* Malaria Program, Naval Medical Research Center, Silver Spring, Maryland;
Henry M. Jackson Foundation, Rockville, Maryland; and
Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland
Correspondence: Dr. Denise L. Doolan, Malaria Program, Naval Medical Research Center, 503 Robert Grant Avenue, Lab 3W16, Silver Spring, MD 20910. E-mail: dooland{at}nmrc.navy.mil
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(IFN-) CD8+ T cell responses by enzyme-linked immunospot assays, and by indirectly evaluating induction of the chemokine monokine induced by IFN- as a marker for IFN--mediated effector function. Our results demonstrate that bystander cell participation, mediated by CD4+ T cell and natural killer (NK) cells, is required for optimal induction of antigen-specific CD8+ T cell effector responses. Our data further establish a novel role for NK cells in the activation of antigen-specific immune responses.
Key Words: chemokines • cytokines • FACS • T lymphocytes • bystander activation
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CD8+ T cell effector responses are characterized by lysis of infected cells through perforin/granzyme- or Fas/FasL-mediated pathways or by the induction of cytokines such as interferon- (IFN-), which has been implicated with an important role in mediating protection against a number of pathogens, including viruses, bacteria, and parasites [11
12
13
14
]. It is thought that IFN- mediates its protective effects by orchestrating a wide range of immunological programs, including induction of genes involved in antigen processing, up-regulation of major histocompatibility complex class I and class II expression, induction of oxygen and nitrogen radicals, and stimulation of chemokine production in vitro and in vivo [15
]. Accordingly, in many systems, detection of IFN- or IFN--producing cells following exposure to antigen is frequently used as a marker for functional effector cell activity.
Herein, we have evaluated the requirement for bystander participation for optimal induction of CD8+ T cell effector responses using a panel of synthetic peptides representing well-defined CD8+ T cell epitopes derived from influenza (FLU), cytomeglavirus (CMV), Epstein Barr Virus (EBV), HIV, and hepatitis B virus (HBV) antigens. Specifically, we focused on IFN- and evaluated antigen-specific CD8+ T cell responses by IFN- enzyme-linked immunospot (ELISPOT) assays by evaluating the biological effects of IFN- production. For the latter, the induction of the chemokine monokine induced by IFN- (MIG) was used as a functional assay for antigen-specific CD8+ T cell-dependent and IFN--mediated responses. These assays allowed us to delineate the requirement for bystander cell participation at the level of induction of CD8+ T cell effector response (IFN- production) and to assess a role for bystander participation for optimal induction of antigen-specific IFN--mediated immune responses (induction of MIG expression).
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(clone B27) and interleukin (IL)-12 (clone C8.6), antibodies to CD122 (clone Mik-2) and CD40 (clone vCD40.4), and control mAb (clone MOPC-21) were purchased from PharMingen (San Diego, CA) and were used at 20 µg/ml. Human MIG and mouse MIG-specific goat immunoglobulin G were purchased from R&D Systems (Minneapolis, MN) and were used at 10 µg/ml. Antibodies were added to PBMC at the initiation of culture and were maintained throughout the experiment.
Synthetic peptides
Synthetic 9-mer or 10-mer peptides representing well-characterized HLA-A*0201-restricted epitopes from the FLU virus matrix protein [16
], CMV phosphoprotein [17
, 18
], HIV gag protein [16
], HBV core antigen [19
], and the HLA-A*03-restricted epitope to the FLU virus nucleoprotein [20
] and HLA-B*08-restricted epitope from the EBV nuclear antigen 3 were purchased from Chiron Corporation (Clayton, VIC, Australia) or Research Genetics (Huntsville, AL) and were purified to >95%. Peptide sequences are detailed in Table 1
.
|
View this table: [in a new window] |
Table 1. Synthetic Peptides
|
ELISPOT assay-producing cells was determined by ELISPOT assay, as described elsewhere [21
]. In brief, sterile 96-well multiScreen-IP MAIP plates (Millipore, Bedford, MA) were coated overnight at 4°C with 50 µl phosphate-buffered saline (PBS) containing 10 µg/ml anti-IFN- mAb (clone 1-D1K; Mabtech, Stockholm, Sweden). Wells were washed six times with RPMI 1640 and blocked for 1 h at room temperature (RT) with 100 µl RPMI 1640 supplemented with 10% FCS. Then, 100 µl input PBMC (5x105–2.5x105 PBMC) was aliquoted together with 100 µl media alone or with test or control peptides at a final concentration of 10 µg/ml. Cultures were incubated for 36 h at 37°C in an atmosphere of 5% CO2. Wells were then washed six times with PBS/0.05% Tween 20 (Sigma Chemical Co.) and incubated for 3 h at RT with 100 µl 1 µg/ml biotinylated anti-IFN- mAb (clone 7B6–1; Mabtech). Wells were again washed six times with PBS/0.05% Tween 20 and incubated for 1 h at RT with 100 µl 1:1000 dilution of streptavidin alkaline phosphatase (Mabtech). Wells were washed six times with PBS/0.05% Tween 20 and three times with PBS and then developed with 100 µl 1:25 diluted alkaline phosphatase substrate (BioRad, Hercules, CA). The colorimetric reaction was stopped after 15 min by extensive washing in tap water, and plates were air-dried. The number of spots corresponding to IFN--producing cells in wells (IFN- spot-forming cells; SFCs) was enumerated with the Zeiss KS ELISPOT system (Carl Zeiss Inc., Thornwood, NY). All assays were performed in triplicate or quadruplicate. Responses were expressed as the number of IFN--secreting cells (SFCs) per 106 PBMC.
