- GM-CSF,3 IFN-␥ (R&D Systems, Minneapolis, MN), and IFN-␣ (Bio- cytes alone by a membrane with pores 0.4 ␮m in diameter. Source International, Camarillo, CA) were used in functional assays (all at 10 ␮g/ml). Isotype-matched mAbs and human IgG1 were obtained from Flow cytometry PharMingen and Sigma Chemical Co. (St. Louis, MO), respectively. Freshly isolated and cultured cells were stained with either FITC-conju- IFN-␥, purchased from R&D Systems, was used at 100 U/ml. gated anti-CD14 or FITC-conjugated anti-CD19 mAb and one of the fol- The following conjugated Abs were used for flow cytometry (purchased lowing PE-conjugated mAb: anti-CD11a, anti-CD54, anti-CD58, anti- from Caltag, South San Francisco, CA, except when indicated): FITC-anti- CD80, anti-CD86, or anti-HLA-DR. When analyzing PBMC, an electronic CD3 (Becton Dickinson), FITC-anti-CD14, FITC-anti-CD19, FITC-anti- gate was set on FITC-stained CD14ϩ or CD19ϩ cells to identify monocytes CD45RA (Immunotech), FITC-anti-CD45RO (Immunotech), FITC-anti- and B cells, respectively. For surface molecules whose levels of expression CD66b (Immunotech), phycoerythrin (PE)-anti-CD11a, PE-anti-CD16 followed a bimodal distribution (CD80 and CD86 on monocytes), the per- (Becton Dickinson), PE-anti-CD54, PE-anti-CD56 (Becton Dickinson), centages of CD80ϩ and CD86high cells were calculated by determining the PE-anti-CD58 (Becton Dickinson), PE-anti-CD80 (Becton Dickinson), PE- percentage of cells that stain above the value of fluorescence obtained with anti-CD86 (PharMingen), or PE-anti-HLA-DR. isotype control mAb (CD80ϩ) or the value of fluorescence of CD86int cells (CD86high). For surface molecules whose levels of expression followed a Cell purifications unimodal distribution, data are expressed as the corrected mean fluores- PBMC were isolated from buffy coats of heparinized blood of healthy cence intensity (MFI). The corrected MFI for PE-conjugated mAb were volunteers donors obtained from the Stanford Blood Bank (Stanford, CA). calculated by subtracting the MFI of the appropriate isotype control from the MFI of each specific mAb. In some experiments, the T. gondii-induced Serologic tests for detection of anti-T. gondii IgG and IgM were performed ⌬ in all samples of blood (20). This allowed for division of the donors into increase in the levels of expression of CD80 and CD86 is expressed as T. gondii-seronegative (negative anti-T. gondii IgG and negative anti-T. MFI, which were calculated by subtracting corrected MFI for CD80 and gondii IgM) and T. gondii chronically infected individuals (positive anti-T. CD86 of uninfected monocytes from corrected MFI for CD80 and CD86 on CD80ϩ and CD86high monocytes, respectively. Sorting of monocytes gondii IgG and negative anti-T. gondii IgM). Ϫ ϩ int high To obtain purified monocytes, PBMC were incubated with the follow- into CD80 , CD80 , CD86 , and CD86 populations was performed ing mAb: anti-CD2, anti-CD3, anti-CD8, anti-CD19, anti-CD56, anti- 18 h after incubation with T. gondii. CD66b, and anti-glycophorin A. After addition of magnetic beads coated T cell proliferation assays with anti-mouse IgG (Dynal, Great Neck, NY), rosetting cells were re- moved with a magnet (Dynal). This resulted in populations that were T cells (5 ϫ 105/ml) were incubated with either T. gondii-infected or un- Ͼ96% pure for monocytes by microscopic examination of Giemsa-stained infected, ␥-irradiated, autologous PBMC with or without TLA (10 ␮g/ml)

