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Cooperation of PD-1 and LAG-3 Contributes to T-Cell Exhaustion in Anaplasma marginale-Infected Cattle Tomohiro Okagawa,a Satoru Konnai,a James R. Deringer,b Massaro W. Ueti,c Glen A. Scoles,c Shiro Murata,a Kazuhiko Ohashi,a Wendy C. Brownb Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japana; Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USAb; Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington, USAc

The CD4ⴙ T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4ⴙ T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OMs), which presumably facilitates the persistence of this rickettsia. In the present study, we hypothesize that T-cell exhaustion following infection is induced by the upregulation of immunoinhibitory receptors on T cells, such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3). OM-specific T-cell responses and the kinetics of PD-1-positive (PD-1ⴙ) LAG-3ⴙ exhausted T cells were monitored in A. marginale-challenged cattle previously immunized with OMs. Consistent with data from previous studies, OM-specific proliferation of peripheral blood mononuclear cells (PBMCs) and interferon gamma (IFN-␥) production were significantly suppressed in challenged animals by 5 weeks postinfection (wpi). In addition, bacteremia and anemia also peaked in these animals at 5 wpi. Flow cytometric analysis revealed that the percentage of PD-1ⴙ LAG-3ⴙ T cells in the CD4ⴙ, CD8ⴙ, and ␥␦ T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3ⴙ ␥␦ T cells was also observed. Importantly, in vitro, the combined blockade of the PD-1 and LAG-3 pathways partially restored OM-specific PBMC proliferation and IFN-␥ production at 5 wpi. Taken together, these results indicate that coexpression of PD-1 and LAG-3 on T cells contributes to the rapid exhaustion of A. marginale-specific T cells following infection and that these immunoinhibitory receptors regulate T-cell responses during bovine anaplasmosis.

A

naplasma marginale is a tick-borne intraerythrocytic rickettsial pathogen that causes acute bacteremia, anemia, and a mortality rate of up to 30% in naive cattle. Cattle surviving acute infection develop a lifelong persistent infection with microscopically undetectable levels of bacteremia that do not cause clinical disease (1). Notably, A. marginale infection is characterized by high levels of bacteremia in the infected animal, with 107 to 109 bacteria/ml of blood during acute infection and a mean of 106 bacteria/ml of blood during persistent infection (2). Immunization of cattle with A. marginale outer membranes (OMs) induced both CD4⫹ T-cell and IgG responses specific for OM proteins and resulted in protection against high-level bacteremia and anemia (3, 4). Previous studies have also shown that cattle immunized with either major surface protein 2 (MSP2) or MSP1a developed antigen-specific CD4⫹ T-cell responses, including memory CD4⫹ T-cell proliferation and interferon gamma (IFN-␥) secretion (5, 6). However, subsequent infection with A. marginale promoted the rapid exhaustion of antigen-specific CD4⫹ T-cell responses prior to the peak of acute infection in immunized cattle. Furthermore, flow cytometric analysis with major histocompatibility complex (MHC)-peptide tetramers revealed that deletion of MSP1a-specific CD4⫹ T cells occurred along with exhaustion of the CD4⫹ T-cell response (6). Induction of T-cell exhaustion required the presence of the priming T-cell epitope on the infecting bacteria, suggesting a requirement of T-cell receptor (TCR) engagement for the loss of antigen-specific T-cell function (7). However, T-cell exhaustion in these models was not associated with an increase in the percentages of either the regulatory T-cell subsets CD4⫹ CD25⫹ FoxP3⫹ T cells and WC1.2⫹ ␥␦ T cells or the cytokines interleukin-10 (IL-10) and transforming

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growth factor ␤ (TGF-␤) (5, 7). Therefore, other mechanisms are likely involved in the induction of CD4⫹ T-cell exhaustion during A. marginale infection. Exhausted T cells are phenotypically characterized by the surface expression of immunoinhibitory receptors such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG3), which are induced by persistent antigenic stimulation via the TCR (8). PD-1 and LAG-3 inhibit TCR signaling and the subsequent induction of effector functions in T cells after binding to their respective ligands, PD ligand 1 (PD-L1) and MHC class II (MHC-II), expressed on antigen-presenting cells (APCs) (9, 10). Previous studies on chronic infections of cattle revealed that the upregulation of bovine PD-1 and LAG-3 in T cells was closely associated with the exhaustion of T-cell responses and disease progression during bovine leukemia virus (BLV) infection and Johne’s disease (11–14). Moreover, blockade of PD-1/PD-L1 and

Received 1 April 2016 Returned for modification 25 April 2016 Accepted 15 July 2016 Accepted manuscript posted online 18 July 2016 Citation Okagawa T, Konnai S, Deringer JR, Ueti MW, Scoles GA, Murata S, Ohashi K, Brown WC. 2016. Cooperation of PD-1 and LAG-3 contributes to T-cell exhaustion in Anaplasma marginale-infected cattle. Infect Immun 84:2779 –2790. doi:10.1128/IAI.00278-16. Editor: C. R. Roy, Yale University School of Medicine Address correspondence to Wendy C. Brown, [email protected] Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.00278-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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LAG-3/MHC-II binding with antagonist antibodies reactivated T-cell functions such as proliferation and cytokine production in vitro (11, 13–16). However, expression of PD-1, LAG-3, and PD-L1 and their functions in cattle undergoing A. marginale infection have not been investigated. This study was designed to test the hypothesis that PD-1 and LAG-3 contribute to the rapid exhaustion of the A. marginalespecific T-cell response that occurs during acute infection with A. marginale. To test this hypothesis, this study investigated the association of OM-specific T-cell responses, clinical signs, and the expression of immunoinhibitory receptors on T cells in OM-immunized cattle challenged with A. marginale. The effect of the blockade of immunoinhibitory pathways on the reactivation of OM-specific T cells was also evaluated. MATERIALS AND METHODS Animals, immunization, and experimental infection. Five age-matched Holstein calves were chosen and shown to be serologically negative for A. marginale by a competitive enzyme-linked immunosorbent assay (ELISA) for MSP5 (VMRD, Pullman, WA). All calves were then immunized subcutaneously four times with 60 ␮g OMs (A. marginale St. Maries strain) in 6 mg saponin at 3-week intervals. Animal experiments were conducted by using an approved Institutional Animal Care and Use Center (Washington State University [WSU], Pullman, WA) protocol. Five months after the final immunization, all cattle were inoculated intravenously with 1.2 ⫻ 103 erythrocytes infected with the homologous strain of A. marginale. Infected erythrocytes were obtained from the blood of a splenectomized calf undergoing acute infection, as described previously (7). The health of each animal was monitored daily, and a blood sample was collected to measure the packed erythrocyte cell volume (PCV) and bacteremia, monitored by light microscopic examination of Giemsa-stained blood smears. Peripheral blood mononuclear cells (PBMCs) were purified from blood samples by using density gradient centrifugation on Histopaque-1077 (Sigma-Aldrich), washed three times with phosphate-buffered saline (PBS) (pH 7.0), and suspended in PBS. PBMCs were used either fresh or following cryopreservation in fetal bovine serum (FBS) containing 10% dimethyl sulfoxide. Lymphocyte proliferation assays and IFN-␥ ELISA. Fresh or cryopreserved PBMCs obtained at various time points before and after immunization and challenge were assayed in triplicate by using 2 ⫻ 105 viable cells/well in complete RPMI 1640 medium as described previously (7). Briefly, PBMCs were stimulated in 96-well round-bottomed plates (Corning Inc., Corning, NY) in a volume of 100 ␮l/well with 1 ␮g/ml of A. marginale St. Maries OMs or membranes prepared from uninfected bovine red blood cells (uRBCs). Bovine T-cell growth factor (TCGF) diluted 1:10 in complete RPMI 1640 medium was also used as a positive control (7). Cells were cultured for 6 days at 37°C in 5% CO2, labeled with 0.25 ␮Ci [3H]thymidine for 18 h, and harvested by using a Harvester96 instrument (Tomtec, Hamden, CT), and radiolabeling was quantified by using a 1450 MicroBeta TriLux liquid scintillation counter (PerkinElmer, Waltham, MA). The results are presented as the mean counts per minute for triplicate wells of cells cultured with antigen or TCGF or as the difference of the mean counts per minute for triplicate wells of cells cultured with OM antigen minus the mean counts per minute for triplicate wells of cells cultured with uRBC antigen (⌬cpm). Additionally, on day 6 before labeling, 50 ␮l of the culture supernatant from each of the triplicate wells was collected and pooled for detection of secreted IFN-␥. IFN-␥ concentrations in supernatants were determined by using a bovine IFN-␥ ELISA (Mabtech, Nacka Strand, Sweden) performed in duplicate according to the manufacturer’s protocol. Flow cytometric analysis of PD-1 and LAG-3. Four-color analysis of PD-1- and LAG-3-expressing T cells was performed by using PBMCs obtained at various time points before or after infection. PBMCs (up to

