JOURNAL OF VIROLOGY, Jan. 2007, p. 434–438
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 1
Proliferative Capacity of Epitope-Specific CD8 T-Cell Responses Is
Inversely Related to Viral Load in Chronic Human
Immunodeficiency Virus Type 1 Infection?
Cheryl L. Day,1,2,3Photini Kiepiela,2Alasdair J. Leslie,1Mary van der Stok,2Kriebashne Nair,2
Nasreen Ismail,2Isobella Honeyborne,1Hayley Crawford,1Hoosen M. Coovadia,2
Philip J. R. Goulder,1,2,3Bruce D. Walker,2,3,4and Paul Klenerman1*
Nuffield Department of Medicine, The Peter Medawar Building for Pathogen Research, University of Oxford, Oxford OX1 3SY,
United Kingdom1; HIV Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu Natal,
Durban 4013, South Africa2; Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS,
Harvard Medical School, Boston, Massachusetts 021153; and Howard Hughes Medical Institute,
Chevy Chase, Maryland 201854
Received 14 August 2006/Accepted 9 October 2006
The relationship between the function of human immunodeficiency virus (HIV)-specific CD8 T-cell re-
sponses and viral load has not been defined. In this study, we used a panel of major histocompatibility complex
class I tetramers to examine responses to frequently targeted CD8 T-cell epitopes in a large cohort of
antiretroviral-therapy-naı ¨ve HIV type 1 clade C virus-infected persons in KwaZulu Natal, South Africa. In
terms of effector functions of proliferation, cytokine production, and degranulation, only proliferation showed
a significant correlation with viral load. This robust inverse relationship provides an important functional
correlate of viral control relevant to both vaccine design and evaluation.
Virus-specific CD8 T cells play an essential role in control of
chronic viral infections (4, 12). The strong association between
HLA class I molecules and protection against disease progres-
sion (5, 10), together with data from models of simian immu-
nodeficiency virus (9, 15), suggest that such responses can be
protective and support current efforts to design T-cell-based
vaccines for human immunodeficiency virus type 1 (HIV-1).
However, despite such T-cell responses, the virus is not con-
trolled long term and disease progression in untreated persons
is almost inevitable. Understanding the mechanisms behind
the failure of CD8 T-cell-based immune containment and how
the T-cell quality, function, and specificity correlate with viral
load is a crucial step in vaccine design.
To address issues of immune control in HIV infection, we
established a cohort of over 600 adults with chronic, untreated
clade C HIV infection from KwaZulu Natal, South Africa (10),
the epicenter of the global HIV pandemic. Comprehensive
screening by a gamma interferon (IFN-?) Elispot assay using
overlapping peptides spanning all expressed viral proteins, to-
gether with high-resolution HLA typing, revealed a number of
peptides frequently targeted by CD8 T cells in the context of
frequently expressed HLA alleles in this cohort (reference 10
and data not shown). From this set we identified eight peptides
with strong linkage between the presence of a specific HLA
allele and the presence of a CD8 T-cell response to the pep-
tide. Major histocompatibility complex class I tetramers corre-
sponding to highly targeted epitopes restricted by HLA-
A*0205, A*3002, B*0801, B*4201, B*8101, and Cw*0304 were
therefore constructed to enable direct visualization of antigen-
specific cells from freshly isolated peripheral blood mononu-
clear cells (PBMC) by flow cytometry (Table 1). A total of 113
subjects, selected based on expressed HLA alleles, were eval-
uated, and a total of eight epitopes examined. The median viral
load of the 113 subjects analyzed was 51,700 HIV-1 RNA
copies/ml plasma (range, ?50 to 784,000), and the median
absolute CD4 count was 329 (range, 23 to 1,273). Information
regarding duration of infection was not available, although all
the patients had established chronic HIV infection at the time
of analysis. The median magnitude of each of the epitope-
specific responses was 0.75% of CD8?T cells (range, 0.01 to
10.68%), with the strongest responses being detected to
B*4201 TL9 and Cw*0304 YL9 (Table 1 and data not shown).