by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. cytocentrifuge preparations. In addition, cytofluorometric analysis indi- as previously described (18). In certain experiments, monocytes were used cated that Ͼ92% of the cells were CD14ϩ, with Ͻ0.5% CD3ϩ, Ͻ0.5% instead of PBMC. Infected cells were added to T cells at a ratio of five T CD19ϩ, Ͻ0.5% CD56ϩ, and Ͻ2% CD66bϩ cells. In certain experiments cells per one infected cell (18). Concentrations of uninfected PBMC or monocytes were purified further by incubation with FITC-conjugated anti- monocytes matched those of infected cells. CTLA-4-Ig, anti-CD80 (L307), CD14 mAb (Becton Dickinson) followed by FACS sorting. This procedure and anti-CD86 (Fun-1) mAb or control Abs were added to either PBMC or resulted in populations of highly purified CD14ϩ monocytes (Ͼ99% by monocytes 30 min before incubation with T cells. Unless indicated, these flow cytometry). For some experiments purified monocytes were incubated reagents were used at 10 ␮g/ml. Cells were cultured in 96-well plates for for 2 days in complete medium (CM) consisting of RPMI 1640 with 10% 7 days, labeled for the final 18 h with [3H]thymidine, and harvested (18). dye test-negative human AB serum (Irvine, Santa Ana, CA) before infec- Radioactivity was measured in a beta scintillation counter (18). Results are tion with T. gondii. This resulted in populations of cells that acquired expressed as the mean counts per minute of [3H]thymidine incorporation of macrophage morphology (21). triplicate wells Ϯ SEM. Data are also presented as stimulation indexes ϩ http://classic.jimmunol.org Resting T cells (Ͼ99% CD3 ) were obtained from nylon wool-nonad- (counts per minute of culture with T. gondii/counts per minute of culture herent PBL that were incubated with anti-CD16 plus anti-CD56 mAb and without T. gondii). subjected to depletion using immunomagnetic beads. To obtain purified populations of CD4ϩ CD45RAϩ TCR-␣␤ϩ, anti-CD8 (OKT8, American Cytokine assays ␥␦ ␦ Type Culture Collection), anti- TCR (anti-TCR 1, gift from Dr. Michael Purified, resting peripheral blood T cells (1 ϫ 106/ml) were incubated with Brenner), anti-CD19 (Coulter Cytometry, Hialeah, FL), and anti-CD45RO purified monocytes (2–5 ϫ 105/ml) that were uninfected, infected with (UCHL-1, Immunotech) were added to the combination of mAb mentioned viable UV-attenuated tachyzoites of T. gondii (8 ϫ 105/ml to 2 ϫ 106/ml), above. Addition of magnetic beads was repeated once. The populations ␮ Downloaded from ϩ ϩ ϩ or incubated with TLA (10 g/ml) in 96-well plates. Supernatants were obtained (Ͼ98% CD4 CD45RA TCR-␣␤ ) did not proliferate in re- Ϫ ϩ collected at 24, 48, and 72 h and stored at 70°C. Concentrations of IL-2, sponse to a recall Ag (tetanus toxoid), whereas unseparated CD4 TCR- IL-4, and IFN-␥ were measured by ELISA (18) in supernatants collected at ␣␤ϩ ϩ ϩ ␣␤ϩ and CD4 CD45RO TCR- cells exhibited significant prolifer- 24, 48, and 72 h, respectively. Data are presented as the mean of triplicate ation in response to this Ag. wells Ϯ SEM. None of the cytokines tested was detected in supernatants obtained from wells that lacked T cells and contained only monocytes with T. gondii and infection or without T. gondii antigenic preparations. T. gondii tachyzoites were obtained from both infected human foreskin Statistical analysis fibroblasts (American Type Culture Collection) and peritoneal cavities of infected mice and exposed to UV light as previously described (22). Nei- Statistical significance was assessed by unpaired Student’s t test. ther uninfected human foreskin fibroblasts nor tachyzoite-free peritoneal lavage fluids from infected mice (after passage through a 0.45-␮m filter) Results mediated changes in expression of the surface molecules tested. In certain Effects of T. gondii on expression of accessory molecules on experiments, tachyzoites were killed by incubation in 1% paraformalde- monocytes and B cells APC express accessory molecules that can enhance Ag-driven T 3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; PE, phycoerythrin; CM, complete medium; TLA, Toxoplasma lysate Ags; high, high level; int, cell responses (23, 24). The effects of T. gondii on the expression intermediate level; MFI, mean fluorescence intensity. of accessory ligands such as CD11a, CD54, CD58, CD80, and The Journal of Immunology 1833

CD86 and on the expression of HLA-DR molecules on monocytes and B cells were analyzed by cytofluorometry. Incubation of PBMC with T. gondii tachyzoites consistently resulted in a striking induction of expression of CD80 and up-regulation of expression of CD86 on monocytes. The levels of expression of these molecules followed a biphasic distribution, giving rise to a subpopulation of monocytes with remarkable levels of expression of CD80 (CD80ϩ) as well as a subpopulation of monocytes with increased levels of expression of CD86 (CD86high; see Fig. 1). The levels of expression of CD54 and HLA-DR on monocytes and of CD54, CD86, and HLA-DR on B cells were also increased in seven of 10 experiments in which PBMC were incubated with the parasite (Ta- ble I). However, the levels of expression of these latter molecules remained in a unimodal distribution. It is interesting to note that the expression of CD80 on B cells was never affected by T. gondii. Whether PBMC originated from individuals with or without Abs to T. gondii did not influence the magnitude of change in the levels of expression of any of the above mentioned molecules ( p Ն 0.2). T. gondii did not induce nonspecific up-regulation of expression of surface molecules on monocytes and B cells, since the levels of expression of CD11a and CD58 on these cells were not increased after incubation with the parasite. Tachyzoites used in these experiments were exposed to UV light to inhibit their intracellular multiplication and prevent destruction of infected cells (18). Changes in the levels of expression of accessory ligands were not caused by treatment with UV light, since unexposed tachyzoites exerted similar effects on the expression of these molecules (data not shown). Thus, T. gondii induced changes in the levels of expression of costimulatory ligands that were particularly striking on monocytes, since there was both induction of expression of CD80 and up-regulation of expression of CD86. Effects of T. gondii on expression of CD80 and CD86 on purified monocytes by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. Given the remarkable changes observed in the expression of costimulatory ligands on monocytes and the importance of macrophages to the immune response against intracellular pathogens (25), our additional studies concentrated on the effects of T. gondii on monocytes/macrophages. Since T cell-mediated cognate (cell contact-mediated) signals can induce the expression of CD80 on B cells (26, 27), experiments were conducted to determine whether changes in the expression of costimulatory ligands on monocytes that were mediated by T. gondii occurred in the absence of lym- http://classic.jimmunol.org phocytes. Incubation of purified monocytes with tachyzoites resulted in the appearance of subpopulations of monocytes with remarkable levels of expression of CD80 molecules (CD80ϩ; Fig. 1A) as well as subpopulations of cells with increased levels of expression of CD86 molecules (CD86high; Fig. 1B). Although the changes in expression of CD80 were striking, levels of expression Downloaded from of CD86 on CD86high monocytes (MFI ϭ 1579.2 Ϯ 70.1) were, on the average, 11.7 times higher than those of CD80 on CD80ϩ FIGURE 1. T. gondii induces the expression of CD80 and up-regulates monocytes (MFI ϭ 139.5 Ϯ 15.4; n ϭ 7). The increase in the the expression of CD86 on purified human monocytes. Changes in levels levels of expression of CD80 and CD86 were not caused by non- of expression of CD80 (A) and CD86 (B) on purified monocytes after specific effects mediated by phagocytosis of foreign particles, incubation with T. gondii tachyzoites. The number within the contour since incubation with latex beads did not alter the expression of graphs indicates the percentage of either CD80ϩ (upper right plus left any of these molecules (data not shown). Incubation of purified quadrants) or CD86high (box) monocytes. C, Effects of incubation with monocytes with increasing numbers of tachyzoites per monocyte increasing concentrations of tachyzoites on the percentages of CD80ϩ and ϩ resulted in a progressive increase in the percentages of CD80 and CD86high monocytes. D, Only viable T. gondii tachyzoites induce CD80 high CD86 monocytes (Fig. 1C). T. gondii tachyzoites also induced and up-regulate CD86 on human monocytes. Monocytes were incubated up-regulation of the expression of CD80 and CD86 molecules on with viable (UV-attenuated) T. gondii tachyzoites (UVTg), paraformalde- monocyte-derived macrophages (data not shown). hyde-killed tachyzoites (PFKTg), or TLA. Purified monocytes were ana- Experiments were conducted to determine whether preparations lyzed by flow cytometry after 48 h of in vitro culture in the absence or the of dead tachyzoites of T. gondii as well as of parasite soluble Ags presence of T. gondii. Results are representative of 10 individual could also induce changes in the expression of CD80 and CD86 on experiments. 1834 CD80/CD86 AND IMMUNITY TO AN INTRACELLULAR PATHOGEN