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2 ⫻ 106) were incubated in PBS containing 10% FBS at room temperature for 15 min to prevent nonspecific reactions. Cells were then stained with an anti-PD-1 monoclonal antibody (MAb) (5D2, rat IgG2a) (11) or a rat IgG2a isotype control (R35-95; BD Biosciences, San Jose, CA) for 20 min at room temperature in PBS containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO), followed by washing and incubation with allophycocyanin-conjugated anti-rat Ig (Southern Biotech, Birmingham, AL) for 20 min at room temperature. Cells were then washed and stained with an anti-LAG-3 MAb (71-2D8, rat IgG1) (14) or a rat IgG1 isotype control (R3-34; BD Biosciences) for 20 min at room temperature. Finally, cells were washed and incubated with an anti-CD4-fluorescein isothiocyanate (FITC) MAb (CC8; AbD Serotec, Oxford, United Kingdom), anti-CD8-FITC MAb (CC63; AbD Serotec), or anti-TCR1-N24 MAb (TCR ␦ chain, GB21A; Washington State University Monoclonal Antibody Center, Pullman, WA), in the presence of an anti-CD3-peridinin chlorophyll protein (PerCP)/Cy5.5 MAb (MM1A; Washington State University Monoclonal Antibody Center) and phycoerythrin (PE)-conjugated anti-rat IgG1 (Southern Biotech) for 20 min at room temperature. MAb MM1A was conjugated with Lightning-Link PerCP/Cy5.5 Tandem Conjugation kits (Innova Biosciences, Cambridge, United Kingdom). MAb GB21A was labeled with FITC-conjugated goat anti-mouse IgG⫹ IgM(H⫹L) (Caltag Laboratories, Burlingame, CA). Cells were then washed and analyzed immediately by using a FACSCalibur instrument (BD Biosciences, San Jose, CA) and FCS Express 4 software (De Novo Software, Glendale, CA). The primary antibodies used in this experiment are shown in Table 1. Flow cytometric analysis of PD-L1. Two-color analysis of PD-L1expressing cells was performed by using PBMCs obtained at various time points before or after infection. PBMCs were then blocked in PBS containing 10% FBS and incubated with an anti-PD-L1 MAb (4G12, rat IgG2a) (15) or a rat IgG2a isotype control (R35-95; BD Biosciences) for 20 min at room temperature. After washing, cells were stained with an antiCD14-PerCP/Cy5.5 MAb (CAM36A; Washington State University Monoclonal Antibody Center) and allophycocyanin-conjugated anti-rat Ig (Southern Biotech) in PBS containing 1% bovine serum albumin for 20 min at room temperature. MAb CAM36A was conjugated by using a Lightning-Link PerCP/Cy5.5 Tandem Conjugation kit (Innova Biosciences). Cells were then washed and immediately analyzed by using a FACSCalibur instrument and FCS Express 4 software. The primary antibodies used in this experiment are shown in Table 1. Flow cytometric analysis of CD95 (Fas) and CD25. Cryopreserved PBMCs collected at the 34 days postinfection (dpi) were thawed, and viable cells obtained following Histopaque-1077 gradient separation were used for flow cytometric analysis. Viable cells were washed and distributed into 96-well plates at ⬃3 ⫻ 105 to 5 ⫻ 105 cells per well. Cells were incubated in PBS containing 10% FBS for 20 min at room temperature to prevent nonspecific reactions. All antibody incubations were performed at room temperature for 20 min. Isotype controls were PBMCs incubated with either mouse IgG1 (MAb Colis69A; WSU Monoclonal Antibody Center) or rat IgG2a and FITC-labeled anti-CD4 MAb CC8. Isotype control mouse IgG1 was stained by using a goat anti-mouse IgG1-PE-Cy5.5 conjugate (catalog number M32018; Invitrogen). Isotype control rat IgG2a was stained by using an anti-rat Ig–allophycocyanin conjugate (Southern Biotech). Cells evaluated for CD95 expression were incubated with anti-PD-1 MAb, anti-human CD95 MAb (IgG1 clone ZB4; EMD Millipore), and FITC–anti-CD4 MAb and stained by using the same MAb-conjugated fluorophores as the ones used to stain isotype control MAbs. Similarly, cells evaluated for CD25 expression were incubated with anti-PD-1 MAb, anti-CD25 MAb (CACT116A; WSU Monoclonal Antibody Center), and FITC–anti-CD4 MAb and stained by using the same MAb-conjugated fluorophores as the ones used to stain isotype control MAbs. Stained cells were fixed in PBS containing 2% formaldehyde. The antibodies are shown in Table 1. A FACSCalibur instrument was used to collect 30,000 to 100,000 events, which were analyzed by using FCS Express 4 software. CD4⫹ cells were gated and evaluated for PD-1-positive

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TABLE 1 Primary MAbs used for flow cytometric analyses in this study Target

Isotype

Clone

Source (reference)

Fluorochrome

Conjugation or labeling (source)

PD-1 and LAG-3 CD4 CD8 TCR1-N24 (␦ chain)

Mouse IgG2a Mouse IgG2a Mouse IgG2b

CC8 CC63 GB21A

AbD Serotec AbD Serotec WSU MAb Center

FITC FITC FITC

CD3

Mouse IgG1

MM1A

WSU MAb Center

PerCp-Cy5.5

PD-1

Rat IgG2a

5D2

In-house (11)

APC

Rat IgG2a isotype control

Rat IgG2a

R35-95

BD Biosciences

APC

LAG-3

Rat IgG1

71-2D8

BD Biosciences

PE

Rat IgG1 isotype control

Rat IgG1

R3-34

BD Biosciences

PE

Conjugated primary antibody Conjugated primary antibody FITC–anti-mouse IgG⫹IgM(H⫹L) antibody (Caltag Laboratories) Lightening-Link PerCP/Cy5.5 conjugation kit (Innova Biosciences) APC-conjugated anti-rat IgG antibody (Southern Biotech) APC-conjugated anti-rat IgG antibody (Southern Biotech) PE-conjugated anti-rat IgG1 antibody (Southern Biotech) PE-conjugated anti-rat IgG1 antibody (Southern Biotech)

