selective depletion of high-avidity hiv-1-specific cd8+ t...

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Selective depletion of high-avidity HIV-1-specific CD8+ T cells following

early HIV-1 infection

Mathias Lichterfeld1,4*

, Xu G. Yu1,4

, Stanley K. Mui1, Katie L. Williams

1, Alicja

Trocha1,2

, Mark A. Brockman1,2

, Rachel L. Allgaier1, Michael T. Waring

1,2,

Tomohiko Koibuchi1,2

, Mary N. Johnston1, Daniel Cohen

3, Todd M. Allen

1, Eric

S. Rosenberg1, Bruce D. Walker

1,2, Marcus Altfeld

1

Running title: High-avidity CD8+ T cells in early HIV-1 infection.

1Partners AIDS Research Center, Massachusetts General Hospital and

Division of AIDS, Harvard Medical School

Boston, MA, USA

2Howard Hughes Medical Institute, Chevy Chase, MD, USA

3Fenway Community Health, Boston, MA, USA

4ML and XGY contributed equally to this work.

Word count: abstract: 176, text: 5577

*Corresponding author:

Xu Yu, MD, MSc

Partners AIDS Research Center

Massachusetts General Hospital

149 13th

Street

Boston, MA 02129, USA

Phone: 617-726-3167

Fax: 617-726-5411

[email protected]

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.01388-06 JVI Accepts, published online ahead of print on 7 February 2007

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Abstract

HIV-1-specific CD8+ T cells in early infection are associated with the dramatic decline

of peak viremia, whereas their antiviral activity in chronic infection is less apparent. The

functional properties accounting for the antiviral activity of HIV-1-specific CD8+ T cell

during early infection are unclear. Using cytokine secretion and tetramer decay assays,

we demonstrate in intra-individual comparisons that the functional avidity of HIV-1-

specific CD8+ T cells was consistently higher in early infection than in chronic infection

in the presence of high-level viral replication. This change of HIV-1-specific CD8+ T cell

avidity between early and chronic infection was linked to a substantial switch in the

clonotypic composition of epitope-specific CD8+ T cells, resulting from the preferential

loss of high-avidity CD8+ T cell clones. In contrast, the maintenance of the initially

recruited clonotypic pattern of HIV-1-specific CD8+ T cells was associated with low-

level set point HIV-1 viremia. These data suggest that high-avidity HIV-1-specific CD8+

T cell clones are recruited during early infection, but are subsequently lost in the presence

of persistent high level viral replication.

Keywords: HIV-1, cellular immunology, HIV-1-specific CD8+ T cells, avidity, T cell

receptor

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Introduction

Early HIV-1 infection is a transient symptomatic illness that is characterized by

extremely high viral loads (28). This high level viremia typically declines dramatically

once a virus-specific CD8+ T cell mediated immune response is mounted. Evidence for a

strong antiviral activity of HIV-1-specific CD8+ T cells during early infection is based on

at least three distinct observations: (i) the temporal coincidence between the first

emergence of HIV-1-specific CD8+ T cells in the peripheral blood and the resolution of

the acute retroviral syndrome (11, 33), (ii) the rapid selection of viral escape mutations in

targeted CD8+ T cell epitopes (26, 43, 48), and (iii) the dramatically accelerated SIV

disease progression that is noted in rhesus macaques following the artificial depletion of

CD8+ cells (52). Early HIV-1 infection therefore offers a unique opportunity to analyze

correlates of protective immunity mediated by HIV-1-specific CD8+ T cells.

In previous studies, it was shown that the total magnitude of HIV-1-specific CD8+ T

cells in acute infection, as determined by interferon-γ secretion assays or tetramer binding

studies, is relatively low and directed against a narrow repertoire of viral epitopes (6, 18,

59), which are typically structured in a clear hierarchical order (61). In contrast,

progressive viremia in chronic infection usually occurs in the presence of strong and

broadly-diversified interferon-γ secreting HIV-1-specific CD8+ T cells (1, 9, 22, 23),

which are only infrequently associated with viral sequence diversification (32),

suggesting limited immune pressure mediated by these responses. These data imply that

HIV-1-specific CD8+ T cells in acute and chronic infection differ in their ability to

contain viral replication, the reasons for which still remain largely unclear.

Recent studies have shown that HIV-1-specific CD8+ T cells in early infection have

strong ex-vivo proliferative activities, while this effector function appears to be

selectively lost during the subsequent disease process in the presence of high-level

viremia (37). This loss of ex-vivo proliferative activity appeared to be at least partially

related to the simultaneous loss of IL-2 producing HIV-1-specific CD4+ T helper cells

(25, 60), but might not necessarily reflect an intrinsic functional deficiency of HIV-1-

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specific CD8+ T cells themselves. Understanding intrinsic differences between HIV-1-

specific CD8+ T cells in acute and chronic HIV-1 infection therefore remains crucial for

the ultimate definition of CD8+ T-cell mediated protective immunity against HIV-1.

In the present study, we comparatively analyzed HIV-1-specific CD8+ T cells in early

and chronic infection, using serial dilutions of antigenic peptides for PBMC stimulation

and four different functional readouts to quantify responding CD8+ T cells. Our data

show that HIV-1-specific CD8+ T cells in early infection have a higher functional avidity

and a clonotypic composition that is different from chronic HIV-1 infection. The

selective depletion of high avidity HIV-1-specific CD8+ T cells during the transition to

chronic progressive HIV-1 infection may contribute to the ultimate inability to

immunologically contain viral replication.

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Materials and Methods

Subjects studied. PBMC samples from 30 HIV-1-infected individuals were obtained and

investigated in this study. Ten individuals with early HIV-1 infection, defined by HIV-1

seroconversion within six months prior to study enrollment, were compared to 10

individuals with chronic HIV-1 infection in a cross-sectional analysis. Individuals with

chronic infection had been infected with HIV-1 for at least three years. In addition, in

another ten individuals identified during early infection, the first detectable HIV-1-

specific CD8+ T cell response was analyzed and longitudinally followed over the

subsequent disease process. Five of these were treated with HAART during early

infection and subsequently exposed to structured treatment interruptions,(29, 50) and the

other five individuals remained completely antiretroviral therapy naive. Study subjects

were recruited from the Massachusetts General Hospital (MGH) and

the Fenway

Community Health Center in Boston. Relevant clinical and demographic data of all study

subjects are summarized in Table 1. The study was

approved by the respective

institutional review boards, and all subjects gave written informed consent to participate.