Table I. Effects of T. gondii tachyzoites on levels of expression of adhesion molecules and HLA-DR on gated monocytes and B cellsa

MFIb MFI 24 h (PBMC ϩ Tg)/MFIb 0h Control T. gondii (PBMC)c

CD14 CD11a 789.0 786.9 731.5 0.7 Ϯ 0.1 CD54 32.0 1004.0 845.1 2.5 Ϯ 0.7 CD58 102.7 254.1 277.6 1.0 Ϯ 0.1 HLA-DR 157.1 793.2 1002.4 1.7 Ϯ 0.6 CD19 CD11a 52.0 76.4 68.0 1.0 Ϯ 0.1 CD54 18.4 50.8 69.1 1.7 Ϯ 0.3 CD58 8.7 25.3 23.1 1.0 Ϯ 0.1 CD80 2.2 5.9 6.1 1.0 Ϯ 0.0 CD86 1.6 12.0 27.3 2.1 Ϯ 0.1 HLA-DR 623.7 1054.0 1602.6 1.8 Ϯ 0.3

a Freshly isolated PBMC and PBMC cultured for 24 h in the absence (control) or presence of T. gondii tachyzoites were stained with either FITC-conjugated anti- CD14 or FITC-conjugated anti-CD19 mAb. An electronic gate was set on FITC- stained CD14 or CD19-positive cells to identify monocytes and B in respectively. MFI for PE-conjugated mAbs were calculated as described in Materials and Methods. b MFI for gated monocytes and B cells from a representative donor. c MFI for gated monocytes and B cells after 24-h incubation with T. gondii (PBMC ϩ Tg) were divided by MFI for cells cultured in the absence of the parasite (PBMC). Results represent a pool of experiments conducted with PBMC from seven T. gondii-seronegative and three T. gondii-seropositive (chronically infected) individuals.

puriﬁed monocytes. Whereas preparations containing viable (UV- attenuated) tachyzoites induced the appearance of CD80ϩ and CD86high monocytes, neither paraformaldehyde-killed tachyzoites nor TLA affected the levels of expression of these molecules (Fig. 1D). Similar results were obtained regardless of whether monocytes originated from individuals with or without Abs to T. gondii.