Mouse IgG1

CAM36A

WSU MAb Center

PerCP-Cy5.5

PD-L1

Rat IgG2a

4G12

In-house (16)

APC

Rat IgG2a isotype control

Rat IgG1

R35-95

BD Biosciences

APC

Mouse IgG2a Mouse IgG1

CC8 CACT116A

AbD Serotec WSU MAb Center

FITC PE-Cy5.5

CD95

Mouse IgG1

ZB4

EMD Millipore

PE-Cy5.5

Mouse IgG1 isotype control

Mouse IgG1

Colis69A

WSU MAb Center

PE-Cy5.5

PD-L1 CD-14

CD25 and CD95 CD4 CD25

(PD-1⫹) and PD-1⫹ CD95⫹ populations or PD-1⫹ and PD-1⫹ CD25⫹ populations. Antibody blockade assays. To examine the effects of blocking MAbs on OM-specific T-cell responses, in vitro blockade assays were performed by using PBMCs cultured in complete RPMI 1640 medium with 10 ␮g/ml of the blocking MAbs anti-PD-L1 (4G12) (16) and anti-LAG-3 (71-2D8) (14) or control rat IgG (Sigma-Aldrich) in the presence of 0.2 ␮g/ml of OMs. The same amount of uRBCs was used as a negative-control antigen. For the positive control, cells were stimulated with TCGF diluted 1:10 in complete RPMI 1640 medium. To determine PBMC proliferation, cells were cultured for 6 days, labeled with [3H]thymidine for 18 h, and harvested as described above, and results are presented as ⌬cpm as described above. In addition, the amount of IFN-␥ in the culture medium at day 6 was determined by an ELISA as described above. Statistical analyses. One-tailed Dunnett’s multiple-comparison test was used to determine significant differences in PBMC proliferation, PBMC IFN-␥ production, and the frequencies of each T-cell subset analyzed by flow cytometry between samples obtained at 0 dpi and those obtained at various time points thereafter. Tukey’s multiplecomparison test was used to compare proliferation and IFN-␥ production between groups at individual time points in proliferation and blockade assays. These statistical tests were performed by using the statistical analysis program MEPHAS (http://www.gen-info.osaka-u .ac.jp/MEPHAS/). One-tailed paired Student’s t test was used to compare frequencies of CD4⫹ PD-1⫹ cells that expressed CD25 or CD95. Differences were considered statistically significant when the P value was ⬍0.05.

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Lightening-Link PerCP/Cy5.5 conjugation kit (Innova Biosciences) APC-conjugated anti-rat Ig antibody (Southern Biotech) APC-conjugated anti-rat Ig antibody (Southern Biotech)

Conjugated primary antibody PE-Cy5.5-conjugated anti-mouse IgG1 (Invitrogen) PE-Cy5.5-congugated anti-mouse IgG1 (Invitrogen) PE-Cy5.5-conjugated anti-mouse IgG1 (Invitrogen)

RESULTS

Rapid exhaustion of T-cell responses to A. marginale OM antigen. Previous studies have shown that A. marginale infection of cattle previously immunized with MSP2 or MSP1a antigens resulted in functional exhaustion and deletion of A. marginale-specific CD4⫹ T cells during acute infection (5, 6). In addition, challenge of OM-immunized cattle with A. marginale also caused a rapid loss of OM-specific PBMC proliferation at 4 to 19 weeks postinfection (wpi) (17). The loss of response was specific for A. marginale, and responses to an unrelated Clostridium vaccine antigen were not impaired. Anaplasma-specific PBMC proliferation is mediated predominantly by CD4⫹ T cells (3, 6, 18, 19). In the present study, significant OM-specific PBMC proliferation was observed following immunization, prior to infection, and at 6 and 13 dpi but was suppressed by 20 dpi and thereafter, with the exception of 62 dpi (Fig. 1A to C). In contrast, responses to TCGF remained significant at all time points, whereas uRBC responses were not significant, as shown at 6 and 34 dpi (Fig. 1B and C and data not shown). The mean level of OM-induced IFN-␥ production by PBMCs was 9.3 ng/ml following immunization but prior to infection and was boosted to 16.2 ng/ml at 6 dpi (Fig. 2A). Thereafter, IFN-␥ production declined and was significantly suppressed in challenged animals at 27, 34, 40, and 125 dpi relative to the preinfection time point (Fig. 2A to C). In contrast, IFN-␥ was still pro-

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FIG 2 Suppression of OM-specific IFN-␥ production in A. marginale-chalFIG 1 Suppression of OM-specific proliferation of PBMCs in A. marginalechallenged cattle. (A) PBMCs were cultured in triplicate with OMs or uRBCs. Results for each individual are presented as the mean for triplicate cultures of cells cultured with OMs minus the mean for triplicate cultures of cells cultured with uRBCs (⌬cpm). Data for individual animals are indicated by symbols. Group means are shown with a bar. An asterisk indicates a significantly lower ⌬cpm than the ⌬cpm at the preinfection (Pre-inf.) time point, where the P value was ⬍0.05. Results obtained for all animals before they were immunized (Pre-imm.) are also shown. (B and C) Mean counts per minute for PBMCs cultured with RPMI 1640 medium only, 10% TCGF, uRBCs, and OMs at 6 dpi (B) and 34 dpi (C). Each circle represents the mean for triplicate cultures of PBMCs from an individual animal, and bars indicate the group mean responses. *, P ⬍ 0.05; n.s., not significant.

duced in response to TCGF but not uRBCs at all time points, as shown at 6 and 34 dpi (Fig. 2B and C and data not shown), showing that cells were viable and that the proliferative response was specific. Therefore, OM-specific T-cell responses in cattle immunized with OMs are exhausted during acute infection with A. marginale, as shown previously (17). Clinical response to infection. To observe the clinical signs in A. marginale-challenged cattle, bacteremia and PCVs were monitored daily during ascending acute infection at 2 to 6 wpi. Bacteremia peaked in all of the challenged animals at 32 to 34 dpi (Fig. 3). Anemia peaked at 38 to 39 dpi (Fig. 3). These clinical signs were the most severe in animal 48411; moderate in animals 48406, 48422, and 48432; and weakest in animal 48453 (Fig. 3). The highest temperatures (ⱖ39.5°C) were also observed at 34 to 35 dpi for all animals (data not shown). Thus, the clinical signs of acute infection observed in A. marginale-challenged animals at ⬃5 wpi were concurrent with the peak of functional exhaustion of OMspecific T cells. Demonstration of persistent infection. To verify that cattle

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lenged cattle. (A) Supernatants were harvested from proliferation assay mixtures on day 6 and pooled for each animal, and the IFN-␥ level was measured by an ELISA. Data for individual animals are indicated by symbols. Results are presented as the mean IFN-␥ levels in duplicate cultures of PBMCs stimulated with 1 ␮g/ml OMs at each time point. Bars indicate group mean responses, and asterisks indicate a significantly lower level of IFN-␥ production than IFN-␥ production at the preinfection (Pre-inf.) time point, where the P value was ⬍0.05. (B and C) IFN-␥ production by PBMCs cultured with RPMI 1640 medium only, 10% TCGF, uRBCs, and OMs at 6 dpi (B) and 34 dpi (C). Each circle represents the mean for duplicate wells for an individual, and bars indicate the group mean responses. *, P ⬍ 0.05; n.s., not significant.