HLA class I typing. HLA class I molecular typing was performed at the Massachusetts

General Hospital Tissue Typing Laboratory or at a commercial laboratory (Dynal

Biotech, Oxford, UK) using sequence-specific PCRs according to standard operational

protocols.

Synthetic HIV-1 peptides. Four hundred and ten peptides, overlapping by 10 amino

acids, which were 13 to 18 amino acids in length and spanned the entire expressed

HIV-1

clade B 2001 consensus sequence (Gag, Pol, Vif, Vpr, Vpu, Rev, Tat, Env, and Nef)

were synthesized at the MGH Peptide Core Facility on an automated peptide synthesizer

(MBS 396; Advanced Chemtech,

Louisville, KY) using Fmoc chemistry. Peptides

corresponding to previously defined HIV-1-specific optimal CD8+ T cell epitopes (12)

were also synthesized according to this procedure.

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Intracellular cytokine staining and multiparameter flow cytometry. PBMC were

separated from whole blood by Ficoll-Hypaque (Sigma, St. Louis, MO) density gradient

centrifugation. Cells (0.5-1 million) were stimulated with 5 pools of overlapping peptides

(final concentration: 4µg/ml) spanning all expressed HIV-1 gene products (Gag, Nef, Pol,

Env, combined pool of Vpr, Vpu, Vif, Tat Rev) or with peptides corresponding to

optimal CD8+ T cell epitopes in serial ten-fold dilutions (0.0001µg/ml-10µg/ml). PBMC

were incubated with anti-CD28 and anti-CD49d antibodies (1µg/ml each, BD

Biosciences, San Jose, CA) for six hours at 37º C, 5% CO2 in the presence of FITC-

labeled CD107a/b antibodies (10µl/ml, BD Biosciences). Brefeldin A (10µg/ml, Sigma)

and Monensin (6µg/ml, BD Biosciences) were added after the initial hour of incubation.

Afterwards, cells were washed with PBS/1% FCS and stained with surface antibodies

(CD8 PerCP-Cy5.5, CD3APC-Cy7, all antibodies from BD Biosciences). Cells were then

washed again, fixed and permeabilized using the Caltag Fixation/Permeabilization kit

(Caltag, Burlingame, CA) and stained for intracellular expression of cytokines (MIP-1β

PE, TNF-α APC, IFN-γ PE-Cy7 (BD Biosciences)). For phenotyping experiments, cells

were initially stained with APC-labeled MHC class I tetramers (Beckmann-Coulter,

Fullerton, CA) and surface antibodies (CD8 Alexa 405 (Caltag), CD38 PerCP-Cy5.5 (BD

Biosciences), CD127-APC-Cy5.5 (R&D Systems, Minneapolis, MN), and TCR Vβ 20, 9,

27, 28 or 10.3 PE antibodies (Immunotech, Marseille, France). Following fixation and

permeabilization, cells were labeled with Ki-67 FITC antibodies (BD Biosciences). Cells

were acquired on an LSRII flow cytometer (BD Biosciences), using the FACS DiVa

software. Electronic compensation was performed with antibody-capture beads (BD

Biosciences) stained separately with individual antibodies used in the test samples. Flow

data were analyzed with the FlowJo software package (Treestar, Ashland, OR). The total

magnitude of HIV-1-specific CD8+ T cell responses was calculated by summing up the

responses to the individual peptide pools following background subtraction.

Tetramer dissociation assays. Overall avidity of epitope-specific CD8+ T cells was

analyzed using tetramer disscociation assays according to a recently published protocol

(58). PBMC suspended in staining buffer (PBS containing 2% bovine serum albumin and

0.2% sodium azide) were stained with PE- or APC-labeled tetramers (Beckmann Coulter)

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refolded with epitopic HIV-1 peptides and FITC-labeled CD8 antibodies (BD

Biosciences) for 45 minutes at room temperature, and then washed three times with

staining buffer. To observe the dissociation of the tetramer, cells were resuspended in

staining buffer with unlabeled tetramer at a hundred fold surplus concentration. At

selected time points, ~0.5×106 cells were withdrawn and fixed in 150 µl 2%

paraformaldehyde/PBS. Cells were then analyzed on a FACSCalibur flow cytometer

(Becton Dickinson, San Jose, CA). The MHC class I tetramer dissociation analysis was

based on the total fluorescence of the tetramer positive population (51). Briefly, the total

fluorescence of the gated CD8+ MHC class I tetramer+ population was calculated and

normalized per gated lymphocyte. This total antigen-specific fluorescence of the CD8+

tetramer+ cell population was then normalized to the total fluorescence at the initial time

point and the graphs were plotted on a logarithmic scale.

Sorting of tetramer+ HIV-1-specific CD8+ T cell populations. Fresh or frozen PBMC

samples were stained with APC- or PE- labeled MHC class I tetramers refolded with

epitopic HIV-1 peptides (Beckman Coulter) and fluorophore-labeled CD8+ antibodies,

followed by decontamination with fixation solution A (Caltag, 1:100 dilution). Tetramer+

CD8+ cells were then sorted on a FACS Aria cell-sorting instrument (BD Biosciences) at

70 pounds per square inch (PSI). Electronic compensation was performed with antibody-

capture beads (BD Biosciences) stained separately with individual antibodies used in the

test samples. The purity of sorted cell populations was consistently higher than 98%.

TCR ββββ chain sequencing. mRNA was extracted from at least 4,000 tetramer-specific

CD8+ T cells using the RNA easy mini kit (Qiagen, Valencia, CA). Anchored RT-PCR

was then performed using a modified version of the SMART (switching mechanism at 5'

end of RNA transcript) procedure and a TCR β chain constant region 3'-primer to obtain

PCR products containing the Vβ chain in addition to the CDR3 region, the Jβ region and

the beginning of the Cβ region (21). Briefly, reverse transcription was carried out at 42º

C for 90 minutes with primers provided for the 5’-RACE reaction in a SMART-RACE

PCR kit (BD Biosciences). First and second round PCR were then performed using

universal 5’-end primers (BD Biosciences) and nested gene-specific 3’-end primers

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annealing to the constant region of the TCR β chain (Cβ outer: 5’-

TGTGGCCAGGCACACCAGTGTGGCC-3’; Cβ inner: 5’-

GGTGTGGGAGATCTCTGCTTCTGA-3’). PCR reaction conditions were as follows:

First run: [95º for 30s, 72º for 2m] for 5 cycles, [95º for 30s, 70º for 30s, 72ºfor 2m] for 5

cycles, [95º for 30s, 60º for 30s, 72º for 1m] for 25 cycles. Second run: [95º for 30s, 60º

for 30s, 72º for 1m] for 30 cycles. The PCR product was ligated into the TOPO TA

cloning vector (Invitrogen, Carlsbad, CA) and used to transform Escherichia coli (Mach

1, Invitrogen). At least 40 colonies were selected, amplified by PCR with M13 primers,

and sequenced by T7 or T3 primers on an ABI 3100 PRISM automated sequencer.

