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Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and
Beckman Coulter, Miami, FL 33116
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SIV/rhesus monkey model for AIDS has provided a powerful system for exploring HIV-1 pathogenesis. Certain isolates of SIV induce an AIDS-like disease in macaques characterized by CD4+ T lymphocyte loss, immunodeficiency, wasting, infections by a variety of opportunistic pathogens, and lymphomas (4, 5). The study of SIV-specific CTL responses has been facilitated by the definition of SIVmac3 CTL epitopes and the MHC class I molecules in rhesus monkeys that present these viral peptide fragments to CD8+ T lymphocytes (6, 7, 8, 9).
Studying the role of CTL in disease pathogenesis has been hindered by the imprecise functional assays traditionally employed in the evaluation of these effector T lymphocytes. However, Altman et al. (10) recently reported that fluorescence dye-coupled tetrameric MHC class I-peptide complexes can specifically bind to subpopulations of epitope-specific CD8+ T cells, facilitating the monitoring of CTL using flow cytometric technology. In fact, we have shown that this technical approach can be applied to the evaluation of SIV-specific CTL in rhesus monkeys (11).
In the present studies, we have characterized the evolution of the virus-specific CTL response during primary SIVmac infection of rhesus monkeys. This has been done in rhesus monkeys expressing the MHC class I allele Mamu-A*01, using both functional and MHC class I/peptide tetramer assays to evaluate effector cells specific for the dominant 9-amino acid SIVmac Gag epitope p11C, C-M.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EDTA-anticoagulated blood samples and lymph node biopsies were obtained from rhesus monkeys (Macaca mulatta) experimentally infected i.v. with 20 animal infectious doses of uncloned SIVmac strain 251. The viral load in the plasma of the infected monkeys was monitored using an Ag-capture assay for SIV Gag p27 protein (Beckman Coulter, Miami, FL). These animals were maintained in accordance with the guidelines of the Committee on Animals for the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals.
Selection of Mamu-A*01+ rhesus monkeys
Rhesus monkeys were screened for the presence of the Mamu-A*01 allele using a PCR-based technique (12). EDTA-anticoagulated whole blood from macaques was subjected to Ficoll diatrizoate density gradient centrifugation (Ficopaque; Pharmacia, Piscataway, NJ) to isolate leukocytes, and the washed cell pellets were resuspended in 200 µl of PBS. DNA extraction was then conducted using a QIAmp Blood Kit (Qiagen, Chatsworth, CA). PCR was performed on 200–500 ng of extracted DNA using allele-specific primers in a 50 µl reaction consisting of 60 mM Tris, 2 mM MgCl2, 15 mM ammonium sulfate, 2 mM dNTPs (0.5 mM each), 5 u Taq polymerase (pH 8.5). Primers A*01/F (5'-GAC AGC GAC GCC GCG AGC CAA-3') and A*01/R (5'-CGCT GCA GCG TCT CCT TCC CC-3') were used at a final concentration of 800 nM each. Two additional primers specific for a conserved MHC class II sequence (based on the macaque homologue of HLA DRB3) were included in the reaction as internal positive controls. Primers 5'MDRB (5'-GCC TCG AGT GTC CCC CCA GCA CGT TTC-3') and 3'MDRB (5'-GCA AGC TTT CAC CTC GCC GCT G-3') were used at a final concentration of 680 nM each. PCR was conducted using a Perkin-Elmer (Norwalk, CT) GeneAmp System 9600 thermocycler. Samples were denatured at 96°C for 2 min followed by 5 cycles of 25 sec at 96°C and 60 sec at 72°C; followed by 21 cycles of 25 sec at 96°C, 50 sec at 67°C, and 45 sec at 72°C; followed by 4 cycles of 25 sec at 96°C, 60 sec at 55°C, and 80 sec at 72°C. PCR products were analyzed by electrophoresis in 2% agarose gels. Ten microliters of each reaction were loaded per lane.
Potential Mamu-A*01+ animals were identified by the presence of two bands, a 685-bp amplified product and a 260-bp band. DNA sequence analysis was then performed on all potential positives to confirm nucleotide sequence identity with the published Mamu-A*01 prototype sequence (6). Before sequencing, amplified DNA was treated with 1 unit per reaction of shrimp alkaline phosphatase and 10 units of exonuclease I for 15 min at 37°C, followed by 15 min at 80°C. The sequencing templates were then purified using a QIAquick PCR purification kit (Qiagen). For each template, 70 ng of DNA were used for PCR sequencing together with 5 pmol of primer. Four PCR primers were used: A*01/F and A*01/R, whose sequences are given above, and A*01-Int2/F (5'-TTC ATT TTC AGT TGA GG-3') and A*01-Int2/R (5'-GGA GGG GTC GTG ACC TGC-3'). Sequencing was conducted at a central sequencing facility on an ABI-373 stretch DNA sequencing machine, using the ABI AmpiTaq FS dye terminator chemistry (Perkin-Elmer). The six animals used in this study that were genotypically Mamu-A*01+, based on the above screening, were also positive by functional CTL assay as described (11).