were persistently infected with A. marginale and not completely protected by OM immunization, after T-cell experiments were completed, an msp5 nested PCR was performed on whole blood. The results clearly demonstrated the presence of msp5 DNA in all five cattle at 196 dpi (see Fig. S1 in the supplemental material). Increased percentages of PD-1ⴙ LAG-3ⴙ T cells in CD4ⴙ and CD8ⴙ T-cell populations in A. marginale-challenged cattle. Immunoinhibitory receptors such as PD-1 and LAG-3 play an immunomodulatory role in T-cell exhaustion during certain chronic infections in cattle (11, 13, 14). We hypothesized that rapid exhaustion of OM-specific T cells is associated with upregulation of PD-1 and LAG-3 on T cells during the acute phase of infection. To test this hypothesis, the cell surface expression of PD-1 and LAG-3 on CD4⫹ and CD8⫹ T cells was investigated by flow cytometric analysis of PBMCs isolated from A. marginale-challenged cattle before and after infection. As shown in Fig. 4A, CD3⫹ CD4⫹ T cells were gated in lymphocytes and then analyzed for the expression of PD-1 and LAG-3. Heavily exhausted T cells display the phenotype of PD-1⫹ LAG-3⫹ T cells (8). In infected animals, the mean percentages of PD-1⫹ LAG-3⫹ CD4⫹ T cells increased sig-

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FIG 3 Bacteremias and PCVs in cattle during acute anaplasmosis. Five cattle were challenged with 1.2 ⫻ 103 A. marginale-infected erythrocytes. Bacteremias and PCVs were assessed daily. Data for individual animals are indicated by symbols. The left axis shows bacteremia, reported as the percentage of infected erythrocytes determined by microscopic evaluation of blood smears. The right axis shows PCVs.

nificantly after 16 dpi and peaked at 34 dpi (Fig. 4B). No remarkable changes were observed for CD4⫹ T cells singly expressing either PD-1 or LAG-3 in these animals, except for a transient increase in the percentage of PD-1⫹ CD4⫹ T cells at 48 dpi (Fig. 4C and D). Likewise, CD3⫹ CD8⫹ T cells were gated in lymphocytes and then analyzed for the expression of PD-1 and LAG-3 (Fig. 5A). In the CD8⫹ T-cell population, the mean percentage of PD-1⫹ LAG-3⫹ CD8⫹ T cells was significantly increased at 34 to 48 dpi (Fig. 5B). No significant changes were observed for the population of PD-1⫹ LAG-3-negative (LAG-3⫺) CD8⫹ T cells (Fig. 5C), but the mean percentage of PD-1⫺ LAG-3⫹ CD8⫹ T cells was also significantly higher in the challenged animals at 34 to 48 dpi (Fig. 5D). However, these changes were not detected in the persistent phase of infection (125 dpi) (Fig. 4 and 5). These results suggest that the coexpression of PD-1 and LAG-3 on CD4⫹ and CD8⫹ T cells contributes to the rapid exhaustion of the T-cell response to A. marginale during acute infection.

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Upregulation of LAG-3 expression on ␥␦ T cells in A. marginale-challenged cattle. In previous studies, ␥␦ T cells proliferated in response to A. marginale and native MSP2 (20), and OMspecific T-cell proliferation was partially dependent on ␥␦ T cells in some A. marginale-infected cattle (18, 20). Because ␥␦ T cells may play a role in the antigen-specific immune response to A. marginale, the expression levels of PD-1 and LAG-3 on ␥␦ T cells were also analyzed in OM-immunized cattle following A. marginale infection. As shown in Fig. 6A, CD3⫹ ␥␦ TCR⫹ T cells were gated in lymphocytes and then analyzed for the expression of PD-1 and LAG-3. In the ␥␦ T-cell population, the mean percentage of PD-1⫹ LAG-3⫹ ␥␦ T cells increased significantly and peaked at 27 to 34 dpi (Fig. 6B). Interestingly, ␥␦ T cells from most cattle did not upregulate PD-1 in the absence of LAG-3 (Fig. 6C) but strongly upregulated the expression of LAG-3 in the absence of PD-1 (Fig. 6D) during acute infection. These results suggest that LAG-3 might be involved in the suppression of ␥␦ T cells during acute infection with A. marginale,

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FIG 4 Expression of PD-1 and LAG-3 on CD4⫹ T cells. (A) Gating strategy and representative dot plots for expression analyses of PD-1 and LAG-3 on CD3⫹

CD4⫹ T cells from peripheral blood of A. marginale-challenged cattle (n ⫽ 5). Values in the quadrants indicate percentages of cells. FSC, forward scatter; SSC, side scatter. (B to D) Percentages of PD-1⫹ LAG-3⫹ T cells (B), PD-1⫹ LAG-3⫺ T cells (C), and PD-1⫺ LAG-3⫹ T cells (D) in CD3⫹ CD4⫹ T cells in peripheral blood. Bars indicate group mean responses. Significantly higher mean percentages of cells expressing the marker(s) than those at preinfection time points (0 dpi), where the P value was ⬍0.05, are indicated by asterisks.

although suppression of ␥␦ T cells during anaplasmosis has not been determined. Upregulation of PD-L1 expression on CD14ⴙ APCs in A. marginale-challenged cattle. A. marginale invades and replicates within mature bovine erythrocytes but does not appear to replicate within cells that express MHC molecules in vivo (1, 21). Thus, A. marginale-infected erythrocytes are likely phagocytosed by macrophages and dendritic cells (DCs) for antigen to be presented to CD4⫹ T cells as exogenous antigens via the MHC-II pathway. The expression level of the immunoinhibitory ligand PD-L1 was examined on CD14⫹ APCs, which included macrophages and DCs, in A. marginale-challenged cattle. As shown in Fig. 7A, CD14⫹ cells were gated in blood monocytes and further analyzed

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for PD-L1 expression. Flow cytometric analysis revealed that the percentage of PD-L1⫹ CD14⫹ cells increased in the challenged animals at 20 to 34 dpi (Fig. 7B), just before and at the peak of T-cell exhaustion and clinical signs (Fig. 1 to 3). Expression of CD25 or CD95 on CD4ⴙ PD-1ⴙ T cells. We wished to determine whether PD-1 is expressed on activated T cells expressing CD25 or cells expressing the death receptor CD95 (Fas). Because MAbs specific for bovine Fas are not commercially available, we used an anti-human CD95 MAb that binds the same epitope as mouse MAb clone CH11 (IgM), which was shown previously to stain bovine corpus luteal cells by flow cytometry (22, 23). Results for four cattle showed that a higher percentage of cells expressed CD25 than CD95 for each animal; between 21 and 57%

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FIG 5 Expression of PD-1 and LAG-3 on CD8⫹ T cells. (A) Gating strategy and representative dot plots for expression analyses of PD-1 and LAG-3 on CD3⫹