Sequences were edited and aligned using Sequencher (Gene Codes Corp., Ann Arbor,

MI) and Se-Al (University of Oxford, Oxford, UK) and compared to the human TCR

genes database (http://imgt.cines.fr:8104/home.html). The TCR clonotype composition

remained consistent in repeated RT-PCR procedures using the same mRNA sample. The

TCR Vβ chain classification system of the international ImMunoGeneTics database

(IMGT) (36) was used throughout the entire manuscript.

Sequencing of autologous virus. Nested PCR for gag, RT and nef on proviral DNA or

plasma viral RNA was performed as previously described (5). PCR fragments were

population sequenced to identify regions of sequence variation. All fragments were

sequenced bi-directionally on an ABI 3100 PRISM automated sequencer. If the height of

the secondary peak at a given residue in the chromatogram was reproducibly more than

25% of the dominant peak, a mixed base was considered present at that position.

Sequencher (Gene Codes Corp., Ann

Arbor, Mich.) and MacVector 4.1 (Oxford

Molecular) were used to edit and align sequences.

Statistics. Data are expressed as means and standard deviations or medians and ranges,

respectively. Generation of dose-response curves and tetramer dissociation curves,

including calculations of EC50-levels and Koff rates, were performed using the Prism

software package (version 2.1 GraphPad Software Corporation, San Diego, CA). Data

sets were tested for normality distribution using the Shapiro-Wilks W test. In case of

normal distributions, statistical hypotheses were tested using paired or unpaired Student t

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tests, otherwise, the Wilcoxon matched pair test or the Mann-Whitney U test were used.

A p-level <0.05 was considered significant.

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Results

Total magnitude of cytokine secreting and degranulating HIV-1-specific CD8+ T cells in

early and chronic HIV-1 infection.

In prior studies, the magnitude of HIV-1-specific CD8+ T cells in early and chronic

infection has been analyzed using interferon-γ Elispot assays (6, 14, 34, 38) or more

recently by CFSE-based proliferation assays (37). Yet, HIV-1-specific CD8+ T cells can

also exert antiviral activities by secreting a variety of additional cytokines or by releasing

cytotoxic enzymes (7, 10). Here, we compared the total magnitude of HIV-1-specific

CD8+ T cells in early and chronic HIV-1 infection, using four different functional

readouts, including the antigen-specific secretion of IFN-γ, TNF-α and MIP-1β as well as

the antigen-dependent degranulation, measured by surface expression of CD107a/b.

These different effector functions were analyzed by multiparameter flow cytometry

following stimulation of PBMC samples with excess concentrations of overlapping HIV-

1 peptides corresponding to all expressed HIV-1 gene products.

In line with previous results (6, 18, 34, 38), the total magnitude of interferon-γ secreting

HIV-1-specific CD8+ T cells was substantially weaker in early HIV-1 infection

compared to chronic infection (figure 1). Importantly, a smaller magnitude of HIV-1-

specific CD8+ T cells in early infection compared to chronic infection was also observed

when HIV-1-specific CD8+ T cells were quantified by flow cytometry according to

secretion of TNF-α, MIP-1β or surface expression of CD107a/b. In addition, the

hierarchy of these different effector functions was similar in early and chronic infection

(MIP-1β > CD107 > IFN-γ > TNF-α). Overall, these data show that for a variety of

different functional readouts, the total magnitude of HIV-1-specific CD8+ T cells is

consistently lower during early HIV-1 infection compared to chronic HIV-1 infection.

Higher functional avidity of HIV-1-specific CD8+ T cells during early HIV-1 infection.

In the above experiments, HIV-1-specific CD8+ T cells were analyzed following

stimulation with excess concentrations of antigenic peptides; however, this approach did

not allow us to discern differences between the functional avidity of these cells, defined

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as the antigen concentration eliciting 50% of the maximal functional response. To

address this, we focused on ten individuals who were identified in early HIV-1 infection

and then longitudinally followed over the ensuing disease course. Five of these study

persons were treated with HAART during early infection and subsequently exposed to a

number of structured treatment interruptions (STI) (29). The other five study individuals

have never received antiretroviral therapy since early infection and exhibited either high-

level viremia (>100.000 copies/ml, 3 persons) or low-level viremia (<6000 HIV-1

copies/ml, 2 persons). The demographic, immunogenetic and clinical characteristics of

the study subjects are summarized in table 1b. In these study persons, the functional

avidity of the earliest detectable immunodominant HIV-1-specific CD8+ T cell response

was analyzed by stimulating PBMC with the epitopic peptide in serial, ten-fold dilutions,

again using the above-mentioned four different functional readouts in a multiparameter

flow cytometric assay. In addition, we longitudinally assessed the functional avidity of

these responses during the subsequent disease process (median: 237.5 days off-therapy,

range: 27-765 days off-therapy following the baseline assessment). Importantly, none of

the targeted epitopes exhibited evidence for viral sequence changes during the time of

observation, as determined by population sequencing of the autologous viruses (data not

shown).