There have been conflicting reports from previous studies in
terms of whether HIV induces cytotoxic T lymphocytes that
are dysfunctional in their ability to produce cytokines upon in
vitro stimulation with peptides (1, 7, 8, 11, 16). By using tet-
ramer staining in conjunction with intracellular cytokine stain-
ing for multiple responses in a large cohort of individuals, we
were able to address what proportion of HIV-specific CD8 T
cells are able to make IFN-? or interleukin-2 (IL-2) in re-
sponse to stimulation with cognate antigen. Representative
data are indicated in Fig. 1a. We found a strong correlation
between the frequency of CD8?tetramer?cells and the fre-
quency of CD8?IFN-??cells following a 6-hour stimulation
with peptide (r ? 0.8202, P ? 0.0001; Fig. 1b). In contrast, CD8
T cells made very little if any detectable IL-2 in the same assay
(range, 0.00 to 0.09% of CD8 T cells), and no correlation was
observed between the frequency of CD8?tetramer?cells and
the frequency of CD8?IL-2?cells (Fig. 1c). We analyzed the
relationship between IFN-? and IL-2 production by HIV-spe-
* Corresponding author. Mailing address: Nuffield Department of
Medicine, The Peter Medawar Building for Pathogen Research, South
Parks Road, Oxford OX1 3SY, United Kingdom. Phone: 44 1865
281885. Fax: 44 1865 281890. E-mail: email@example.com
?Published ahead of print on 18 October 2006.
cific CD8?T cells and viral load and found no significant
correlations (Fig. 1d and e). Furthermore, no significant cor-
relation was observed between the fraction of tetramer?cells
that produce IFN-? and viral load (P ? 0.4011; data not
We next addressed the ability of HIV-specific CD8 T cells to
degranulate upon stimulation with cognate antigen. PBMC
were stimulated with peptide in the presence of anti-CD107a
antibodies for 6 h, followed by surface staining with anti-CD8
and anti-CD107a antibodies (3). Tetramer staining was per-
formed in parallel with the CD107a assay on the same samples,
and a significant positive correlation was found between the
frequency of CD8?tetramer?cells and the frequency of CD8?
CD107a?cells following stimulation with the cognate peptide
FIG. 1. Frequency of IFN-?- and IL-2-producing HIV-specific CD8?T cells is not associated with plasma HIV viral load. (a) Representative
intracellular cytokine staining and tetramer staining data from a Cw*0304?individual with a Cw*0304-restricted YL9 response. Freshly isolated
PBMC were stimulated for 6 h with YL9 peptide in the presence of brefeldin A and stained intracellularly with IFN-? and IL-2 antibodies. PBMC
from the same PBMC preparation were stained concurrently with Cw*0304 YL9 tetramer. (b and c) The percentage of CD8?tetramer?cells is
correlated with the percentage of CD8?IFN-??cells (b) but not with the percentage of CD8?IL-2?cells (c). (d and e) One hundred twenty-five
tetramer and intracellular cytokine staining pairs were evaluated for the correlations between tetramer (populations, ?0.03% of CD8 T cells) and
IFN-? production and tetramer and IL-2 production. Neither the frequency of CD8?IFN-??cells (d) nor the frequency of IL-2?cells (e)
correlated with HIV plasma viral load.
TABLE 1. Description of major histocompatibility complex
class I tetramers
Median ex vivo
aPercentage of CD8?tetramer?cells.
VOL. 81, 2007 NOTES435
(P ? 0.0001; Fig. 2a). However, when the frequency of CD8?
CD107a?cells was analyzed in relation to viral load of these
subjects, no correlation was observed (Fig. 2b).
Recent studies indicate that the proliferative capacity of
HIV-specific CD8 T cells is gradually lost following acute in-
fection and is thus compromised during chronic infection in
individuals with high-level viremia (13, 14, 17). In order to
further investigate the proliferative capacity of CD8 T cells
specific for dominant clade C epitopes, we labeled freshly
isolated PBMC with 0.5 ?M carboxyfluorescein diacetate, suc-
FIG. 2. HIV-specific CD8?T cells maintain the ability to degranulate upon short-term stimulation with specific peptide, although this is not
linked to HIV plasma viral load. (a) A significant correlation exists between the frequency of CD8?tetramer?cells and the frequency of CD8?