CD8⫹ T cells from peripheral blood of A. marginale-challenged cattle (n ⫽ 5). Values in the quadrants indicate percentages of cells. (B to D) Percentages of PD-1⫹ LAG-3⫹ T cells (B), PD-1⫹ LAG-3⫺ T cells (C), and PD-1⫺ LAG-3⫹ T cells (D) in CD3⫹ CD8⫹ T cells in peripheral blood. Bars indicate group mean responses. Significantly higher mean percentages of cells expressing the marker(s) than those at preinfection time points (0 dpi), where the P value was ⬍0.05, are indicated by asterisks.

of CD4⫹ PD-1⫹ cells expressed CD25, whereas 8 to 50% of cells expressed the death receptor CD95 (Table 2). The percentage of cells expressing CD25 was significantly higher than that of cells expressing CD95, where the P value was ⬍0.05. Partial reactivation of OM-specific T-cell responses by blockade of the PD-1 and LAG-3 pathways. Because we observed increases in the percentage of T cells that expressed immunoinhibitory receptors and the percentage of APCs that expressed PD-L1 during acute infection, leading to the onset of CD4⫹ T-cell exhaustion, it was important to determine if blockade of these receptor-ligand interactions could restore T-cell function ex vivo. To address this, OM-specific PBMC proliferation and IFN-␥ production were assessed in the presence of blocking anti-PD-L1 and

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anti-LAG-3 MAbs by using PBMCs isolated from infected animals at 34 dpi, when T-cell exhaustion and clinical signs occurred. Although OM-specific PBMC proliferation was enhanced by antiLAG-3 MAb alone and in combination with anti-PD-L1 MAb, these differences were not significant (Fig. 8A). Additionally, OMspecific IFN-␥ production also tended to be reactivated in PBMCs treated with anti-PD-L1 MAb (mean of 13.1-fold versus control IgG) and anti-LAG-3 MAb (mean of 18.8-fold versus control IgG) (Fig. 8B). However, dual blockade using both anti-PD-L1 and anti-LAG-3 MAbs significantly increased IFN-␥ production in response to OM stimulation over that of control IgG (mean of 35.3fold versus control IgG) (Fig. 8B). Overall, these results indicate that OM-specific T-cell responses can be partially reactivated by

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FIG 6 Expression of PD-1 and LAG-3 on ␥␦ T cells. (A) Gating strategy and representative dot plots for expression analyses of PD-1 and LAG-3 on CD3⫹

␥␦ T cells from peripheral blood of A. marginale-challenged cattle (n ⫽ 5). Values in the quadrants indicate percentages of cells. (B to D) Percentages of PD-1⫹ LAG-3⫹ T cells (B), PD-1⫹ LAG-3⫺ T cells (C), and PD-1⫺ LAG-3⫹ T cells (D) in CD3⫹ ␥␦ T cells in peripheral blood. Bars indicate group mean responses. Significantly higher mean percentages of cells expressing the marker(s) than those at preinfection time points (0 dpi), where the P value was ⬍0.05, are indicated by asterisks.

the combined blockade of PD-1 and LAG-3 pathways in A. marginale-infected cattle. DISCUSSION

Pathogens that cause persistent infection or chronic disease have evolved mechanisms to evade both the innate and adaptive immune responses of their hosts (24). A. marginale has developed several mechanisms for evading host adaptive immunity (25). First, A. marginale undergoes extensive antigenic variation in immunodominant MSP2 and MSP3 (26, 27). Persistent infection by this rickettsia is established and maintained in large part through antigenic variation. Second, genes of the biosynthetic pathways for lipopolysaccharide (LPS) and peptidoglycan production are

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absent in A. marginale (28). These two pathogen-associated molecular patterns are known to be conserved in most Gram-negative bacteria and activate Toll-like receptors and nucleotide oligomerization domain receptors in host cells (29). Finally, a high antigen load during acute and persistent infections leads to a dramatic exhaustion of CD4⫹ T-cell responses specific for A. marginale immunogens, contributing to bacterial persistence (5, 6, 17, 18). However, the molecular mechanisms underlying the exhaustion of A. marginale-specific CD4⫹ T-cell responses have remained unclear. To elucidate the associated molecular mechanisms, we investigated whether PD-1/PD-L1 and LAG-3/MHC-II pathways mediate the development of T-cell exhaustion during acute infection of cattle with A. marginale.

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FIG 7 Expression of PD-L1 on CD14⫹ APCs. (A) Gating strategy and representative dot plots for expression analyses of PD-L1 on CD14⫹ monocytes from

peripheral blood of A. marginale-challenged cattle (n ⫽ 5). Values in the quadrants indicate percentages of cells. (B) Percentage of PD-L1⫹ cells in CD14⫹ APCs in peripheral blood. Bars indicate group mean responses. Significantly higher mean percentages of cells expressing PD-L1 than at preinfection time points (0 dpi), where the P value was ⬍0.05, are indicated by asterisks.

A previous study found that MSP1a-specific CD4⫹ T cells underwent progressive exhaustion, characterized by the loss of MSP1a T-cell epitope-specific proliferation and IFN-␥ secretion following infection, which led to the deletion of epitope-specific T cells monitored with MHC class II tetramers (6). Moreover, the exhaustion of immunization-induced MSP1a-specific CD4⫹ T cells after infection required the presence of the T-cell epitope on the infecting bacteria (7). In the present study, both an increase in the percentage of PD-1⫹ LAG-3⫹ CD4⫹ T cells and an exhaustion of OM-specific PBMC responses were observed in A. marginalechallenged animals at 5 wpi. Furthermore, these changes were concurrent with the peak of bacteremia and anemia in these animals and independent of the level of bacteremia in individual cattle. We have similarly observed that there is a loss of response in OM vaccinates following challenge regardless of whether we stimulated the T cells ex vivo with 0.2 or 1 ␮g/ml antigen (this study) or 10 ␮g/ml antigen (5, 7, 17). When stimulations with 1 and 10 ␮g/ml antigen were compared, there was little difference in the response or lack thereof following challenge (6). Because A. marginale-specific proliferation of PBMCs is CD4 TABLE 2 Frequencies of CD4⫹ PD-1⫹ cells that express CD25 or CD95 Frequency (%) of CD4⫹ PD-1⫹ T cells that expressa: Animal

CD25

CD95

48406 48411 48422 48453

31 29 21 57

8 27 9 50

a PBMCs were obtained at day 34 postinfection and cryopreserved. Thawed cells were used for flow cytometric analysis.