Experimental results for the subject AC-15 are depicted in figure 2A/B. In this intra-

individual analysis, the B8-FL8 peptide concentration eliciting 50% of the maximal

interferon-γ response was 0.004µg/ml during the initial assessment and 0.026µg/ml in

chronic infection, thus indicating a substantial avidity change between the initially-

mounted CD8+ T cells response and the same epitope-specific response in the same study

individual during chronic infection. A similar difference between the functional avidity of

interferon-γ secreting HIV-1-specific CD8+ T cells in early and chronic infection was

observed in the other study subjects undergoing STIs (figure 2B) or experiencing high

level viremia in the absence of antiretroviral therapy (figure 2C). Interestingly, the

difference between the functional avidity of interferon-γ secreting HIV-1-specific CD8+

T cells in early and chronic infection was lowest in the two study individuals who, in the

background of the protective HLA class I alleles HLA-B57 or –B27, spontaneously

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controlled HIV-1 viremia after early infection to levels below 6,000 copies/ml (figure

2D). Overall, in the ten subjects who were studied, the functional avidity of HIV-1-

specific CD8+ T cells for secretion of interferon-γ, MIP-1β and antigen-specific

degranulation (CD107a/b expression) was significantly higher at the time of early

infection compared to chronic infection (figure 2E). There was also a trend for a higher

functional avidity of TNF-α secreting CD8+ T cells in early infection compared to

chronic infection (figure 2E), however, antigen-specific TNF-α secretion below the level

of detection by flow cytometry precluded this analysis in study persons AC-131, AC-177,

AC-132, AC-160 and AC-121. Importantly, for all CD8+ T cell effector functions tested,

the smallest difference between the functional avidity in early and chronic infection was

observed in the two study individuals spontaneously achieving low-level viremia after

early infection (figure 2E). Taken together, these results show in intra-individual

comparisons that HIV-1-specific CD8+ T cells emerging during early infection have a

higher functional avidity compared to those CD8+ T cells targeting the same viral epitope

in progressive chronic infection.

Slower tetramer dissociation rate of HIV-1-specific CD8+ T cells in early infection

compared to chronic infection.

The higher functional avidity of HIV-1-specific CD8+ T cells in early HIV-1 infection

might reflect a higher overall avidity of the TCR/peptide-MHC class I interaction in early

infection, which can be analyzed using a tetramer drop-off assay (51). Figure 3 shows the

tetramer dissociation curves for all ten study individuals. In study subjects undergoing

STIs (figure 3A) or maintaining high-level viremia in the absence of antiretroviral

therapy (figure 3B), we consistently observed a slower dissociation of the tetramer (lower

dissociation rate constant Koff) in early infection compared to chronic infection.

Corresponding to the functional avidity data described above, the difference between

tetramer dissociation rates in early and chronic infection was least pronounced in the two

HLA-B57 or –B27 expressing individuals achieving spontaneous low-level viral loads

following early infection (figure 3C). Overall, the difference between the tetramer

dissociation rate constants during early and chronic infection in the ten study individuals

was statistically significant (figure 3D). Thus, in intra-individual comparisons, these

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results indicate a significantly higher avidity of the TCR/pMHC interaction of HIV-1-

specific CD8+ T cells recruited in early infection compared to chronic progressive

infection, while a substantially smaller degree of avidity changes was observed in persons

achieving control of viral replication at low levels following early infection.

Evolution of the TCR clonotype repertoire between early and chronic HIV-1 infection.

To determine if the observed avidity differences between HIV-1-specific CD8+ T cells in

early and chronic infection corresponded to changes in their clonal composition, we next

conducted a longitudinal analysis of the clonotypic repertoire of the respective HIV-1-

specific CD8+ T cell populations. In order to overcome the limitations of previously-

described (41) Vβ-chain-specific immunostainings for the clonotypic assessment of HIV-

1-specific CD8+ T cells, we determined their clonotypic composition by TCR β chain

sequencing using a PCR amplification technique without bias for selected TCR Vβ

chains to ensure that epitope-specific clonotypes were represented in the PCR product

with a relative frequency reflecting that in the originally sorted cell population (48).

Figure 4 (table 2) summarizes the clonotypic TCR repertoires of the epitope-specific

CD8+ T cell populations analyzed during early and chronic HIV-1 infection in the ten

study subjects. In the study subjects exposed to STIs (figure 4A), the dominant CD8+ T

cell clonotypes recruited during early infection were subsequently either deleted and

replaced by alternative clonotypes (AC-02, AC-14, AC-09) or preserved but substantially

reduced in their overall contribution to the clonotypic repertoire (AC-15). In study

individual AC-46, one co-dominant clonotype detected during early infection persisted

until chronic infection, while the other co-dominant clonotypes were eliminated after

early infection. In addition, we observed in these individuals that clonotypes which were

rare or hardly detectable during early infection became dominant during chronic infection

(AC-46, AC-09). In untreated study individuals with continuous high-level viremia

(figure 4B), the original clonotypic composition either entirely changed (AC-131), or the

dominance of initially recruited clonotypes was subsequently reduced due to the

expansion of subdominant clonotypes that were completely (AC-177) or almost

completely (AC-132) undetectable during the first assessment. Moreover, in study

subjects AC-177 and AC-132, clonotype preservation after early infection was not related

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to clonal persistence, but resulted from a considerable underlying switch in the

composition of nucleotypes encoding for identical CDR3 amino acid sequences (table 2),

as previously observed in the context of TCRs specific for an immunodominant influenza

virus epitope (31). Finally, in line with our previous data on CD8+ T cell avidity in the

two HLA-B57 or –B27 positive study individuals achieving spontaneous control of

viremia at low levels, the initially-recruited clonotypic repertoire remained fairly

consistent after early infection in these two study persons, with a preservation of three

(AC-160) or four (AC-121) clonotypes (figure 4C), both on the level of the amino acid

and nucleic acid CDR3 sequences (table 2). Overall, these data indicate a substantial

switch of the TCR clonotype repertoire after early HIV-1 infection in individuals

experiencing high viral setpoints, while control of viral replication at low levels after

early infection was associated with a largely conserved pattern of the originally-recruited

clonotypic TCR repertoire.

Selective loss of high-avidity HIV-1-specific CD8+ T cells after early infection

We next explored the potential mechanisms accounting for the selective loss or

persistence of specific CD8+ T cell clones after early infection. In three of our study

subjects, we had the opportunity for a direct flow-cytometric comparison of the avidity of

HIV-1-specific CD8+ T cell clonotypes persisting or disappearing after early infection,

using tetramers dissociation assays in conjunction with TCR Vβ chain specific

antibodies. In study subject AC-46, we compared the B8-FL8-specific CD8+ T cell

clonotypes using Vβ9 (6.5% of all clonotypes in early infection, 0% of clonotypes in

chronic infection) or Vβ20.1 (25% of all clonotypes in early infection, 31.6% of

clonotypes in chronic infection), in study individual AC-09, we compared the A2-IV9-

specific CD8+ T cell clonotypes using Vβ20.1 (38% of clonotypes in early infection, 0%

of clonotypes in chronic infection) or Vβ10.3 (7.6% of clonotypes in early infection, 62%

in chronic infection) and in study person AC-14, we analyzed the B8-FL8-specific

clonotypes using Vβ27 (42.5% of all clonotypes in early infection, 0% in chronic

infection) or Vβ28 (7.9% of all clonotypes in early infection, 5.8% in chronic infection)

(table 2). The individual contribution of these clonotypes to the overall magnitude of

epitope-specific CD8+ T cells during early infection, as determined by TCR sequencing,

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closely matched the proportion of tetramer-positive CD8+ T cells using the

corresponding TCR Vβ chains, as measured by flow cytometry in cell samples collected

from the same time point (figure 5A). Using tetramer dissociation assays, we found that

in each of these three study persons, the above-mentioned clonotypes persisting after

early infection had a lower avidity compared to the corresponding clonotypes being

deleted after early infection (figure 5B). These results demonstrate that high avidity

CD8+ T cell clonotypes generated during early infection were preferentially lost in

chronic infection, while CD8+ T cell clonotypes with intermediate or lower TCR avidity

persisted.