Staining and phenotypic analysis of p11C, C-M-specific CD8+ T lymphocytes
The preparation of soluble tetrameric Mamu-A*01/p11C, C-M
complex has been described previously (11). Phycoerythrin (PE)-labeled
ExtrAvidin (Sigma, St. Louis, MO) or Alexa 488-labeled NeutrAvidin
(Molecular Probes, Eugene, OR) was mixed with biotinylated
Mamu-A*01/p11C, C-M complex at a 1:4 molar ratio to produce the
tetramers. The mAbs used for this study were directly coupled to FITC,
PE-texas red (ECD), or allophycocyanin (APC). The following mAbs were
used: anti-CD8
(Leu2a)-FITC and anti-CD62L (Leu8)-PE (Becton
Dickinson, San Jose, CA), anti-CD8ß(2ST8–5H7)-ECD,
anti-CD11a (25.3.1)-PE, anti-CD28 (4B10)-PE, anti-CD45RA
(2H4)-PE, anti-CD49d (HP2/1)-PE, and anti-HLA-DR (I3)-PE
(Beckman Coulter), anti-CD95 (DX2)-PE (Caltag, Burlingame, CA). The
mAb FN18, which recognizes rhesus monkey CD3, a gift from Dr. D.
M. Neville Jr. (National Institutes of Health, Bethesda, MD), was
directly coupled to APC. The three reagents: Alexa 488-coupled
tetrameric Mamu-A*01/p11C, C-M complex, anti-CD8ß-ECD, and
anti-rhesus monkey CD3-APC were used with anti-CD11a-PE,
anti-CD28-PE, anti-CD45RA-PE, anti-CD49d-PE,
anti-CD62L-PE, anti-CD95-PE, or anti-HLA-DR-PE to perform
four-color flow cytometric analyses. Because nearly all of the
tetrameric Mamu-A*01/p11C, C-M complex-binding T cells express the
CD8ß molecule, all the analyses were performed by gating on
CD8ß+ CD3+ cells. Therefore, the
lymphocytes referred to as CD8+ T cells are gated
CD8ß+ CD3+ cells. The PE-coupled
tetrameric Mamu-A*01/p11C, C-M complex was used with
anti-CD8-FITC or annexin V-FITC (PharMingen, San Diego, CA) in
conjunction with anti-CD8ß-ECD and anti-rhesus monkey
CD3-APC. The tetramer staining of CD8ß+ cells was
performed on gated CD3+ cells, since the CD8ß-specific
mAb used in this study binds occasionally to NK cells of rhesus
monkeys. Alexa 488 or PE-coupled tetrameric Mamu-A*01/p11C, C-M complex
(0.5 µg) was used in conjunction with the directly labeled mAbs to
stain 100 µl of fresh whole blood, 5 x 105 single
cells from lymph nodes, or 5 x 105 lymphocytes
isolated by density gradient centrifugation over Ficoll diatrizoate
following in vitro culture. Peripheral lymph nodes of uninfected and
infected monkeys, obtained by standard biopsy procedures, were
carefully teased to generate single cell suspensions. Samples were
analyzed on a Coulter EPICS Elite ESP as described previously
(11). Data presentation was performed using WinMDI software version 2.7
(Joseph Trotter, La Jolla, CA) and Microsoft PowerPoint software
version 4.0c (Microsoft, Redmond, WA).