by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. Role of infection of monocytes with T. gondii and of IFN-␣, IFN-␥, and GM-CSF on induction of expression of CD80 and up-regulation of expression of CD86 molecules Experiments were conducted to determine whether induction of the expression of CD80 and up-regulation of the expression of CD86 caused by T. gondii were due to infection of monocytes and/or to the secretion of cytokines known to affect the expression of these molecules on monocytes/macrophages. Purified mono- FIGURE 2. Neutralizing mAb against GM-CSF, but not against IFN-␣ cytes incubated with the parasite were sorted by FACS into http://classic.jimmunol.org or IFN-␥, partially inhibits the T. gondii-mediated induction of CD80 and ϩ Ϫ high int CD80 , CD80 , CD86 , and CD86 populations. Microscopic up-regulation of CD86. Purified monocytes were incubated with saturating examination of these populations revealed that whereas at least concentrations of anti-IFN-␣, anti-IFN-␥, or anti-GM-CSF mAb or with ϩ 50% (59 Ϯ 8%) of CD80 and CD86high monocytes had evidence isotype control mAb before addition of T. gondii. The percentages of of intracellular tachyzoites (mostly degenerated organisms), the CD80ϩ and CD86high monocytes (A), the ⌬ MFI for CD80 (B), and the ⌬ ϩ percentages of CD80Ϫ and CD86int monocytes with intracellular MFI for CD86 (C) on CD80 and CD86high cells, respectively, were de- tachyzoites were never more than 2% (1.5 Ϯ 0.5%; n ϭ 3). termined by flow cytometry as described in Materials and Methods. Nei- Downloaded from Since IFN-␣, IFN-␥, and GM-CSF have been reported to induce ther isotype control nor anti-cytokine mAb affected the percentage of monocytes with intracellular T. gondii. Results are representative of three the expression of CD80 and/or to up-regulate the expression of independent experiments. CD86 on monocytes/macrophages (9, 10, 26–29), we performed experiments to determine whether the effects of T. gondii on the levels of expression of these costimulatory ligands were mediated by these cytokines. Incubation of purified monocytes with saturat- ϩ high Յ ing concentrations of neutralizing mAb against IFN-␣ or IFN-␥ inhibition) on CD80 and CD86 monocytes, respectively ( p did not affect either the T. gondii-mediated induction of CD80ϩ 0.04; Fig. 2, A–C). At the concentration used, anti-GM-CSF mAb and CD86high monocytes (Fig. 2A) or the increase in the levels of completely ablated the GM-CSF-mediated induction of expression expression of CD80 and CD86 on CD80ϩ and CD86high cells, of CD80 on monocytes (data not shown). Even though our results respectively (Fig. 2, B and C; n ϭ 3). Although a neutralizing mAb suggest that GM-CSF is involved in the T. gondii-mediated induc- against GM-CSF did not significantly affect the percentages of tion of expression of CD80 and the up-regulation of expression of CD80ϩ and CD86high monocytes, this mAb induced a modest, but CD86, experiments performed with Transwell inserts indicate that consistent, inhibition of parasite-mediated increase in the levels of it is unlikely that soluble factors (such as GM-CSF) alone are expression of CD80 (30.6 Ϯ 5% inhibition) and CD86 (18.8 Ϯ 2% sufficient to mediate these effects. Incubation of monocytes with T. The Journal of Immunology 1835

gondii tachyzoites separated from monocytes alone by a membrane permeable to particles Յ0.4 ␮m in diameter resulted in induction of the expression CD80 and up-regulation of the expression of CD86 only on monocytes directly exposed to the parasite (data not shown). The changes in levels of expression of these costimulatory ligands were not attributable to LPS, since preparations of T. gondii did not contain detectable levels of LPS (Ͻ10 pg/ml by amebocyte Limulus assays) and addition of a mAb against CD14 (LPS receptor) that neutralizes the effects of LPS on human monocytes (30) did not inhibit the effects of T. gondii on the levels of expression of these costimulatory ligands (data not shown). Taken together, these experiments indicate that infection of monocytes with viable tachyzoites of T. gondii results in induction of remarkable levels of expression of CD80 molecules and up-regulation of expression of CD86 molecules. In addition, our results also suggest that GM- CSF has a secondary role, probably by potentiating the effects of T. gondii infection on the expression of these molecules. Since IFN-␥ is consistently produced upon exposure of human PBMC to T. gondii (31), experiments were conducted to determine whether, in the presence of lymphocytes, IFN-␥ modulated the T. gondii-induced changes in the expression of CD80 and CD86 on human monocytes. As shown in Figure 3, A and B, addition of anti-IFN-␥ mAb to PBMC incubated with T. gondii did not result in a significant inhibition of the induction of CD80ϩ and CD86high monocytes or a significant decrease in the levels of expression of these molecules on CD80ϩ and CD86high monocytes ( p Ն 0.2; n ϭ 4). Similar results were obtained regardless of the serologic status of the donor. At the concentration used, anti-IFN-␥ ablated the IFN-␥-mediated induction of expression of CD80 and up-regulation of expression of CD86 on monocytes (Fig. 3C). Thus, these results indicate that, even in the presence of lymphocytes, the early induction of CD80 and up-regulation of CD86 on human monocytes triggered by T. gondii are not mediated by IFN-␥. by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. Kinetics of T. gondii-mediated induction of expression of CD80 and up-regulation of expression of CD86 molecules on purified monocytes The presence of costimulatory ligands during the early phases of T cell-APC interaction would appear to be required to affect T cell responses. Thus, experiments were conducted to determine the kinetics of the T. gondii-mediated changes in the levels of expression of CD80 and CD86 on purified monocytes. Figure 4A demon- http://classic.jimmunol.org strates that a significant increase in the percentages of CD80ϩ and CD86high monocytes was detected 12 h after incubation with T. gondii tachyzoites and reached a peak at 24 h. Similarly, when the FIGURE 3. The T. gondii-dependent early induction of expression of ϩ high MFI of CD80 and CD86 cells were analyzed, maximum lev- CD80 on gated human monocytes is not mediated by IFN-␥ even in the ϩ high els of expression of these molecules on the CD80 and CD86 presence of lymphocytes. The percentages of CD80ϩ and CD86high mono- populations were observed at 24 h (Fig. 4B). Thus, these data cytes (A) and ⌬ MFI for CD80 and CD86 (B) on CD80ϩ and CD86high Downloaded from indicate that T. gondii triggers a rapid induction of expression of cells, respectively, were determined by flow cytometry. Neither isotype CD80 and up-regulation of expression of CD86 molecules on control nor anti-IFN-␥ mAb affected the percentage of PBMC with intra- monocytes. It is interesting to note that CD80ϩ and CD86high cells cellular T. gondii. C, MFI for CD80 and CD86 on gated monocytes before ␥ ␥ underwent a progressive decrease in the levels of expression of and after incubation with rIFN- plus either anti-IFN- mAb or isotype control mAb. Results shown are representative of four independent exper- CD14 (Fig. 1, A and B). In contrast to dendritic cells derived by iments using cells from two T. gondii-seronegative and two T. gondii- incubation of monocytes with GM-CSF plus IL-4, T. gondii-in- seropositive (chronically infected) individuals. duced CD80ϩ and CD86high monocytes remained CD1aϪ (data not shown). Ags (18). We used this experimental system to assess the func- Role of CD80 and CD86 molecules on T cell proliferation in tional significance of CD80 and CD86 molecules on the response response to T. gondii of resting T cells to T. gondii. T cells from T. gondii-seronegative We have previously demonstrated that both peripheral blood rest- donors were stimulated with autologous T. gondii-infected PBMC ing T cells from healthy T. gondii chronically infected donors as in the presence of CTLA-4-Ig, a chimeric protein with high affinity well as those from T. gondii-seronegative donors proliferate when for CD80 and CD86 that blocks the interaction between these mol- incubated with either parasite-infected cells or T. gondii-soluble ecules and CD28 (19). Figure 5A shows that CTLA-4-Ig induced 1836 CD80/CD86 AND IMMUNITY TO AN INTRACELLULAR PATHOGEN