We subsequently assessed whether these high avidity epitope-specific CD8+ T cell

clones differed from the persisting lower avidity clones in their activation status and the

expression of the receptor for IL-7, a cytokine that has recently been identified to be

responsible for the maintenance of CD8+ T cells and their transformation into memory

cells (27). Interestingly, in these three study subjects, the epitope-specific CD8+ T cell

clonotypes that were lost in chronic infection had a lower surface expression of the IL-7

receptor α chain (CD127), a higher expression of the activation marker CD38 (17) and a

higher intracellular expression of the proliferation-associated antigen Ki-67 (figure 5C)

during early infection. Taken together, these data show that during early HIV-1 infection,

clonal subsets of HIV-1-specific CD8+ T cells with higher avidity are more strongly

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Discussion

A striking feature of the natural HIV-1 disease process is the dramatic decline of HIV-1

viremia during early HIV-1 infection. A number of observations suggest that this

decrease of viral replication is mediated by HIV-1-specific CD8+ T cells, however, the

precise characteristics of these cells accounting for their apparent antiviral activities are

still unknown. Here, we conducted a detailed analysis of the functional properties of

HIV-1-specific CD8+ T cell responses during early and chronic HIV-1 infection, using

both a cross-sectional and longitudinal study design as well as assays to evaluate the

avidity and the clonotypic composition of HIV-1-specific CD8+ T cell populations.

Our results indicate that HIV-1-specific CD8+ T cells in early infection, despite being

lower in magnitude compared to chronic infection, have a higher avidity by multiple

functional readouts, which was associated with a slower tetramer dissociation rate. In

addition, the reduced avidity of CD8+ T cells in chronic infection was linked to a

substantial switch in the clonotypic composition of epitope-specific CD8+ T cells

compared to early infection. Interestingly, the avidity and clonotypes of HIV-1-specific

CD8+ T cells were largely preserved after early infection in individuals who

spontaneously controlled HIV-1 replication at low-level viral set points, suggesting an

association between the conservation of the originally-recruited pattern of HIV-1-specific

CD8+ T cell clonotypes and the degree of spontaneous viral control. Finally, our data

indicate on the level of the CD8+ T cell clonotype that high avidity epitope-specific

CD8+ T cell populations lost after early infection have higher degrees of activation

compared to lower avidity CD8+ T cells clonotypes specific for the same epitope that

persist after early infection. These data suggest that high avidity HIV-1-specific CD8+ T

cell clones recruited during early infection are more prone to clonal deletion, thus

contributing to a defective memory response in chronic infection.

Previous studies have mainly relied on interferon-γ secretion to characterize and

enumerate HIV-1-specific CD8+ T cells during early HIV-1 infection (6, 14, 15, 18, 44).

In addition to the identification of preferential viral targets (14, 38), these studies

indicated a substantially smaller magnitude and breadth of HIV-1-specific CD8+ T cells

in early infection compared to chronic infection, but did not allow for quantification of

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subsets of HIV-1-specific CD8+ T cells with alternative functional activities. Assessing a

combination of different functional readouts, our data indicate that in addition to

interferon-γ secreting HIV-1-specific CD8+ T cells, the total magnitude of HIV-1-

specific CD8+ T cells with antigen-specific secretion of TNF-α, MIP-1β or expression of

CD107a/b is also lower in early infection compared to chronic infection. Moreover, the

hierarchy of effector functions during early infection was similar to that seen in chronic

infection and closely resembled the functional pattern of CD8+ T cells observed during

chronic HIV-1-infection by other investigators (8). Overall, these data indicate that

quantitative characteristics of HIV-1-specific CD8+ T cells and the specificities of their

cytokine secretion profile are unlikely to account for the significant decline of HIV-1

viremia during early HIV-1 infection.

In this study, we observed a preferential recruitment of HIV-1-specific CD8+ T cells with

high functional avidities and slow tetramer dissociation rates during early infection. The

dominance of high avidity CD8+ T cell responses in early HIV-1-infection may be

related to the fact that these CD8+ T cells can sense their HIV-1 cognate epitopes at the

first stage of systemic antigenemia, providing these high avidity clones with a kinetic

selection advantage over CD8+ T cell clones with lower avidity (30). Furthermore, data

from in vivo studies in animal models suggested that the functional avidity of CD8+ T

cells is linked to their antiviral activity (20, 53, 55): For instance, the adoptive transfer of

high avidity LCMV-specific CD8+ T cells resulted in complete clearance of the

infection, while viral persistence was noticed following transfer of low-avidity CD8+ T

cells (3, 24). In addition, high avidity SIV-specific CD8+ T cells preferentially selected

for viral escape mutations during early SIV infection in epitopes targeted by high-avidity

SIV-specific CD8+ T cells (43, 57). The fact that no viral epitope diversification within

tested CTL epitopes occurred in our study subjects most likely reflects structural

constraints that make it difficult for the virus to escape rapidly at these positions, because

in many of our study persons, viral sequence diversification consistent with escape

mutations was detected within the respective epitopes at a later time point (after the time

points chosen for our analysis, data not shown). Overall, these data suggest that high

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avidity HIV-1-specific CD8+ T cells recruited during early infection might significantly

contribute to the dramatic decline of HIV-1 viremia during early infection.

In order to analyze the fate of these high avidity HIV-1-specific CD8+ T cell responses

primed during early infection, we performed a longitudinal analysis of the clonotypic

composition of epitope-specific CD8+ T cell populations in early and chronic infection.