Cytotoxicity assay
Autologous B-lymphoblastoid cell lines (B-LCL) were used as target cells in functional CTL assays. B-LCL were incubated with 5 µg/ml of p11C, C-M (CTPYDINQM), or the negative control peptide p11B (ALSEGCTPYDIN) for 90 min during 51Cr labeling. For effector cells, PBMC or single cells isolated from lymph nodes of monkeys chronically infected with SIVmac were cultured for 3 days at 2 x 106 cells/ml with Con A (5 µg/ml) (Sigma), washed, and then maintained for another 7–11 days in medium supplemented with human rIL-2 (20 U/ml) (Hoffman-La Roche, Nutley, NJ). Alternatively, PBMC or single cells isolated from lymph nodes were cultured for 3 days at a density of 3 x 106 cells/ml in the presence of 1 µg/ml of the peptide p11C, C-M. Cells were then maintained for another 7–11 days in medium supplemented with human rIL-2 (20 U/ml), as described above. PBMC or lymph node cells cultured according to one of these two protocols were then centrifuged over Ficoll diatrizoate and assessed as effector cells in a standard 51Cr release assay using U-bottom microtiter plates containing 104 target cells with effector cells at different E:T ratios. All wells were established and assayed in duplicate. Plates were incubated in a humidified incubator at 37°C for 4 h. Specific release was calculated as: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. Spontaneous release was <20% of maximal release with detergent (1% Triton X-100; Sigma) in all assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The kinetics and magnitude of the virus-specific CTL response
during primary SIVmac infection was assessed in rhesus monkeys.
Peripheral blood was prospectively sampled from six
Mamu-A*01+ monkeys after i.v. infection with 20 animal
infectious doses of SIVmac and analyzed by flow cytometry using
tetrameric Mamu-A*01/p11C, C-M complex (tetramer) to quantitate p11C,
C-M-specific CTL responses. The evolution of tetramer-binding
CD8+ peripheral blood T cells after SIVmac infection in two
representative animals is shown in Fig. 1
. Tetramer-binding CD8+ T
cells were detected in the peripheral blood of both monkeys by day 11
after infection and reached a peak at day 13. In the six monkeys
studied, the peak of the tetramer-binding CD8+ T cells
ranged from 1.3 to 8.3% (Figs. 1 and 5). In five of these six animals,
decreases were then demonstrated in the percent tetramer-binding
CD8+ T cells.
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). In both monkeys, the detection of
tetramer-binding CD8+ T cells in freshly obtained whole
blood specimens correlated with the detection of functional CTL
activity in those same cell populations at all evaluated time points
during the period of primary infection.
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Since SIVmac replication occurs predominantly in lymph nodes of
infected monkeys, we sought to characterize the CTL response in that
anatomic compartment during primary infection. Lymph node and
peripheral blood specimens were obtained from four infected animals on
days 13 and 21 following infection. This facilitated comparing CTL in
lymph nodes with those in PBL during the peak and 1 wk after the peak
of virus replication. Tetramer-binding CD8+ T lymphocytes
were readily demonstrated in all of the sampled lymph nodes.
Interestingly, the percent tetramer-binding CD8+ T cells
detected in the lymph nodes of the four evaluated animals at both
sampling times was smaller than the percent detected in PBL (Table II). Functional effector Gag
epitope-specific CTL were also demonstrated in these sampled lymph node
lymphocytes from the two animals that were evaluated (Table III).
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The phenotypes during primary SIVmac infection of both the
tetramer-binding and nonbinding lymph node and peripheral blood
CD8+ T cells were analyzed using four-color flow cytometry.
The expression by these cells of CD11a, CD28, CD45RA, CD49d, CD62L,
CD95, and MHC class II-DR was investigated in samples obtained at
various days following infection. The staining patterns of
tetramer+ CD8+ T cells from a representative
animal obtained before and 13 days after infection are shown in Fig. 2, and graphically displayed data from
four animals are shown in Fig. 3. At 13
days following infection, when the Gag epitope-specific CTL response
was maximal in the evaluated monkeys, the tetramer-binding
CD8+ T lymphocytes in PBL and lymph nodes uniformly
expressed the activation-associated adhesion molecules CD11a and CD49d,
as well as the Fas molecule CD95. Expression of the naive
lymphocyte-associated molecules CD45RA (bright) and CD62L were low,
with median percent cell positivity of 7.6% and 7.4%, respectively,
in PBL; and 28% and 17%, respectively, in lymph nodes. The
tetramer-binding CD8+ T cells were heterogeneous in their
expression of the signal transduction molecule CD28, but expression was
higher in lymph nodes (median of 66%) than in PBL (median of 35%).
MHC class II-DR expression was also greater in lymph nodes than PBL,
with a median positivity of 57% in lymph nodes and 21% in PBL (Fig. 3).
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). The CD8+ peripheral blood
T cells showed an increase in staining for CD11a (from a median of
39–68%), CD49d (from a median of 27–56%), and CD95 (from a median
of 35–70%). The CD8+ peripheral blood T cells
demonstrated a decrease in staining for CD28 (from a median of
68–45%), CD45RA (from a median of 66–40%), and CD62L (from a median
of 62–37%). Changes of this magnitude were not observed in
CD8+ T cells from lymph nodes (Fig. 4).