critical costimulatory ligands for T. gondii-mediated proliferation of T cells from seronegative individuals, and that whereas CD80 plays a role only in the T cell proliferation mediated by infected cells, CD86 plays a role in both proliferation mediated by cells infected with T. gondii and that mediated by cells pulsed with parasite-soluble Ags. Costimulatory signals appear to be particularly relevant in the initiation of the immune response by naive T cells (32). We have recently demonstrated that both resting CD4ϩ CD45RAϩ T cells from T. gondii-seronegative adults and CD45RAϩ T cells from T. gondii-seronegative newborns proliferate in response to T. gondii.4 Therefore, experiments were performed to study the roles of CD80 and CD86 molecules in the response to T. gondii by CD4ϩ CD45RAϩ T cells from seronegative donors. The proliferative response of these T cells to T. gondii-infected PBMC was partially inhibited by anti-CD80 mAb (24.2 Ϯ 1.4% inhibition; p Յ 0.001), was almost completely inhibited by anti-CD86 mAb (83.1 Ϯ 6.8% inhibition; p Յ 0.0001), and was ablated by the combination of these two mAb (98.1 Ϯ 1.8% inhibition; p Յ 0.0001; Fig. 5D). Thus, these results indicate that the proliferation of presumably unprimed T cells in response to T. gondii-infected cells is dependent on both CD80 and CD86 molecules. To determine the role of costimulation through CD28 in the response of T cells from individuals previously exposed to T. gondii, experiments similar to those described above were performed using T cells from healthy, chronically infected donors. As shown in Figure 6A, anti-CD86 mAb significantly inhibited the proliferation of T cells in response to T. gondii-infected PBMC (58.5 Ϯ 14.0% inhibition; p Յ 0.02; n ϭ 4). Incubation with anti-CD80 Ϯ FIGURE 4. T. gondii tachyzoites mediate rapid induction of CD80 and mAb resulted in variable partial inhibition (17.7 7.4% inhibi- ϭ up-regulation of CD86 on purified monocytes. A, The percentages of tion; p 0.01–0.1) of the T cell proliferation in response to T. CD80ϩ and CD86high monocytes were determined by flow cytometry at gondii-infected PBMC. The combination of anti-CD80 plus anti- different time points after incubation with either tachyzoites or CM alone. CD86 mAb resulted in additive inhibitory effect (84.4 Ϯ 4.1% B, The MFI of CD80 and CD86 on CD80ϩ and CD86high monocytes, inhibition; p Յ 0.01), leading to a dramatic reduction in T cell by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. respectively, at different time points after incubation with the parasite. proliferation. Figure 6B shows that anti-CD86 mAb inhibited pro- These results are compared with the MFI of CD80 and CD86 on monocytes liferation of T cells in response to TLA (67.5 Ϯ 11.4% inhibition; before in vitro culture or on monocytes cultured with CM alone. The MFI p Յ 0.001), and there was no inhibitory effect exerted by anti- of CD80 and CD86 on uninfected monocytes remained constant between CD80 mAb (2.7 Ϯ 2.7% inhibition; p Ն 0.1) and no additive effect the 12 and 48 h points of in vitro culture. Results are representative of three observed after combining anti-CD80 plus anti-CD86 mAbs individual experiments. (76.1 Ϯ 13.3% inhibition; p Յ 0.0003). Thus, CD80 and CD86 are also important costimulatory ligands in the in vitro proliferative response to T. gondii by T cells from previously exposed a dose-dependent inhibition of the T. gondii-mediated T cell pro- individuals. http://classic.jimmunol.org liferation. To determine the individual roles of CD80 and CD86 in the proliferative response of T cells, anti-CD80 and/or anti-CD86 Roles of CD80 and CD86 molecules in the T cell cytokine mAb were added to T cells stimulated with the parasite. Incubation production in response to T. gondii with either anti-CD80 ( p Յ 0.03) as well as anti-CD86 mAb ( p Յ We studied whether the T. gondii-induced changes in the levels of 0.008) resulted in a statistically significant inhibition of the T cell expression of CD80 and CD86 on monocytes modulated cytokine proliferative response to T. gondii-infected autologous PBMC production by T cells in response to the parasite. Whereas there Downloaded from (Fig. 5B). Anti-CD80 mAb reduced the proliferative response by was no significant production of IFN-␥ when resting T cells from 30.1 Ϯ 13.0%; anti-CD86 induced a 74.1 Ϯ 5.4% inhibition of this T. gondii-seronegative individuals were incubated with uninfected, response (n ϭ 4). Furthermore, when anti-CD80 was used in com- untreated monocytes, T cells secreted significant amounts of this bination with anti-CD86 mAb, the proliferative response was ab- cytokine when incubated with T. gondii-infected monocytes (Fig. rogated (96.0 Ϯ 2.1% inhibition; p Յ 0.01; Fig. 5B; n ϭ 4). In 7A; n ϭ 3). Interestingly, stimulation of T cells with monocytes contrast, anti-CD86 mAb ( p Յ 0.02), but not anti-CD80 mAb incubated with paraformaldehyde-killed tachyzoites or TLA did ( p Ն 0.4), significantly inhibited T cell proliferation in response to not result in the production of significant amounts of IFN-␥. This TLA (Fig. 5C). Anti-CD86 mAb induced a 76.8 Ϯ 17.4% inhibi- was observed despite the fact that T cells proliferated not only in tion of T cell proliferation in response to TLA, and incubation with response to T. gondii-infected monocytes but also in response to the combination of anti-CD80 and anti-CD86 did not result in any monocytes that had phagocytosed killed parasites and to mono- further inhibition (76.8 Ϯ 19.4% inhibition; p Յ 0.008) of TLA- cytes incubated with TLA (Fig. 7A; n ϭ 3). The lack of production mediated T cell proliferation (n ϭ 4). Similar results were obtained of IFN-␥ in response to incubation with monocytes and either when T cells were stimulated with monocytes, including T. gondii- ϩ ϭ infected highly purified CD14 monocytes (n 3; data not 4 Subauste, C. S., R. de Waal Malefyt, and J. S. Remington. 1998. ␣␤ T cell response shown). These results demonstrate that both CD80 and CD86 are to Toxoplasma gondii in previously unexposed individuals. J. Immunol. In press. The Journal of Immunology 1837