These studies indicated a considerable switch of the clonotypic repertoire of HIV-1-

specific CD8+ T cells between early and chronic infection, although the persistence of

individual clonotypes was observed in eight out of ten study subjects analyzed and we

cannot exclude low-frequency persistence of some of the initially-recruited clonotypes

below the level of detection. Thus, our data suggest that the observed alterations of

epitope-specific CD8+ T cell avidity between early and chronic infection were at least

partially due to the elimination of high avidity CD8+ T cell clones initially recruited

during early infection and their subsequent replacement by CD8+ T cell clones with

lower avidity, although changes in the TCR signaling cascade between early and chronic

HIV-1 infection might have also contributed (54). In the presence of high viral loads, this

loss of high avidity CD8+ T cells can apparently occur rapidly after early infection, as in

study-subject AC-132, in whom a substantial decrease of HIV-1-specific CD8+ T cell

avidity, together with a prominent alteration of the TCR clonotype pattern, was observed

within 27 days after the initial assessment. Moreover, in the study subjects undergoing

STIs, brief exposures to high viral loads during off-therapy periods seemed to be

sufficient to lead to a considerably lower HIV-1-specific CD8+ T cell avidity. Thus, our

data indicate that the previously described fluctuations of the clonotypic repertoire of

HIV-1-specific CD8+ T cells (41) can be prominently shaped by their antigenic avidity,

as recently suggested for the clonotypic repertoire of EBV-specific CD8+ T cells (19,

47). Moreover, our results show that the previously reported loss of certain HIV-1-

specific (46, 56) or SIV-specific (48) CD8+ T cell clones after early infection can be

associated with significant changes in their functional avidity. However, our experiments

were only performed in HIV-1-specific CD8+ T cell responses recognizing

immunodominant epitopes, and it is well possible that differences in avidity and

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clonotype composition between early and chronic infection are less pronounced in

subdominant HIV-1-specific CD8+ T cell populations.

What are the mechanisms that govern the elimination or persistence of HIV-1-specific

CD8+ T cells after early infection? Our data demonstrate that high avidity CD8+ T cell

clones that were no longer detectable after early infection exhibited higher degrees of

activation, while the persisting clones with lower avidity had a lower degree of

activation, measured by CD38- and Ki-67-expression ex vivo. Thus, persistence or

elimination of initially recruited CD8+ T cells, at least under conditions of chronic

antigenic stimulation, appears to be linked to the degree of cellular activation, which

seems to depend on functional avidity of the lymphocyte. This view is supported by

previous studies in animal models of LCMV infected mice: In these studies, it was shown

that high dose viral infection not only led to an almost complete switch in the clonotypic

repertoire of epitope-specific T cells between acute and chronic infection (39), but also

resulted in the emergence of dominating T cells with intermediate-to-low avidity TCRs in

chronic infection (49). One mechanism potentially accounting for the preferential

deletion of high avidity CD8+ T cells in the presence of high level antigenemia could be

their increased sensitivity to induction of apoptosis by high levels of peptide-MHC

complexes, through a mechanism involving induction of TNF-α and TNF-α receptor II

(2, 4). In contrast, in different animal models of chronic viral or bacterial infections, low-

dose antigenic challenge was associated with a more stable clonotypic TCR pattern and

the ultimate evolution of dominant T cells with high avidity TCRs (13, 51). Our data

support a similar model in which extremely high HIV-1 loads during early HIV-1

infection lead to the overactivation and subsequent depletion of high avidity HIV-1-

specific CD8+ T cells. This appears to result in a domination of intermediate-to-low

avidity CD8+ T cells in chronic infection, because T cells, unlike B lymphocytes, cannot

take advantage of the mechanism of somatic mutation to create new cells with higher

avidity receptors during the longitudinal course of an immune response. In contrast, in

the two study subjects who spontaneously achieved a low level set point viremia in the

absence of STI, we observed an almost undetectable difference in receptor avidity

between early and chronic infection, together with substantial persistence (yet differential

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contributions) of recruited CD8+ T cell clonotypes, suggesting that the persistence of

HIV-1-specific CD8+ T cell clones after early infection critically depends on the levels of

viral control that these CD8+ T cells and the other components of the innate and adaptive

immune system are able to achieve.

When comparing short-lived high avidity HIV-1 epitope-specific CD8+ T cell clones

with long-term persisting low-avidity clones specific for the same epitope in the same

individual during early infection, we observed that these subsets of CD8+ T cells

strikingly differ in their surface expression of the IL-7 receptor α chain (CD127). IL-7

has recently been identified as a cytokine responsible for the maintenance of antigen-

specific CD8+ T cells (27, 35) and their transformation into functional antigen-specific

memory responses, a process that appears to be selectively disturbed in chronic HIV-1

infection (45). Our study suggests that the characteristic reduction of CD127 expression

(16, 40, 42, 45) on HIV-1-specific CD8+ T cells originates early in early HIV-1 infection

and that the early loss of CD127 expression on the highly activated high avidity CD8+ T

cell clones might co-determine the inability of these clones to persist during the ensuing

disease process. Since this analysis was only performed in three study persons, the

investigation of factors contributing to the impairment of an effective antigen-specific

memory response in chronic HIV-1 infection needs further elucidation in future studies.

In summary, our data suggest that high avidity CD8+ T cell clones are recruited early in

HIV-1-infection, but are subsequently deleted in the presence of high level viremia,

resulting in the persistence of intermediate-to-low avidity CD8+ T cells in chronic

infection. Persistence of epitope-specific CD8+ T cell clonotypes was associated with

low viral set-points, lower activation levels during early infection and elevated expression

of the IL-7 receptor α chain (CD127). These data provide insight into the mechanisms

that govern the recruitment, maintenance and elimination of HIV-1-specific CD8+ T cells

and contribute to the understanding of events leading to the ultimate inability to

immunologically control HIV-1 infection.

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Acknowledgement

This study was supported by the National Institutes of Health (to M. A., X. G. Y., E. S.

R., B. D. W), the Doris Duke Charitable Foundation (to X. G. Y., E. S. R., B. D. W.), the

Howard Hughes Medical Institute (to B. D. W.). The authors declare no competing

financial interests.

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Figure legends

Figure 1: Lower total magnitude of cytokine secreting and degranulating HIV-1-

specific CD8+ T cells in early HIV-1 infection compared to chronic infection. Data

reflect the total magnitude of CD8+ T cells secreting IFN-γ, TNF-α, MIP-1β or

expressing CD107a/b on the cell surface following stimulation with overlapping peptides

corresponding to the entire HIV-1 proteome in individuals with early (n=10) and chronic

HIV-1 infection (n=10). Results are presented as Box-Whisker plots, indicating the

median and the 10th

, 25th

, 75th

and 90th

percentile.