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We then sought to assess the role of the tetramer-binding
CD8+ T cells in the immunopathogenesis of primary SIVmac
infection by characterizing the temporal relationship between the
emergence of this cellular response and the clearance of virus in the
monkeys. The six Mamu-A*01+ rhesus monkeys were concurrently
analyzed following SIVmac infection for viral load by measuring viral
Gag p27 Ag in plasma and tetramer-binding CD8+ peripheral
blood T cells (Fig. 5
). In all six
animals, the peak of viremia was observed 9–11 days following
infection. A rapid fall in viral load occurred thereafter, with plasma
p27 level undetectable by day 27 following infection. Consistently, the
beginning of viral clearance coincided with the emergence of the
tetramer-binding CD8+ T cell response (Fig. 5).
Large percent of tetramer+ CD8+ T cells bind annexin V at time of peak CTL response during primary infection
The kinetics of the p11C, C-M-specific CTL response in these monkeys was characterized by the rapid appearance and then loss of tetramer-binding CD8+ T cells. We sought to determine whether the loss of the tetramer-binding cells reflected the death of the specific lymphocytes or their anatomic redistribution. To do this, we evaluated the binding to these cells of annexin V, a molecule that binds to cells early after apoptotic or necrotic events are initiated (13).
The pattern of annexin V-binding to CD8+ peripheral blood T
cells during primary infection from two representative animals is shown
in Fig. 6A, and the data
obtained in studying four animals is summarized in Fig. 6B.
A higher percent of annexin V-binding cells was detected in the
tetramer+ CD8+ T cell subpopulation (range,
from 33% to 75%) than in the remaining CD8+ T cells
(range, from 3.3% to 15%) at the time of maximum CTL expansion in all
four animals. An increased binding of the annexin V to
tetramer-CD8+ T cells (median 9.6%)
during the peak of CTL expansion is also demonstrated in Fig. 6, A and B.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The previous demonstration of a Vß-restricted CD8+ T cell response in the first weeks following HIV-1 infection has been interpreted as indicating that the early CD8+ T cell response elicited by the virus can be quite restricted in its clonality (3). We have shown a similar expansion of Vß-restricted CD8+ T lymphocytes in the peripheral blood and lymph nodes of rhesus monkeys 2 wk following infection with SIVmac (15). Such findings are fully concordant with the demonstration in the present study that 1.3–8.3% of all circulating CD8+ T lymphocytes 13 days following infection share a recognition specificity for a single peptide/MHC class I complex. These data provide strong evidence that the early clonally restricted CD8+ T lymphocyte responses represent virus-specific CTL.
We have previously reported that SIVmac-specific CTL were detected 4–6 days following infection (16), while the present study shows that such cells emerge 11 days after infection. This difference does not appear to be attributable to differences in techniques employed for detecting these CTL, since, in both studies, Gag peptide-stimulated PBL were assessed as effector cells. Rather, this difference may be due to the fact that different virus challenge stocks were employed in these experiments. Stocks of SIVmac may differ in the amount of early virus replication that they initiate, resulting in differences in the early antigenic load that drives the expansion of CTL populations.
Recent studies of MHC class I/Gag peptide-binding CD8+ T lymphocytes in chronically SIVmac-infected rhesus monkeys using four-color flow cytometric analysis have indicated that these CTL are activated memory cells (11, 17). Moreover, these studies also demonstrated that the tetramer-binding CD8+ T cells are relatively homogeneous in their expression of the surface molecules associated with this functional status. The flow cytometric analyses in the present study indicate that the tetramer+ CD8+ T lymphocytes that arise during primary SIVmac infection are similar to those seen in chronic infection, both in activation status and homogeneity.
The circulating CD8+ T lymphocyte pool dramatically expands following AIDS virus infections (3, 18). The function of the cells in this expanded pool has remained unclear. In this study, during the period of primary infection, a sizeable proportion of tetramer-, CD8+ peripheral blood and lymph node T lymphocytes expressed activation-association molecules. It is possible that a substantial fraction of these CD8+ T lymphocytes are activated "bystander cells" with no virus recognition specificity (19, 20). However, the tetramer technology employed in the present study allowed us to evaluate CTL with specificity for only a single epitope of SIVmac. Based on analyses of CTL responses to other viruses in mice and humans (21, 22, 23, 24, 25), it is reasonable to suppose that the Gag epitope recognized by these monkeys represents only one of a number of CTL specificities. Many of the expanded tetramer, CD8+ T lymphocytes expressing activation-associated molecules might be SIVmac-specific CTL that recognize viral epitopes other than p11C, C-M. In fact, if one postulates that SIVmac infection elicits CTL with only 3–4 dominant epitope specificities, virtually all of the expansion in the circulating CD8+ T lymphocyte compartment in these monkeys could be accounted for by virus-specific CTL. Moreover, since the p11C, C-M-specific CD8+ T cells probably represent only a portion of the entire Gag-specific CTL response in these monkeys, a precise quantitative correlation between the size of this epitope-specific CTL response and the early viral load may not be apparent.