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mediated (3, 6, 18, 19), it is likely that the coexpression of PD-1 and LAG-3 on CD4⫹ T cells partially mediates the rapid exhaustion and apoptosis of these T cells and facilitates bacterial persistence during acute infection. In fact, 8 to 50% of CD4⫹ PD1⫹ T cells from the day 34 time point expressed the death receptor Fas, suggesting that these cells could be on the way to undergoing apoptosis. The expression of Fas and PD-1 on T cells has been shown to correlate with increased T-cell apoptosis (30, 31). However, approximately one-third to one-half of the CD4 T cells expressing PD-1 at the peak of A. marginale infection also expressed CD25, the IL-2 receptor ␣ chain and a marker associated with differentiated effector cells (32). This indicates that these cells are effector T cells and could be rescued by antibody blockade. Unexpectedly, the percentage of PD-1⫹ LAG-3⫹ CD8⫹ T cells also increased and peaked in three animals (48406, 48411, and 48453) at 5 to 6 wpi. Although T-cell subset depletion studies showed that CD8⫹ T cells do not respond to A. marginale antigen following immunization or infection (6, 18, 19), CD8⫹ T cells expressed an exhausted phenotype following infection in this study. However, the antigen specificity and effector function of these PD-1⫹ LAG-3⫹ CD8⫹ T cells in infected animals require clarification. Cattle have a large number of ␥␦ T cells in their T-cell population, comprising up to 80% of circulating T cells in young calves and 10% to 20% of circulating T cells in adult cattle (28, 29). Bovine ␥␦ T cells have cytolytic and cytokine secretory properties similar to those of ␣␤ T cells but also have the potential to recruit cells via chemokines and perform functionally as professional APCs, thereby playing dual roles in innate and adaptive immunity (33, 34). In addition, human ␥␦ T cells have been shown to express PD-1 following antigenic stimulation (35). Human PD-1⫹ ␥␦ T

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FIG 8 Reactivation of OM-specific T-cell responses by dual blockade of the PD-1 and LAG-3 pathways. PBMCs were isolated from the blood of A. marginale-challenged cattle at 34 dpi and cultured in triplicate for 7 days with 0.2 ␮g/ml OM antigen and no antibody or 10 ␮g/ml of one or two blocking antibodies, including anti-PD-L1 MAb, anti-LAG-3 MAb, and control rat IgG, to reach a total concentration of 20 ␮g/ml IgG in the presence of OM antigen. Medium and uRBC antigen were included as negative controls. (A) PBMC proliferation presented as ⌬cpm values for each animal after subtracting the mean counts per minute for cells cultured with uRBCs from the mean counts per minute for cells cultured with OMs. (B) Supernatants were harvested from the proliferation assay mixture on day 6 and pooled, and IFN-␥ production for each animal was measured by an ELISA in duplicate. Bars indicate group mean responses. Asterisks indicate significant differences between control IgG and anti-LAG-3 MAb plus anti-PD-L1 MAb, where the P value was ⬍0.05.

cells but not PD-1⫺ ␥␦ T cells have decreased IFN-␥ production and cytolytic function (35). Our study demonstrated a dramatic upregulation of LAG-3 on ␥␦ T cells in peripheral blood of challenged animals at 3 to 9 wpi. LAG-3 expression is normally upregulated on ␣␤ T cells by long-term TCR and Th1 cytokine stimulation (10). In the present study, LAG-3⫹ ␥␦ T cells may be induced by massive antigenic stimulation and IFN-␥ production during the first few weeks of acute infection. Although the immu-

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nomodulatory mechanisms via the LAG-3 pathway in ␥␦ T cells are still unclear, effector functions of LAG-3⫹ ␥␦ T cells may be impaired compared to those of LAG-3⫺ ␥␦ T cells, but this awaits further experimentation. In this study, the percentages of PD-1⫹ LAG-3⫹ T cells increased during acute infection but were not consistently increased in the persistent phase of infection, although OM-specific responses remained suppressed at 125 dpi. This observation suggests that other inhibitory receptors may be playing a role the maintenance of T-cell exhaustion during persistent infection. Further studies are needed to investigate other immunoinhibitory receptors such as TIM-3 (36) and CTLA-4 (37). Another explanation for this result is that the population of OM-specific T cells is very small, such that changes in PD-1 and LAG-3 expression are undetectable in total T-cell subpopulations during persistent infection. Support for this was shown in our studies using MSP1aspecific MHC class II tetramer staining, which revealed rapid deletion, to preimmunization levels, of epitope-specific CD4⫹ T cells in peripheral blood at the peak of bacteremia following infection (6). Studies incorporating MHC class II tetramer analysis in A. marginale-challenged cattle will be important in the future to investigate PD-1 and LAG-3 expression levels in A. marginalespecific CD4⫹ T cells (6, 38). PD-L1 is expressed by many cell types but not by erythrocytes (39). In A. marginale-infected animals, CD14⫹ APCs strongly express PD-L1, enabling interaction with PD-1⫹ T cells. PD-L1 expression on CD14⫹ APCs was upregulated during the early phase of acute infection (at 20 to 34 dpi). Expression of PD-L1 on murine APCs is induced by cytokine signaling, such as IFN-␥, IFN␣/␤, IL-4, and granulocyte-macrophage colony-stimulating factor signaling, or Toll-like receptor signaling by LPS (9, 40). Because A. marginale lacks LPS, cytokine stimulation is likely to be important for upregulating PD-L1 during acute infection. It was shown previously that IFN-␥ produced by HIV type 1-specific CD4⫹ T cells in response to viral antigen induced PD-L1 upregulation on monocytes (41). Thus, PD-L1 upregulation is potentially induced by IFN-␥ produced by A. marginale-specific CD4⫹ T cells in response to bacterial growth during the acute phase of infection before the CD4⫹ T-cell response is exhausted. IFN-␥ produced by CD4⫹ Th1 cells may play an important role in activating macrophages to produce bactericidal molecules, such as nitric oxide, that help control acute bacteremia (1, 2). Therefore, the enhancement of IFN-␥ production by a blockade strategy might decrease bacteremia and clinical signs during the acute phase of infection. The ligand for LAG-3, MHC class II, was not examined in this study because previous work from our group determined that 90 to 100% of CD14⫹ CD11b⫹ APCs expressed MHC class II molecules when cells were obtained from cattle that were healthy or infected with Mycobacterium avium subsp. paratuberculosis (14). Furthermore, we reported previously that at the peak of bacteremia after A. marginale infection, APCs are fully functional for antigen presentation, suggesting that there are sufficient MHC class II molecules to present antigen to responsive CD4 T cells (5) and presumably to interact with LAG-3. In vitro blockade of the PD-1/PD-L1 and LAG-3/MHC-II interactions confirmed an effect of the blocking MAbs on increasing proliferation as well as IFN-␥ production, but the effect on proliferation was not statistically significant. The reasons for this discrepancy are not known. However, the lack of significant increases