Figure 2: Higher functional avidity of HIV-1-specific CD8+ T cells in early infection

than in chronic HIV-1 infection. (A): Dot plots reflecting the proportion of IFN-γ

secreting CD8+ T cells following stimulation with the HIV-1 epitopic peptide B8-FL8 in

serial 10-fold dilutions in study individual AC-15 during early and chronic infection.

Percentages indicate the proportion of gated CD8+ IFN-γ+ lymphocytes. (B-D): Intra-

individual analysis of the functional avidity of interferon-γ secreting HIV-1-specific

CD8+ T cells recognizing the same viral epitope in early and chronic infection. Dose-

response curves indicate the proportion of CD8+ T cell secreting IFN-γ, following

peptide stimulation in serial 10-fold dilutions. Data were normalized (maximum=1).

Dashed lines and triangles correspond to chronic infection; solid lines and squares reflect

data obtained during early infection. Panel B shows data from individuals started on

antiretroviral therapy during early infection and subsequently undergoing STIs. Panels C

and D reflect data from study persons who remained completely untreated after early

infection in the presence of high level (C) or low level viremia (D). (E): Epitopic peptide

concentrations eliciting 50% of the maximum of the indicated functional activity in the

ten study individuals during early and chronic infection. Antigen-specific TNF-α

secretion was below the threshold of detection by flow cytometry in five study

individuals.

Figure 3: Slower tetramer dissociation of HIV-1-specific CD8+ T cells in early

infection compared to chronic infection. Tetramer dissociation curves of HIV-1-

specific CD8+ T cells in early (squares) and chronic (triangles) HIV-1 infection in study

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persons treated during early infection and subsequently undergoing STIs (A) or

remaining completely untreated after early infection in the presence of high level (B) or

low level viremia (C). (D): Dissociation rate constants (Koff) of HIV-1-specific CD8+ T

cells during early and chronic infection in the ten study persons described in the text.

Figure 4: Clonotypic composition of HIV-1-specific CD8+ T cell populations during

early and chronic HIV-1 infection. (A): Clonotypic composition of HIV-1-specific

CD8+ T cells during early and chronic infection in individuals with STIs. (B-C):

Clonotypic composition of HIV-1-specific CD8+ T cells during early and chronic HIV-1

infection in five study persons who remained treatment-naïve after early infection in the

presence of high-level (B) or low-level viremia (C). Each fraction corresponds to one Vβ

clonotype detected by TCR sequencing. Clonotypes labeled in black or gray were

detected both during early and chronic infection, while clonotypes labeled in white were

exclusively detected during early or chronic infection.

Figure 5: Tetramer dissociation and phenotypic characteristics of HIV-1-specific

CD8+ T cell clonotypes persisting or disappearing after early HIV-1-infection. (A):

Dot plots indicating the proportion of tetramer+ CD8+ T cells with specific TCR Vβ

chain usage during early infection in the three indicated study persons. Gating was

performed according to FSC/SSC characteristics of the lymphocyte population and CD8

expression. (B): Intra-individual comparison of tetramer dissociation rates of HIV-1-

specific CD8+ T cell clonotypes persisting or disappearing after early infection. (C):

Histograms (presented on a log scale) indicating the expression of CD127, CD38 and Ki-

67 during early HIV-1 infection in HIV-1-specific clonotypes persisting or disappearing

after early infection. Dotted lines correspond to HIV-1-specific CD8+ T cell clones being

eliminated after early infection, solid lines correspond to CD8+ T cell clones persisting

after acute HIV-1 infection. Scattered lines indicated background staining intensity (no

antibodies). In study subject AC-46, B8-FL8-specific CD8+ T cell using Vβ9 (6.5% of

all clonotypes in early infection, 0% of clonotypes in chronic infection) were compared to

those using Vβ20.1 (25% of all clonotypes in early infection, 31.6% of clonotypes in

chronic infection). In study individual AC-09, A2-IV9-specific CD8+ T cell clonotypes

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using Vβ20.1 (38% of clonotypes in early infection, 0% of clonotypes in chronic

infection) were compared to those using Vβ10.3 (7.6% of clonotypes in early infection,

62% in chronic infection). In study person AC-14, we analyzed the B8-FL8-specific

clonotypes using Vβ27 (42.5% of all clonotypes in early infection, 0% in chronic

infection) or Vβ28 (7.9% of all clonotypes in early infection, 5.8% in chronic infection)

(table 2).

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Table 1: Demographical and clinical characteristics of the study individuals.

a) cross-sectional analysis

Study cohort Age

(median, range)

Sex

(m:f ratio)

Ethnicity CD4+ T cell count

(median, range)

HIV-1 RNA

(median, range)

Individuals with early infection

(N=10)

36 years

(27-49)

10:0 9 Caucasian,

1 Hispanic

454 cells/µl

(237-784)

151500 copies/ml

(2500-750000)

Individuals with chronic infection

(N=10)

37 years (29-51) 9:1 8 Caucasian

2 African

American

547.5 cell/µl

(68-1106)

27350 copies/ml

(1743-175000)

b) longitudinal analysis

Patient ID Age

(years)

Sex Ethnicity HLA class I

type

Antiretroviral

treatment and

subsequent

STIs*

CD4+ T cell

count and HIV-1

RNA at initial

presentation

Time of T cell analysis in

chronic infection

CD4+ T cell count

and HIV-1 RNA at

the time of analysis

in chronic infection

Epitope

studied

(sequence)

AC-15 40 m Caucasian A1,3; B7,8;

Cw7

Yes 413 cells/µl

27000 cpm

Day 1752 p. p.

(765 days off therapy)

542 cells/µl

12200 cpm

B8-FL8

(FLKEKGGL)


Cw7,10

Yes 981 cells/µl

951000 cpm

Day 686 p. p.

(186 days off-therapy)

969 cells/µl

10860 cpm

B8-FL8

(FLKEKGGL)

AC-46 49 m Caucasian A1,26; B8, 51;

Cw7, 15

Yes n. d.

123000 cpm

Day 1040 p. p.


562 cells/µl

20300 cpm

B8-FL8

(FLKEKGGL)

AC-02 42 m Caucasian A11,29;

B8,44;

Cw4

Yes n. d.

4,85*106 cpm

Day 941 p. p.


625 cells/µl

18400 cpm

B8-FL8

(FLKEKGGL)

AC-09 35 m Hispanic A2,31; B61;

Cw10

Yes 365 cells/µl

>750000 cpm

Day 904 p. p.