CD107a?cells following a 6-hour stimulation with the specific peptide. (b) The frequency of CD8?CD107a?cells does not correlate with HIV
plasma viral load (P ? 0.1989). Forty-six CD107a degranulation assays where the tetramer?frequency was ?0.03% of CD8 T cells were evaluated
for the correlations between ex vivo frequency of tetramer?cells and HIV-1 viral load.
FIG. 3. Proliferative capacity of virus-specific CD8?T cells is inversely correlated with plasma HIV viral load. (a) Representative CFSE
proliferation assay from a B*4201?individual with low viral load (top panels) and a B*4201?individual with high viral load (bottom panels). The
left two panels indicate the proliferation of B*4201 TL9?cells after 6 days in the absence of antigenic stimulation, whereas the right two panels
indicate the percentage of B*4201 TL9?cells following a 6-day incubation with TL9 peptide. The percentage in the upper right quadrant of the
CD8-versus-tetramer plots indicates the percentage of CD8?tetramer?cells. The tetramer-versus-CFSE plots are gated on CD8?tetramer?cells,
and the percentage in the upper left quadrant indicates the percentage of CFSEloCD8?tetramer?cells. (b) The frequency of tetramer?CFSElo
cells following a 6-day stimulation with peptide in vitro correlates inversely with HIV viral load but not with absolute CD4 count (n ? 89).
436 NOTESJ. VIROL.
cinimidyl ester (CFSE), and stimulated them for 6 days in the
presence or absence of 0.2 ?g/ml of the specific peptide. Rep-
resentative staining showing proliferation of TL9-specific
CD8?cells from a B*4201?individual with low viral load (top
panel) and a B*4201?individual with high viral load (bottom
panel) is shown in Fig. 3a. The proliferative response to each
of the eight tetramer epitopes described in this study was
tested in a total of 89 samples, and we found a strong inverse
correlation between the percentage of CD8?tetramer?CFSElo
cells and the viral load of these subjects (P ? 0.0008; Fig. 3b,
left panel). Although there were variations between prolifera-
tive responses within individual epitopes, when analyzed indi-
vidually, we did not observe a significant difference in prolif-
erative capacity between responses to the eight different
epitopes (analysis of variance, P ? 0.2012). Despite the fact
that we observed a significant inverse correlation between the
proliferative capacity of tetramer?cells and viral load, we did
not see a significant correlation between the absolute CD4
count and proliferative capacity of tetramer?cells in these
subjects (P ? 0.1872; Fig. 3b, right panel). Multivariate regres-
sion analysis confirmed that the inverse correlation between
proliferation and viral load was independent of CD4 count
(P ? 0.007; data not shown).
HIV infection is associated with a strong adaptive cellular
immune response, and yet the majority of infected persons
progress to AIDS in the absence of treatment. In this study, we
assessed the impact of T-cell function on viral load in persons
with untreated clade C HIV infection from KwaZulu Natal,
South Africa, where the seroprevalence of HIV infection is
over 35% in some age groups. Using a panel of eight HLA
class I tetramers to frequently targeted epitopes in this popu-
lation, we find that the proliferative capacity of epitope-specific
responses may be a correlate of viral control in chronic HIV
Previous studies have addressed similar relationships but
have been done in small cohorts and for the most part have not
been linked to viral load. To our knowledge this is the largest
study of its kind, and the data set is unique in that it is derived
from a highly characterized, untreated population of adults
with chronic HIV clade C infection. This analysis is not entirely
comprehensive, in that not all responses in each individual
were assessed. However, the responses analyzed represent the
dominant response against HIV in this population.