In the setting of chronic SIVmac infection of rhesus monkeys, the percent of total lymphocytes in the peripheral blood and in lymph nodes that are CTL are not significantly different (17). However, as shown in this study, during primary infection, CTL are present in smaller numbers in lymph nodes than in PBL. Moreover, a smaller percent of the CD8+ T lymphocytes in lymph nodes than in PBL express activation-associated molecules. These two observations can be explained by the trafficking of CD8+ T lymphocytes, upon activation, from the lymph nodes into the peripheral blood. However, it is also possible that substantial numbers of CTL are present in lymphatic tissues distinct from the peripheral node-bearing areas sampled in the present study.
Previous studies have suggested that the abrupt decrease in number of virus-specific CTL that occurs after initial expansion during primary infection may be due to apoptosis (26). This apoptosis is presumably initiated as virus load decreases, with the elimination of much of the antigenic stimulation that drove the initial expansion of the cell population. The large number of annexin V-binding, tetramer+ CD8+ T lymphocytes observed during primary infection with SIVmac suggests that this lymphocyte subpopulation dies. However, the binding of cells to annexin V, by itself, does not allow us to differentiate between apoptotic and necrotic events.
The present experiments illustrate the power of the tetramer technology for studying epitope-specific subpopulations of CTL in vivo. These observations do not address the mechanism by which CTL are acting to contain SIVmac replication, whether they do so by a lytic process or by the production of chemokines and other soluble factors. However, in documenting with quantitative precision that the clearance of SIVmac correlates temporally with the emergence of virus-specific CTL, these findings lend further credence to the notion that CTL are important in containing AIDS virus replication.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marcelo J. Kuroda, Division of Viral Pathogenesis, RE-102, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215. E-mail address:
3 Abbreviations used in this paper: SIVmac, SIV/macaque; PE, phycoerythrin; ECD, phycoerythrin-texas red; APC, allophycocyanin.
Received for publication November 17, 1998. Accepted for publication February 8, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß and 
T-cell networks and their roles in natural resistance to viral infections. Immunol. Rev. 159:79.[Medline]
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M. R. Reynolds, E. Rakasz, P. J. Skinner, C. White, K. Abel, Z.-M. Ma, L. Compton, G. Napoe, N. Wilson, C. J. Miller, et al. CD8+ T-Lymphocyte Response to Major Immunodominant Epitopes after Vaginal Exposure to Simian Immunodeficiency Virus: Too Late and Too Little J. Virol., July 15, 2005; 79(14): 9228 - 9235. [Abstract] [Full Text] [PDF]
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B. L. Lohman, J. A. Slyker, B. A. Richardson, C. Farquhar, J. M. Mabuka, C. Crudder, T. Dong, E. Obimbo, D. Mbori-Ngacha, J. Overbaugh, et al. Longitudinal Assessment of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Gamma Interferon Responses during the First Year of Life in HIV-1-Infected Infants J. Virol., July 1, 2005; 79(13): 8121 - 8130. [Abstract] [Full Text] [PDF]
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G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. McClure, M. B. Feinberg, and S. I. Staprans Divergent Host Responses during Primary Simian Immunodeficiency Virus SIVsm Infection of Natural Sooty Mangabey and Nonnatural Rhesus Macaque Hosts J. Virol., April 1, 2005; 79(7): 4043 - 4054. [Abstract] [Full Text] [PDF]
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) and the p27 Ag in the plasma (
) of
six Mamu-A*01+ monkeys (88, 87, 253, 191, 575, and 348)
were analyzed prospectively following SIVmac infection.
CD8+ T cells are cells gated on CD8
) for LN.
CD8+ T cells are cells gated on CD8
)
CD8+ T cells from four Mamu-A*01+ rhesus
monkeys (191, 253, 87, and 88) were determined at different
times following SIVmac infection. CD8+ T cells are cells
gated on CD8