FIGURE 6. Both CD80 and CD86 act as costimulatory ligands for T. gondii-induced proliferation of T cells obtained from seropositive donors. Autologous PBMC infected with tachyzoites (A) or pulsed with TLA (B) were incubated with mAb before addition of T cells. Data shown are representative of four individual experiments.

by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. killed parasites or TLA was not caused by an inherent inability of these stimuli to trigger IFN-␥ production, since T cells from healthy individuals chronically infected with T. gondii produced considerable amounts of IFN-␥ after stimulation with monocytes plus either killed tachyzoites (Ն945 pg/ml) or TLA (Ն582 pg/ml; n ϭ 3). These results raised the possibility that induction of expression of CD80 and/or up-regulation of expression of CD86 were involved in the production of IFN-␥ by T cells from seronegative http://classic.jimmunol.org individuals. In this regard, production of IFN-␥ by these T cells in response to T. gondii-infected monocytes was significantly inhibited by anti-CD86 (40.8 Ϯ 5.2% inhibition; p Յ 0.04). Incubation with anti-CD80 mAb resulted in variable partial inhibition (7.5 Ϯ 7.0% inhibition; p ϭ 0.02–0.1) of IFN-␥ production; the combination of CD80 plus anti-CD86 mAb resulted in an additive in- Downloaded from hibitory effect (80.2 Ϯ 1.4% inhibition; p Յ 0.002), leading to almost complete ablation of IFN-␥ production (n ϭ 3; Fig. 7B). Similar results were obtained using highly purified CD14ϩ monocytes (data not shown). In addition, production of IFN-␥ by CD4ϩ CD45RAϩ T cells from seronegative individuals was inhibited by either anti-CD80 (69.47 Ϯ 6.1% inhibition; p Յ 0.001) or anti- CD86 mAb (53.0 Ϯ 5.1% inhibition) and was ablated by the com- FIGURE 5. Both CD80 and CD86 act as costimulatory ligands for T. bination of mAbs (100% inhibition p Յ 0.001; n ϭ 2; Fig. 7C). gondii-induced proliferation of T cells obtained from seronegative donors. Taken together, these results indicate that the early production of A, T. gondii-infected autologous PBMC were incubated with different con- IFN-␥ by presumably unprimed T cells is dependent on the ex- centrations of either CTLA-4-Ig or human IgG1 before addition of T cells. pression of CD80 and CD86 on monocytes. Autologous PBMC infected with tachyzoites (B) or pulsed with TLA (C) were incubated with mAb before addition of T cells. D, Purified CD4ϩ We attempted to determine whether T. gondii-mediated changes CD45RAϩ TCR-␣␤ cells were incubated with infected PBMC in the pres- in expression of costimulatory ligands affect T cell production of ence or the absence of mAb. Data shown are representative of four indi- IL-2 and IL-4 in response to the parasite. Stimulation of T cells vidual experiments. with monocytes plus PHA resulted in the secretion of measurable 1838 CD80/CD86 AND IMMUNITY TO AN INTRACELLULAR PATHOGEN