Functional studies were performed for analysis of IFN-? and
IL-2 secretion, proliferation, and degranulation. IFN-? secre-
tion and degranulation appeared to be well maintained follow-
ing a short-term stimulation with peptide, with no evidence of
a significant functional defect. In contrast, IL-2 secretion was
weak throughout, with little secretion even at relatively low
viral loads. The proliferative capacity of epitope-specific CD8?
T cells showed a clear inverse correlation with viral load. These
data therefore provide a functional assay of HIV tetramer?
cells that is associated with viral load in a large, untreated,
chronically infected cohort. PBMC from HIV-infected individ-
uals with a wide range of viral loads were assayed, and a
substantial number of relevant tetramer responses were eval-
uated. Thus, the result appears robust and biologically signif-
icant—analysis of proliferative function using a combined
CFSE tetramer assay is technically relatively simple and pro-
vides critical information relevant to both vaccine design and
evaluation. In particular, these data highlight the importance
of assessing the proliferative capacity of HIV-specific T cells in
addition to quantifying the number of IFN-?-producing cells
when evaluating the function of vaccine-induced cellular im-
Overall these data provide important support for the anal-
ysis of the role of proliferative function of antigen-specific CD8
T cells as a key measure of the exhaustion of T cells in chronic
viral infections in general and HIV in particular. The mecha-
nism behind this functional loss is not yet fully established,
although recent data on the expression of the inhibitory recep-
tor PD-1 (2, 6) may provide important links between function,
phenotype, and disease progression.
This work was supported by a Royal Society postdoctoral fellowship
(C.L.D.), the Wellcome Trust (P.K. and P.J.R.G.), and the Doris Duke
Charitable Foundation (B.D.W.).
We thank Chantal de Pierres, Nompumelelo Mkhwanazi, Eshia S.
Moodley, Zenele Mncube, and Sharon Reddy for technical assistance.
1. Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King,
G. S. Ogg, H. M. Spiegel, C. Conlon, C. A. Spina, D. V. Havlir, D. D.
Richman, A. Waters, P. Easterbrook, A. J. McMichael, and S. L. Rowland-
Jones. 2000. HIV-specific CD8?T cells produce antiviral cytokines but are
impaired in cytolytic function. J. Exp. Med. 192:63–75.
2. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe,
G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 T
cells during chronic viral infection. Nature 439:682–687.
3. Betts, M. R., J. M. Brenchley, D. A. Price, S. C. De Rosa, D. C. Douek, M.
Roederer, and R. A. Koup. 2003. Sensitive and viable identification of anti-
gen-specific CD8? T cells by a flow cytometric assay for degranulation.
J. Immunol. Methods 281:65–78.
4. Borrow, P., H. Lewicki, B. H. Hahn, G. M. Shaw, and M. B. Oldstone. 1994.
Virus-specific CD8?cytotoxic T-lymphocyte activity associated with control
of viremia in primary human immunodeficiency virus type 1 infection. J. Vi-
5. Carrington, M., G. W. Nelson, M. P. Martin, T. Kissner, D. Vlahov, J. J.
Goedert, R. Kaslow, S. Buchbinder, K. Hoots, and S. J. O’Brien. 1999. HLA
and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage. Science
6. Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S.
Reddy, E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, Z. Mncube, J.
Duraiswamy, B. Zhu, Q. Eichbaum, M. Altfeld, E. J. Wherry, H. M. Coovadia,
P. J. R. Goulder, P. Klenerman, R. Ahmed, G. J. Freeman, and B. D. Walker.
2006. PD-1 expression on HIV-specific T cells is associated with T cell
exhaustion and disease progression. Nature 443:350–354.
7. Goepfert, P. A., A. Bansal, B. H. Edwards, G. D. Ritter, Jr., I. Tellez, S. A.
McPherson, S. Sabbaj, and M. J. Mulligan. 2000. A significant number of
human immunodeficiency virus epitope-specific cytotoxic T lymphocytes de-
tected by tetramer binding do not produce gamma interferon. J. Virol.