Intracellular cytokine staining for MIG
Intracellular cytokine detection of MIG expression was determined as described previously [22
]. Briefly, PBMC (5x105–2.5x105) were cultured in a total volume of 200 µl complete medium in a 96-well round-bottom plate at 37°C in an atmosphere of 5% CO2. Synthetic peptides were added a final concentration of 10 µg/ml. Cytokine secretion inhibitors such as brefeldin-A (BFA) or monesin were not added to cultures unless otherwise noted. Following overnight culture (16 h), PBMC were washed once in cold Dulbecco’s PBS and stained with mAb to CD14 (Becton Dickinson, San Jose, CA). PBMC were permeabilized with Cytofix/Cytoperm (PharMingen) and stained intracellularly with PE-conjugated mAb to MIG or IFN- (0.4 µg antibody/106 cells; PharMingen). Samples were acquired on a Becton Dickinson FACSCALIBUR. For each analysis, at least 25,000 events were acquired, and cells within the monocyte/macrophage population were gated based on forward-scatter and side-scatter characteristics. Gated cells were analyzed, and the frequency of CD14-positive cell expression of MIG was determined using the Cellquest software (Becton Dickinson).
Whole blood stimulation and staining
Whole blood stimulation protocols were performed essentially as described elsewhere [23
]. Heparinized blood (1 ml; fresh, collected within 24 h before use) was added to 15 ml round-bottom polypropylene tubes, and 20 µg/ml peptide was added to each tube and stimulated for 6 h. In some experiments, BFA (10 µg/ml) was added at the initiation of culture or during the last 3 h of stimulation. In some instances, neutralizing antibodies to IFN- were added at the initiation of culture, before addition of BFA at 3 h. Following 6 h of stimulation, cultures were treated with 2 mM EDTA for 15 min at RT before lysing red blood cells and fixing samples using BDBioscience’s (San Jose, CA) FACS lysing solution, according to the manufacturer’s instructions. Samples were permeabilized for 30 min at RT using BDBioscience’s permeabilization solution. At this point, each tube was split, one aliquot was stained with antibodies to CD8 and IFN-, and the other aliquot was stained with antibodies to CD14 and MIG, and evaluated by flow cytometry.
Statistical analysis
Unless otherwise stated, all assays were performed in triplicate or quadruplicate. The significance of group differences for the MIG and ELISPOT assays was calculated using the Student’s t-test, two-tailed (Microsoft Excel Version 8.0, Microsoft Corp., Redmond, WA). Responses were considered positive if the response to test peptide (FLU, CMV, EBV) was significantly different (P<0.05) as compared with the response to negative-control peptides (HIV or HBV) and if the stimulation index (SI=response with test peptide/response with control peptide) was greater than 2.0.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
ELISPOT, we also evaluated the biological effects of IFN- production by detecting antigen-specific, IFN--mediated induction of MIG expression. Previous studies from our laboratory have established that induction of MIG expression by CD14+ monocytes/macrophages serves as a sensitive and predictive measure of IFN- production from antigen-specific CD8+ T cells [22
]. This assay provides an indirect measure of IFN- production from antigen-specific cells following biological amplification of IFN- responses. Representative flow cytometry data demonstrating antigen-specific induction of IFN- and MIG in antigen-stimulated cultures are shown in Figure 1
. Antigen-specific induction of MIG expression was dependent on IFN- production and secretion from activated CD8+ T cells; addition of specific inhibitors of cytokine secretion, such as BFA at the initiation of culture-inhibited induction of MIG expression but not IFN- production and induction of MIG, was abrogated by treatment with neutralizing antibodies to IFN-. Depletion of CD8+ T cells inhibited antigen-specific induction of IFN- and MIG.