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in proliferation during blockade may be due to the large variation between animals. For all but one animal (animal 48453), the proliferative response to OM antigen increased in the presence of anti-LAG-3 MAb or anti-LAG-3 and anti-PD-L1 MAbs. It is also possible that there are too few antigen-specific T cells remaining in some cattle to measure a proliferative response, even in the presence of blocking antibodies, as proliferation is less sensitive than the IFN-␥ ELISA. This is supported by the dramatic deletion of tetramer-staining antigen-specific CD4 T cells following infection (6). Additionally, there is a well-described hierarchy in the loss of different effector functions of T cells during exhaustion due to chronic infection, where IL-2 production is lost first, while IFN-␥ secretion is more resistant to exhaustion and is lost later (8). Thus, restoration of these functions following blockade may also behave in a differential manner, which could explain the significant restoration of IFN-␥ but not proliferative responses. In another study with OM-vaccinated cattle that developed an exhausted T-cell response following infection, we were unable to detect IL-2 in supernatants of antigen-stimulated T cells before and after infection was cleared with antibiotic therapy, even though proliferation and IFN-␥ and tumor necrosis factor alpha (TNF-␣) production were partially but significantly restored upon the clearance of bacteremia (17). Thus, we did not attempt to measure IL-2 levels in the present study. IL-10 is an inhibitory cytokine that is known to play a role in the exhaustion of antigen-specific T-cell responses during chronic infection (8). IL-10 was not examined in the present study, but it was examined in a previous study where MSP2-immunized cattle were infected with Anaplasma and the MSP2-specific response became exhausted. In that study, nonresponding PBMCs obtained at the peak of infection and stimulated ex vivo with MSP2 antigen produced significantly less IL-10 than did responding PBMCs obtained preinfection (5). This finding suggests that IL-10 production in response to antigen stimulation during the exhausted response did not explain the inability of CD4 T cells to proliferate. In summary, we provide evidence that the coexpression of PD-1 and LAG-3 contributes to the induction of functional exhaustion of A. marginale-specific CD4⫹ T cells during acute infection with this pathogen. In vitro blockade of PD-1 and LAG-3 pathways using specific antibodies partially restores OM-specific T-cell responses. In a mouse model of Plasmodium infection, in vivo blockade of PD-1/PD-L1 and LAG-3/MHC class II pathways improved the clinical outcome of malaria by enhancing CD4⫹ T-cell function (42). Additional in vivo studies could determine antibacterial effects of blocking the inhibitory pathways by antibody treatment in cattle infected with A. marginale. This rapid CD4⫹ T-cell exhaustion is a unique feature of A. marginale infection among cattle and can be an effective model for further investigation of bovine immunoinhibitory receptors and T-cell exhaustion. ACKNOWLEDGMENTS We thank Emma Karel, Ralph Horn, James Allison, and Sara Davis for excellent animal handling and technical assistance. This research was supported by NIH NIAID grant AI053692 (to W.C.B.), USDA-NIFA grant 2010-65119-20456 (to W.C.B.), USDA ARS CRIS project number 5348-32000-033-00D (to G.A.S. and M.W.U.), JSPS KAKENHI grant number 25257415 (to S.K.), and a grant from the Science

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and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry, Japan (grant 26058B to S.K.).

FUNDING INFORMATION This work, including the efforts of Wendy C. Brown, was funded by United States Department of Agriculture-NIFA (2010-65119-20456). This work, including the efforts of Massaro W. Ueti, was funded by USDA-ARS CRIS (5348-32000-033-00D). This work, including the efforts of Glen A. Scoles, was funded by USDA-ARS-CRIS (5348-32000033-00D). This work, including the efforts of Satoru Konnai, was funded by JSPS KAKENHI (25257415). This work, including the efforts of Satoru Konnai, was funded by Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry, Japan (26058B). This work, including the efforts of Wendy C. Brown, was funded by HHS | National Institutes of Health (NIH) (AI053692).

REFERENCES 1. Brown WC. 2012. Adaptive immunity to Anaplasma pathogens and immune dysregulation: implications for bacterial persistence. Comp Immunol Microbiol Infect Dis 35:241–252. http://dx.doi.org/10.1016/j.cimid .2011.12.002. 2. Palmer GH, Rurangirwa FR, Kocan KM, Brown WC. 1999. Molecular basis for vaccine development against the ehrlichial pathogen Anaplasma marginale. Parasitol Today 15:281–286. http://dx.doi.org/10.1016/S0169 -4758(99)01469-6. 3. Brown WC, Shkap V, Zhu D, McGuire TC, Tuo W, McElwain TF, Palmer GH. 1998. CD4⫹ T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge. Infect Immun 66:5406 – 5413. 4. Brown WC, Zhu D, Shkap V, McGuire TC, Blouin EF, Kocan KM, Palmer GH. 1998. The repertoire of Anaplasma marginale antigens recognized by CD4⫹ T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3. Infect Immun 66:5414 –5422. 5. Abbott JR, Palmer GH, Kegerreis KA, Hetrick PF, Howard CJ, Hope JC, Brown WC. 2005. Rapid and long-term disappearance of CD4⫹ T lymphocyte responses specific for Anaplasma marginale major surface protein-2 (MSP2) in MSP2 vaccinates following challenge with live A. marginale. J Immunol 174:6702– 6715. http://dx.doi.org/10.4049/jimmunol .174.11.6702. 6. Han S, Norimine J, Palmer GH, Mwangi W, Lahmers KK, Brown WC. 2008. Rapid deletion of antigen-specific CD4⫹ T cells following infection represents a strategy of immune evasion and persistence for Anaplasma marginale. J Immunol 181:7759 –7769. http://dx.doi.org/10 .4049/jimmunol.181.11.7759. 7. Brown WC, Turse JE, Lawrence PK, Johnson WC, Scoles GA, Deringer JR, Sutten EL, Han S, Norimine J. 2015. Loss of immunization-induced epitope-specific CD4⫹ T cell response following Anaplasma marginale infection requires the presence of the T-cell epitope on the pathogen but is not associated with an increase in lymphocytes expressing known regulatory cell phenotypes. Clin Vaccine Immunol 22:742–753. http://dx.doi .org/10.1128/CVI.00168-15. 8. Wherry EJ. 2011. T cell exhaustion. Nat Immunol 12:492– 499. 9. Keir ME, Francisco LM, Sharpe AH. 2007. PD-1 and its ligands in T-cell immunity. Curr Opin Immunol 19:309 –314. http://dx.doi.org/10.1016/j .coi.2007.04.012. 10. Sierro S, Romero P, Speiser DE. 2011. The CD4-like molecule LAG-3, biology and therapeutic applications. Expert Opin Ther Targets 15:91– 101. http://dx.doi.org/10.1517/14712598.2011.540563. 11. Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. 2013. Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro. Vet Res 44:59. http://dx.doi.org/10.1186/1297-9716-44-59. 12. Shirai T, Konnai S, Ikebuchi R, Okagawa T, Suzuki S, Sunden Y, Onuma M, Murata S, Ohashi K. 2011. Molecular cloning of bovine lymphocyte activation gene-3 and its expression characteristics in bovine leukemia virus-infected cattle. Vet Immunol Immunopathol 144:462– 467. http://dx.doi.org/10.1016/j.vetimm.2011.08.018. 13. Konnai S, Suzuki S, Shirai T, Ikebuchi R, Okagawa T, Sunden Y, Mingala CN, Onuma M, Murata S, Ohashi K. 2013. Enhanced expres-

Infection and Immunity

iai.asm.org

2789

Okagawa et al.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24. 25.

26.

27.