8. Goulder, P. J., Y. Tang, C. Brander, M. R. Betts, M. Altfeld, K. Annamalai,
A. Trocha, S. He, E. S. Rosenberg, G. Ogg, C. A. O’Callaghan, S. A. Kalams,
R. E. McKinney, Jr., K. Mayer, R. A. Koup, S. I. Pelton, S. K. Burchett, K.
McIntosh, and B. D. Walker. 2000. Functionally inert HIV-specific cytotoxic
T lymphocytes do not play a major role in chronically infected adults and
children. J. Exp. Med. 192:1819–1832.
9. Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E.
Irwin, J. T. Safrit, J. Mittler, L. Weinberger, L. G. Kostrikis, L. Zhang, A. S.
Perelson, and D. D. Ho. 1999. Dramatic rise in plasma viremia after CD8?
T cell depletion in simian immunodeficiency virus-infected macaques. J. Exp.
10. Kiepiela, P., A. J. Leslie, I. Honeyborne, D. Ramduth, C. Thobakgale, S.
Chetty, P. Rathnavalu, C. Moore, K. J. Pfafferott, L. Hilton, P. Zimbwa, S.
Moore, T. Allen, C. Brander, M. M. Addo, M. Altfeld, I. James, S. Mallal, M.
Bunce, L. D. Barber, J. Szinger, C. Day, P. Klenerman, J. Mullins, B.
Korber, H. M. Coovadia, B. D. Walker, and P. J. Goulder. 2004. Dominant
influence of HLA-B in mediating the potential co-evolution of HIV and
HLA. Nature 432:769–775.
11. Kostense, S., G. S. Ogg, E. H. Manting, G. Gillespie, J. Joling, K. Vanden-
berghe, E. Z. Veenhof, D. van Baarle, S. Jurriaans, M. R. Klein, and F.
Miedema. 2001. High viral burden in the presence of major HIV-specific
CD8?T cell expansions: evidence for impaired CTL effector function. Eur.
J. Immunol. 31:677–686.
12. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky,
VOL. 81, 2007NOTES 437
C. Farthing, and D. D. Ho. 1994. Temporal association of cellular immune Download full-text
responses with the initial control of viremia in primary human immunode-
ficiency virus type 1 syndrome. J. Virol. 68:4650–4655.
13. Lichterfeld, M., D. E. Kaufmann, X. G. Yu, S. K. Mui, M. M. Addo, M. N.
Johnston, D. Cohen, G. K. Robbins, E. Pae, G. Alter, A. Wurcel, D. Stone,
E. S. Rosenberg, B. D. Walker, and M. Altfeld. 2004. Loss of HIV-1-specific
CD8? T cell proliferation after acute HIV-1 infection and restoration by
vaccine-induced HIV-1-specific CD4? T cells. J. Exp. Med. 200:701–712.
14. Migueles, S. A., A. C. Laborico, W. L. Shupert, M. S. Sabbaghian, R. Rabin,
C. W. Hallahan, D. Van Baarle, S. Kostense, F. Miedema, M. McLaughlin,
L. Ehler, J. Metcalf, S. Liu, and M. Connors. 2002. HIV-specific CD8? T
cell proliferation is coupled to perforin expression and is maintained in
nonprogressors. Nat. Immunol. 3:1061–1068.
15. Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A.
Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, J. Ghrayeb,
M. A. Forman, D. C. Montefiori, E. P. Rieber, N. L. Letvin, and K. A.
Reimann. 1999. Control of viremia in simian immunodeficiency virus infec-
tion by CD8? lymphocytes. Science 283:857–860.
16. Shankar, P., M. Russo, B. Harnisch, M. Patterson, P. Skolnik, and J.
Lieberman. 2000. Impaired function of circulating HIV-specific CD8?T
cells in chronic human immunodeficiency virus infection. Blood 96:3094–
17. Zimmerli, S. C., A. Harari, C. Cellerai, F. Vallelian, P. A. Bart, and G.
Pantaleo. 2005. HIV-1-specific IFN-gamma/IL-2-secreting CD8 T cells sup-
port CD4-independent proliferation of HIV-1-specific CD8 T cells. Proc.
Natl. Acad. Sci. USA 102:7239–7244.
438NOTES J. VIROL.