 View larger version (33K): [in a new window] |
Figure 1. Antigen-specific induction of IFN- and MIG. Samples of whole blood or whole blood depleted of CD8+ T cells were incubated for 6 h with HIV or CMV peptide (20 µg/ml). BFA (10 µg/ml) was added at the initiation of culture (CMV+BFA) or during the last 3 h of incubation (HIV/BFA or CMV/BFA). In some cultures, neutralizing antibodies to IFN- (20 µg/ml) were added at the initiation of culture ( IFN-). Cells were then processed as described in Materials and Methods and stained with antibodies to (A) CD8 and IFN- or (B) CD14 and MIG and were analyzed by flow cytometry. Numbers represent the mean of duplicate staining wells and indicate the frequency of CD8+ T cells or CD14+ monocytes/macrophages staining positive for IFN- or MIG, respectively. Similar results were obtained using PBMC from different volunteers.
|
 View larger version (19K): [in a new window] |
Figure 2. Requirement for bystander participation for induction of MIG. PBMC or PBMC depleted of CD8+ T cells (CD8-), CD4+ T cells (CD4-), or NK cells (NK-) were incubated with 20 µg/ml HIV or FLU peptide (A) or were cultured with autologous APC pulsed with HIV or FLU peptides (B). Following 16 h of culture, induction of MIG expression was evaluated as described in Materials and Methods. Representative data of repeated experiments using PBMC from same and different volunteers.
|
responses, we wanted to determine if bystander participation of CD4+ T cells and NK cells was required for induction of antigen-specific IFN- production using peptide-pulsed APC. As shown in Figure 3
, depletion of CD4+ T cells or NK cells also decreased the number of IFN- SFCs in cultures stimulated with peptide-pulsed APC, concomitant with a decreased induction of MIG expression in parallel cultures. We consistently observed a requirement for CD4+ T cells and NK cells for optimal induction of CD8+ T cell responses to the FLU, CMV, or EBV peptides in each of the eight volunteers used in these studies.
 View larger version (19K): [in a new window] |
Figure 3. Requirement for bystander participation for antigen-specific CD8+ T cell responses. Parallel cultures of PBMC or PBMC depleted of CD8+ T cells, CD4+ T cells, or NK cells from two volunteers (vol. #5, A and B; vol. #6, C and D) were cultured with peptide-pulsed APC for 16 h and were evaluated for IFN- SFCs by ELISPOT (A and C) or MIG expression (B and D). Similar results were obtained in a separate experiment using PBMC from a different volunteer.
|
 View larger version (21K): [in a new window] |
Figure 4. Pathways for bystander participation in CD8+ T cell responses. PBMC from two volunteers (vol. #4, A; vol. #8, B) were stimulated with EBV, FLU, or HIV peptides and were treated with neutralizing antibodies to IL-2 or IL-12 or blocking antibodies to CD40 or CD122 (all used at 20 µg/ml). After 16 h of incubation, cells were evaluated for MIG expression. Numbers in parentheses indicate percent inhibition of MIG expression compared with untreated cultures. Similar results were obtained using PBMC from same and different volunteers.
|
-mediated effector function. We hypothesized that as MIG is a chemokine known to selectively attract activated lymphocytes [24
], MIG may contribute to antigen-specific immune responses by specifically recruiting cells to participate in antigen-specific responses. To evaluate this, PBMC from volunteers known to respond to a given peptide were cultured with neutralizing antibodies to MIG and evaluated for MIG expression. As shown in Figure 5
, addition of neutralizing antibodies to human MIG (hMIG) significantly inhibited antigen-specific induction of MIG expression (44% in vol. #4, and 90% in vol. #8). However, MIG expression was not inhibited in cultures treated with antibodies to murine MIG (mMIG), which served as an isotype control. Preincubation of cultures with neutralizing antibodies to hMIG immediately before staining or following fixation and permeabilization did not decrease MIG staining (data not shown), indicating that neutralizing antibodies did not interfere with intracellular staining of MIG.
 View larger version (16K): [in a new window] |
Figure 5. Role for MIG expression during antigen-specific immune responses. PBMC from two volunteers (vol. #6 and vol. #4) were stimulated with HIV, EBV, or CMV peptides in media alone or in the presence of neutralizing antibodies to hMIG (10 µg/ml) or antibodies to murine MIG (mMIG, 10 µg/ml) as an isotype control. MIG expression was evaluated following 16 h of stimulation as described previously. Numbers in parentheses indicate percent inhibition of MIG expression compared with untreated cultures. The addition of neutralizing antibodies before staining or following fixation/permeabilization of cells did not decrease the MIG staining (data not shown).