sion of LAG-3 on lymphocyte subpopulations from persistently lymphocytotic cattle infected with bovine leukemia virus. Comp Immunol Microbiol Infect Dis 36:63– 69. http://dx.doi.org/10.1016/j.cimid.2012.09.005. Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R, Kawaji S, Tanaka S, Kagawa Y, Murata S, Mori Y, Ohashi K. 2016. Bovine immunoinhibitory receptors contribute to the suppression of Mycobacterium avium subsp. paratuberculosis-specific T-cell responses. Infect Immun 84:77– 89. http://dx.doi.org/10.1128/IAI.01014-15. Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K. 2011. Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1 blockade. Vet Res 42:103. http://dx.doi.org/10.1186/1297-9716-42 -103. Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. 2014. Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytes. Immunology 142: 551–561. http://dx.doi.org/10.1111/imm.12243. Turse JE, Scoles GA, Deringer JR, Fry LM, Brown WC. 2014. Immunization-induced Anaplasma marginale-specific T-lymphocyte responses impaired by A. marginale infection are restored after eliminating infection with tetracycline. Clin Vaccine Immunol 21:1369 –1375. http://dx.doi.org /10.1128/CVI.00246-14. Han S, Norimine J, Brayton KA, Palmer GH, Scoles GA, Brown WC. 2010. Anaplasma marginale infection with persistent high-load bacteremia induces a dysfunctional memory CD4⫹ T lymphocyte response but sustained high IgG titers. Clin Vaccine Immunol 17:1881–1890. http://dx .doi.org/10.1128/CVI.00257-10. Abbott JR, Palmer GH, Howard CJ, Hope JC, Brown WC. 2004. Anaplasma marginale major surface protein 2 CD4⫹-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR. Infect Immun 72:7360 –7366. http://dx.doi.org/10.1128/IAI.72.12.7360 -7366.2004. Lahmers KK, Norimine J, Abrahamsen MS, Palmer GH, Brown WC. 2005. The CD4⫹ T cell immunodominant Anaplasma marginale major surface protein 2 stimulates ␥␦ T cell clones that express unique T cell receptors. J Leukoc Biol 77:199 –208. http://dx.doi.org/10.1189/jlb .0804482. Wamsley HL, Alleman AR, Johnson CM, Barbet AF, Abbott JR. 2011. Investigation of endothelial cells as an in vivo nidus of Anaplasma marginale infection in cattle. Vet Microbiol 153:264 –273. http://dx.doi.org/10 .1016/j.vetmic.2011.05.035. Duncan A, Forcina J, Birt A, Townson D. 2012. Estrous cycle-dependent changes of Fas expression in the corpus luteum: influence of keratin 8/18 intermediate filaments and cytokines. Reprod Biol Endocrinol 10:90. http: //dx.doi.org/10.1186/1477-7827-10-90. Fadeel B, Thorpe CJ, Yonehara S, Chiodi F. 1997. Anti-Fas IgG1 antibodies recognizing the same epitope of Fas/APO-1 mediate different biological effects in vitro. Int Immunol 9:201–209. http://dx.doi.org/10.1093 /intimm/9.2.201. Finlay BB, McFadden G. 2006. Anti-immunology: evasion of the host immune system by bacterial and viral pathogens. Cell 124:767–782. http: //dx.doi.org/10.1016/j.cell.2006.01.034. Brown WC, Barbet AF. 2016. Persistent infections and immunity in ruminants to arthropod-borne bacteria in the family Anaplasmataceae. Annu Rev Anim Biosci 4:177–197. http://dx.doi.org/10.1146/annurev -animal-022513-114206. Meeus PFM, Brayton KA, Palmer GH, Barbet AF. 2003. Conservation of a gene conversion mechanism in two distantly related paralogues of Anaplasma marginale. Mol Microbiol 47:633– 643. http://dx.doi.org/10.1046 /j.1365-2958.2003.03331.x. Brayton KA, Meeus PFM, Barbet AF, Palmer GH. 2003. Simultaneous variation of the immunodominant outer membrane proteins, MSP2 and

2790

iai.asm.org

28.

29. 30.

31.

32. 33.

34.

35.

36.

37.

38.

39. 40.

41.

42.

MSP3, during Anaplasma marginale persistence in vivo. Infect Immun 71:6627– 6632. http://dx.doi.org/10.1128/IAI.71.11.6627-6632.2003. Brayton KA, Kappmeyer LS, Herndon DR, Dark MJ, Tibbals DL, Palmer GH, McGuire TC, Knowles DP. 2005. Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. Proc Natl Acad Sci U S A 102:844 – 849. http://dx.doi.org/10.1073/pnas.0406656102. Ulevitch RJ, Mathison JC, da Silva Correia J. 2004. Innate immune responses during infection. Vaccine 22(Suppl 1):S25–S30. http://dx.doi .org/10.1016/j.vaccine.2004.08.013. Petrovas C, Chaon B, Ambrozak DR, Price DA, Melenhorst JJ, Hill BJ, Geldmacher C, Casazza JP, Chattopadhyay PK, Roederer M, Douek DC, Mueller JP, Jacobson JM, Kulkarni V, Felber BK, Pavlakis GN, Katsikis PD, Koup RA. 2009. Differential association of programmed death-1 and CD57 with ex vivo survival of CD8⫹ T cells in HIV infection. J Immunol 183:1120 – 1132. http://dx.doi.org/10.4049/jimmunol.0900182. Badr G, Sayed D, Alhassa IM, Elsayh KI, Ahmed EA, Alwasel SH. 2011. T lymphocytes from malnourished infants are short-lived and dysfunctional cells. Immunobiology 216:309 –315. http://dx.doi.org/10.1016/j .imbio.2010.07.007. Malek TR, Castro I. 2010. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 33:153–165. http://dx .doi.org/10.1016/j.immuni.2010.08.004. Guzman E, Price S, Poulsom H, Hope J. 2012. Bovine ␥␦ T cells: cells with multiple functions and important roles in immunity. Vet Immunol Immunopathol 148:161–167. http://dx.doi.org/10.1016/j.vetimm.2011 .03.013. Baldwin CL, Telfer JC. 2015. The bovine model for elucidating the role of ␥␦ T cells in controlling infectious diseases of importance to cattle and humans. Mol Immunol 66:35– 47. http://dx.doi.org/10.1016/j.molimm .2014.10.024. Iwasaki M, Tanaka Y, Kobayashi H, Murata-Hirai K, Miyabe H, Sugie T, Toi M, Minato N. 2011. Expression and function of PD-1 in human ␥␦ T cells that recognize phosphoantigens. Eur J Immunol 41:345–355. http: //dx.doi.org/10.1002/eji.201040959. Okagawa T, Konnai S, Ikebuchi R, Suzuki S, Shirai T, Sunden Y, Onuma M, Murata S, Ohashi K. 2012. Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection. Vet Res 43:45. http://dx.doi.org/10.1186/1297-9716-43-45. Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Nishimori A, Kohara J, Mingala CN, Murata S, Ohashi K. 2015. Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection. Vet Immunol Immunopathol 163:115–124. http://dx.doi.org/10 .1016/j.vetimm.2014.10.006. Norimine J, Han S, Brown WC. 2006. Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4⫹ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers. Immunogenetics 58:726 –739. http://dx.doi.org/10.1007/s00251-006-0140-3. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. 2008. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 26:677–704. http://dx.doi .org/10.1146/annurev.immunol.26.021607.090331. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, Tanno Y, Shin T, Tsuchiya H, Pardoll DM, Okumura K, Azuma M, Yagita H. 2002. Expression of programmed death 1 ligands by murine T cells and APC. J Immunol 169:5538 –5545. http://dx.doi.org/10.4049/jimmunol.169.10.5538. Porichis F, Hart MG, Zupkosky J, Barblu L, Kwon DS, McMullen A, Brennan T, Ahmed R, Freeman GJ, Kavanagh DG, Kaufmann DE, Doms RW. 2014. Differential impact of PD-1 and/or interleukin-10 blockade on HIV-1-specific CD4 T cell and antigen-presenting cell functions. J Virol 88:2508 –2518. http://dx.doi.org/10.1128/JVI.02034-13. Butler NS, Moebius J, Pewe LL, Traore B, Doumbo OK, Tygrett LT, Waldschmidt TJ, Crompton PD, Harty JT. 2012. Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection. Nat Immunol 13:188 –195. http://dx.doi.org/10.1038/ni.2180.

Infection and Immunity

October 2016 Volume 84 Number 10

Cooperation of PD-1 and LAG-3 Contributes to T-Cell Exhaustion in Anaplasma marginale-Infected Cattle.

The CD4(+) T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhausti...
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