on CD80 and CD86, but, more importantly, our data indicate that changes in the levels of expression of these costimulatory ligands on monocytes were associated with the production of IFN-␥ by T cells from unexposed individuals. Our results are of particular relevance to immunity against intracellular organisms, since production of IFN-␥ as a result of the interaction between infected monocytes/macrophages and T cells would lead to activation of these phagocytic cells, enabling them to act as major effectors of anti- microbial defense. It is well recognized that cognate signals and certain cytokines can induce or up-regulate the expression of CD80 and CD86 on APC. We demonstrate that induction of the expression of CD80 and up-regulation of the expression of CD86 on human monocytes caused by T. gondii occurred in the absence of significant concentrations of lymphocytes and was not mediated by IFN-␣ or IFN-␥, which are cytokines capable of inducing the expression of CD80 and up-regulating the expression of CD86 on monocytes/macrophages (9, 10, 29). In these same experiments, GM-CSF was found to be involved in the process of induction of the expression of CD80 and up-regulation of the expression of CD86 triggered by T. gondii. These latter results suggest that, similar to what has been reported for dendritic cells (33), GM-CSF may have immuno- modulatory activity on infected monocytes that, through promoting up-regulation of costimulatory ligands, may lead to an enhanced immunostimulatory function of these APC. In addition to the cytokines mentioned above, we have recently studied TNF-␣, IL-1␣, and IL-12 and demonstrated that neutralizing Abs against these cytokines failed to inhibit the T. gondii-mediated changes in the expression of CD80 and CD86 on monocytes (C. S. Subauste, unpublished observations). Despite the results that we obtained after neutralization of GM-CSF, cell-sorting experiments as well as data obtained with Transwell inserts indicated that infection of monocytes with viable tachyzoites and not soluble factors appeared to play the primary role in induction of the expression of by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. CD80 and up-regulation of the expression of CD86. Although not all the CD80ϩ and CD86high monocytes contained intracellular tachyzoites 18 h after incubation with T. gondii, our microscopic examination of monocytes incubated with the parasite is consistent with this conclusion. Monocytes can rapidly eliminate intracellular tachyzoites so that the percentage of infected monocytes 18 to 24 h after challenge with T. gondii will be remarkably lower than the percentage of infected monocytes 1 h after challenge (34). FIGURE 7. T. gondii-induced production of IFN-␥ by T cells is depen- T. gondii-mediated up-regulation of expression of CD86 on hu- http://classic.jimmunol.org dent on both CD80 and CD86. A, Purified resting T cells from seronegative donors were incubated with uninfected/untreated monocytes or with mono- man monocytes has been described recently (35). The authors re- cytes plus viable tachyzoites (Tg), paraformaldehyde-killed tachyzoites ported that the expression of this molecule followed a unimodal (PFKTg), or TLA. Uninfected monocytes or infected monocytes preincu- distribution, and that this up-regulation appeared to be inhibited by bated with mAb were used to stimulate purified resting T cells (B)or an anti-IFN-␥ polyclonal Ab. However, in our studies, double purified resting CD4ϩ CD45RAϩ TCR-␣␤ cells (C) from seronegative staining with anti-CD14 and anti-CD86 mAb allowed us to dem- donors. The detection limit of the assay was 78 pg/ml. Data shown are onstrate that up-regulation of CD86 occurred only in a subpopu- Downloaded from representative of three individual experiments. *SI, stimulation index. lation of monocytes. Furthermore, we demonstrated that IFN-␥ did not play an important role in this up-regulation, since an increase in the levels of expression of CD86 occurred in populations of concentrations of IL-2 and either low or undetectable concentra- highly purified monocytes and was not affected by a neutralizing tions of IL-4. However, neither of these cytokines was detected in anti-IFN-␥ mAb. supernatants from T. gondii-stimulated T cells (data not shown). Except in experimental models using cells transfected with CD80 or transgenic mice expressing CD80 on pancreatic ␤ cells Discussion (36, 37), it has been difficult to demonstrate significant costimu- We have established that T. gondii caused a rapid induction of the lation by CD80 molecules (12, 38). The predominant costimula- expression of CD80 and up-regulation of the expression of CD86 tory activity of CD86 molecules may be explained, at least in part, on human monocytes. These changes were observed after infection by the fact that after APC activation, CD86 is expressed earlier and of monocytes with viable tachyzoites and not after phagocytosis of at higher levels than CD80 (39, 40). In contrast, our results dem- killed tachyzoites or incubation with T. gondii-soluble Ags. These onstrate that infection of monocytes with T. gondii results in a costimulatory ligands played a crucial role in the T cell response rapid induction of CD80 and up-regulation of CD86, indicating triggered by T. gondii. Not only was T cell proliferation dependent that both costimulatory ligands can be present in the early phases The Journal of Immunology 1839