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-producing cells by ELISPOT assays and by indirectly evaluating the biological effects of antigen-specific IFN- production by detecting expression of the MIG chemokine. Through their ability to recruit distinct cell types to sites of inflammation, chemokines are important immune mediators for induction of protective immune responses, and the specific induction of chemokines may provide another marker for effector cell responses, as demonstrated by us previously [22
]. Here, the participation of bystander cells was only apparent when peptide-pulsed APC were used to present antigen to CD8+ T cells; the addition of soluble peptide to cultures did not result in enhanced MIG. These differences may be a result of the presentation of antigens to CD8+ T cells by other cell types, including B cells, CD4+ T cells, and dendritic cells, which may not require additional help for optimal induction of CD8+ T cell responses. Although it is known that DC can serve as potent APC for CD8+ T cells [27 ], it remains to be determined if peptides presented to CD8+ T cells by other cell lineages require additional help.
Studies were also conducted to begin to delineate the mechanism(s) by which bystander participation mediated by CD4+ and NK cells provides help for CD8+ T cell responses. It is now well-established that CD4+ T cells may provide T cell help via CD40L:CD40 pathways [28 ]. More recently, it has been reported that CD40L can be expressed by NK cells [29 , 30 ]. In our studies, the addition of antibodies to CD40 significantly inhibited antigen-specific responses. Additionally, antibodies to the IL-2 receptor (CD122) and neutralizing antibodies to IL-12 also inhibited responses; a slight decrease in response was observed in cultures treated with neutralizing antibodies to IL-2. The apparent inconsistency in the degree of responses between the addition of neutralizing antibodies against IL-2 and antibodies to the IL-2 receptor remains to be determined. However, IL-15 is also known to bind CD122 [31 ], and it is possible that the addition of antibodies to CD122 may be inhibiting IL-2 and IL-15 activity in these cultures [32 ].
Our observation that addition of neutralizing antibodies to human MIG but not mMIG inhibited antigen-specific IFN- effector function additionally suggests that chemokine recruitment may have a role in the bystander induction of optimal antigen-specific responses. Indeed, recent studies have implicated MIG as an important immune effector molecule in its own right. Like IFN-inducible protein-10 and IFN-inducible T cell chemoattractant, MIG binds to a common receptor, CXCR3, which is expressed on the surface of activated/memory T cells and NK cells [33
]. MIG has been shown to enhance NK cell-mediated cytotoxicity and to mediate antitumor and antiviral responses in vivo [34
, 35
]. Neutralization of MIG has also been shown to prolong graft survival in vivo [36
], and MIG mRNA can be detected in a variety of different organs following IFN- administration, including liver, thymus, lung, and spleen [37
] or in the liver and spleen of mice following infection with Plasmodium yoelii or T. gondii [37
]. Thus, MIG has been shown to have important participation in antigen-specific, IFN--mediated, immune responses.
The consistent requirement for bystander participation of CD4+ T cells and NK cells for optimal induction of antigen-specific CD8+ T cell IFN- production and the requirement of these cells for CD8+ T cell-mediated effector function as demonstrated by induction of MIG expression in vitro may also be manifested in vivo. This may have particular relevance in immunodeficient individuals, such as individuals infected with HIV. The loss of CD4+ T cells is considered a marker for progression to AIDS, and qualitative defects in CD4+ T cell helper function may occur before CD4+ T cell loss [38
, 39
]. A recent study demonstrating marked, increased antigen-specific CD8+ CTL effector function in simian immunodeficiency virus-infected rhesus macaques following transfusion with autologous anti-CD3/CD28-activated CD4+ T cells provides functional in vivo support of this view [40
]. In addition, Rosenberg et al. [41
] reported that HIV-infected patients who are able to control viremia in the absence of antiviral therapies maintain vigorous HIV-specific CD4+ T cell responses, suggesting that one mechanism by which HIV-specific CD4+ T cells contribute to the antiviral immunity may be a result of increased CD8+ CTL precursor activity. Besides CD4+ T cells, impairment of NK cell function has also been reported in HIV-infected individuals [42
].
Finally, in this report, we establish a novel role for NK cells in mediating antigen-specific CD8+ T cell effector function. The mechanism by which cells of the NK lineage provide bystander help is intriguing. Clearly, this is not a result of antigen-specific induction of immune responses. It may be possible that NK cells may provide augmentation of immune responses through expression of CD40L or through production of specific cytokines that augment antigen-specific immune responses. Additional studies are underway to address this issue. Altogether, these studies support the hypothesis that bystander participation of CD4+ T cells and NK cells is important for optimal induction of CD8+ T cell effector function.
Received July 5, 2002; revised August 21, 2002; accepted August 23, 2002.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  C. Brando, L. A. Ware, H. Freyberger, A. Kathcart, A. Barbosa, S. Cayphas, M.-A. Demoitie, P. Mettens, D. G. Heppner, and D. E. Lanar Murine Immune Responses to Liver-Stage Antigen 1 Protein FMP011, a Malaria Vaccine Candidate, Delivered with Adjuvant AS01B or AS02A Infect. Immun., February 1, 2007; 75(2): 838 - 845. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