of the immune response to an intracellular pathogen. Moreover, that nonclonal recognition of a micro-organism by monocytes can our studies indicate that CD80 molecules provided significant co- affect the nature of the T cell response to the offending pathogen. stimulation under conditions in which the expression of CD80 was In addition, given that IFN-␥ production by NK cells can be en- induced in human monocytes (infection with viable tachyzoites). hanced by costimulation through CD28 (48, 49), it is likely that One of the hallmarks of the immune response elicited by T. microbially mediated induction/up-regulation of costimulatory li- gondii is the production of IFN-␥. Indeed, this cytokine is a major gands can further promote the generation of protective immunity mediator of protection against the parasite (16, 41). The critical through stimulation of secretion of IFN-␥ by NK cells. role of IFN-␥ in the immune response to intracellular pathogens The results of our present study illustrate the importance of a makes identification of factors required for production of this cy- microbially induced increase in the levels of expression of co- tokine one of the most important questions in the study of immu- stimulatory ligands for the production of protective cytokines (i.e., nity to infectious organisms. We have recently demonstrated that IFN-␥). Of interest in this regard is the evidence that certain in- both human ␣␤ and ␥␦ T cells from T. gondii-seronegative indi- tracellular organisms, such as Leishmania donovani and Mycobac- viduals secrete significant amounts of IFN-␥ after in vitro stimu- terium tuberculosis, either fail to up-regulate or actually decrease lation with the parasite (18) (see Footnote 4). Our data indicate that the expression of these molecules (50, 51). These effects on co- IFN-␥ production by human T cells from unexposed individuals is stimulatory ligands may represent strategies used by the pathogens associated with a T. gondii-induced increase in the levels of ex- to avoid recognition, induce anergy, or cause immunosuppression. pression of costimulatory ligands on infected monocytes. IFN-␥ Thus, intracellular organisms can have a major influence on anti- was produced only when T cells were incubated with monocytes microbial immunity through regulation of costimulation. that displayed high levels of expression of CD80 and CD86 Our demonstration that tachyzoites, but not parasite-soluble (monocytes infected with viable tachyzoites). Moreover, anti- Ags, are capable of inducing these changes and that the tachyzoites CD80 plus anti-CD86 mAb almost completely inhibited T cell have to be viable to affect the levels of expression of CD80 and production of IFN-␥. Of particular relevance to the events in the CD86 on monocytes suggest that the process of induction of CD80 initiation of the immune response to intracellular pathogens are our and up-regulation of CD86 in monocytes may be multifactorial. results with CD4ϩ CD45RAϩ cells, which indicate that presum- Signals triggered within monocytes by the presence of viable in- ably naive CD4ϩ T cells require CD80 and CD86 for the produc- tracellular organisms, factors released by viable intracellular par- tion of IFN-␥ in response to T. gondii-infected monocytes. These asites and/or interactions between the intact pathogen and mole- data are of importance to the induction of protective immunity to cules on the surface of monocytes may be involved in this process. the parasite, since the early production of IFN-␥ may confer pro- Identification of the mechanisms by which microbes induce the tection to the host not only because of direct effects of this cytokine expression of CD80 and up-regulate the expression of CD86 are of on the growth of intracellular tachyzoites (41), but also because paramount importance, since they may tie molecular events that IFN-␥ appears to play a role in promoting the generation of a Th1 occur during the interaction of pathogens with APC with the out- cytokine pattern (42). come of infections. Comparison of the molecular events triggered It is well established that proliferation and IL-2 secretion by in monocytes by infection with T. gondii with those triggered by naive T cells are dependent on the presence of costimulatory sig- pathogens that fail to increase levels of expression of costimula- by guest on October 1, 2021. Copyright 1998 Pageant Media Ltd. nals (32). Indeed, we have demonstrated that the proliferation of tory ligands may provide an understanding of the mechanisms by CD4ϩ CD45RAϩ T cells in response to T. gondii is ablated by which monocytes can direct T cell responses. Finally, our results anti-CD80 plus anti CD86 mAb. However, our results indicate that have important implications for the efforts to establish a vaccine these costimulatory ligands also play a central role in the parasite- against intracellular pathogens, since they suggest that vectors that triggered proliferation of T cells from chronically infected indi- induce/up-regulate the expression of costimulatory ligands on viduals. In this regard, human CD45ROϩ memory T cells undergo APC will help generate a protective (IFN-␥-dependent) immune optimal anti-CD3-mediated proliferation in the presence of CD80- response. transfected cells (43). Our model provides a clear example of the capacity of mono- http://classic.jimmunol.org Acknowledgments cytes to discriminate among different microbial preparations and illustrates that the T cell cytokine response is affected by the type We express our appreciation to J. Remington for generous support and for critical review of the manuscript. We thank G. Deepe, Jr., and of microbial preparation that elicits the immune response. In the L. Lanier for helpful suggestions, and P. Linsley and M. Brenner for pro- case of T. gondii, this pathogen-monocyte-T cell interaction would viding reagents. result in IFN-␥ production in situations (noxious stimulus; i.e., infection with viable tachyzoites) where an IFN-␥-dependent cell- Downloaded from References mediated response would be appropriate, whereas no such a response would be triggered when encountering nonviable parasite 1. Janeway, C. A., Jr. 1992. The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol. Today 13:11. preparations (harmless stimulus). A probable in vivo correlate to 2. Gimmi, C. D., G. J. Freeman, J. G. Gribben, K. Sugita, A. S. Freedman, our observations can be drawn from the demonstration that infec- C. Morimoto, and L. M. Nadler. 1991. 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Cloning of B7-2: a CTLA-4 the generation of a protective immune response through IFN- counter-receptor that costimulates human T cell proliferation. Science 262:909. production by NK cells (47). It has also been proposed that the 6. Linsley, P. S., and J. A. Ledbetter. 1993. The role of CD28 receptor during T cell mechanisms used by the innate immune system to recognize responses to antigen. Annu. Rev. Immunol. 11:191. 7. Freeman, G. J., F. Borriello, R. J. Hodes, H. Reiser, J. G. Gribben, J. W. Ng, pathogens would determine the type of adaptive immunity elicited J. Kim, J. M. Goldberg, K. Hathcock, G. Laszlo, L. A. Lombard, S. Wang, (46). Our results support this proposal, since they provide evidence G. S. Gray, L. M. Nadler, and A. H. Sharpe. 1993. Murine B7-2, an alternative 1840 CD80/CD86 AND IMMUNITY TO AN INTRACELLULAR PATHOGEN

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