361 cells/µl

30277 cpm

A2-IV9

(ILKEPVHGV)

AC-131 32 m Caucasian A1, 3; B8,15;

Cw7

No 475 cells/µl

>750000 cpm

Day 477 p. p.

n. d.

175000 cpm

B8-EI8

(EIYKRWII)

AC-177 44 m Caucasian A1,2; B8; Cw

7

No 363 cells/µl

26800 cpm

Day 162 p. p.

217 cells/µl

427000 cpm

B8-EI8

(EIYKRWII)

AC-132 30 m Caucasian A3,68; B

14,44; Cw8, 16

No 815 cells/µl

5,6*106 cpm

Day 27 p. p.

790 cells/µl

115000 cpm

A3-QK10

(QVPLRPMTYK)


Cw1,7

No n. d.

468000 cpm

Day 205 p. p.

874 cells/µl

2830 cpm

B27-KK10

(KRWIILGLNK)

AC-121 37 m Hispanic A1, 24; B35,

57; Cw7,12

No 858 cells/µl

12600 cpm

Day 562 p. p. n. d.

5940 cpm

B57-KF11

(KAFSPEVIPMF)

* For details on the length of antiretroviral treatment, length of treatment interruptions and the evolution of HIV-1 RNA and CD4+ T

cells counts, see (29). (p.p.= post presentation, cpm= copies per milliliter)

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Table 2: Clonotypic composition of epitope-specific CD8+ T cells during early and chronic HIV-1 infection (Nucleotide data are provided when multiple nucleotypes encoding for identical CDR3 amino acid sequences were detected in clonotypes persisting between early and chronic infection.)

Study Person Primary HIV - 1 Infection Chronic HIV -1 Infection

Epitope Vβ CDR3 Jβ frequency Vβ CDR3 Jβ frequency

AC-46 29.1 CSV WGTGKTYEQY FG-2.7 19/75

B8-FL8 29.1 CSV WGEGRSYEQY FG-2.7 5/75

20.1 CSA TILAGVPYGEQY FG-2.7 17/75 20.1 CSA TILAGVPYGEQY FG-2.7 24/76

ACGATCCTAGCGGGAGT T CCCTATGGGGAGCAGTAC 17/17 ACGATCCTAGCGGGAGT T CCCTATGGGGAGCAGTAC 23/24

ACGATCCTAGCGGGAGT C CCCTATGGGGAGCAGTAC 1/24

20.1 CSA TILAGVPYGGQH FG-2.7 1/75

20.1 CSA SEGTSSYEQY FG-2.7 1/75

7.3 CASS FDREVTGELF FG-2.2 7/75

7.3 CASS PDGGNTEAF FG-1.2 2/75

9 CASS VGAGTEAF FG-1.1 5/75

10.3 CAI SESGYGGPPGANVLT FG-2.6 1/75

10.3 CTI SESGYRGPPGANVLT FG-2.6 1/75

10.3 CAI SEPGYRGPPGANVLT FG-2.6 1/75

10.3 CAI SESGYRGPPGANVLT FG-2.6 1/75

28 CAS RPTDRNTGELF FG-2.2 3/75

28 CASS LAAGGPYEQY FG-2.7 1/75

7.9 CASS PPSGSYEQY FG-2.7 1/75

7.9 CASS LESGQRPYEQY FG-2.7 1/75

19 CASS IGPLEGNEQF FG-2.1 2/75 19 CASS IGPLEGNEQF FG-2.1 1/76

27 CASS WGQGQLSYEQY FG-2.7 2/75 27 CASS WGQGQLSYEQY FG-2.7 35/76

5.4 CASS YRGQGNYGYT FG-1.2 1/75

6.3 CASS YERGMNTEAF FG-1.1 1/75

6.5 CASS MGQGATEAF FG-1.1 1/75

6.6 CASS YPMGANEKLF FG-1.4 1/75

27 CAS RIGQGTVGELF FG-2.2 3/76

29.1 CSV DNSYEQY FG-2.7 3/76

29.1 CSV VGLESSYEQY FG-2.7 2/76

29.1 CSV GENTEAF FG-1.1 1/76

7.2 CASS IFGSPFNQPQH FG-1.5 4/76

19 CAT LREGPTTGELF FG-2.2 3/76

AC-14 27 CASS LGQGLANYGYT FG-1.2 17/40

B8-FL8 20.1 CSA RPMAASGLTYEQY FG-2.7 6/40

6.1 CASS EYGAAVYEQY FG-2.7 8/40

7.3 CASS LNLRGSSGNTIY FG-1.3 5/40

28 CASS SNPGTSGGYSYEQY FG-2.7 3/40 28 CASS SNPGTSGGYSYEQY FG-2.7 3/51

7.9 CASS GSPGLAGEQT FG-2.1 1/40

6.3 CASS LLGQYNEQF FG-2.1 22/51

6.3 CASS LLGQCNEQF FG-2.1 1/51

6.3 CASS FHPGQGASYSNQPQH FG-1.5 11/51

6.6 CASS YERGGLPKNIQY FG-2.4 10/51

27 CASS LGQGRSLGHEQY FG-2.7 3/51

28 CASS PRDRVIEDTQY FG-2.1 1/51

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Table 2: Clonotypic composition of epitope-specific CD8+ T cells during primary and chronic HIV-1 infection (Nucleotide data are provided when multiple nucleotypes encoding for identical CDR3 amino acid sequences were detected in clonotypes persisting between early and chronic infection.)



AC-09 20.1 CSA RQYRTDMNTEAF FG-1.1 15/39

A2-IV9 29.1 CSV EVGAGETQY FG-2.5 7/39

29.1 CSV EDLGNEQF FG-2.3 1/39

29.1 CSA ETSDSYEQY FG-2.7 1/39

6.6 CASS YEWGGLPKNIQY FG-2.4 7/39

27 CAS RLVGIGNSPLH FG-1.6 5/39

10.3 CAI SEDGGETQY FG-2.5 3/39 10.3 CAI SEDGGETQY FG-2.5 29/47

10.3 CAI SGDGGETQY FG-2.5 1/47

10.3 CTI SEWGGETQY FG-2.5 1/47

28 CASS FGGDEQH FG-1.5 4/47

28 CASS LGGDTQY FG-2.1 3/47

28 CASS LGGDEQY FG-2.7 1/47

28 CASS FGGDTQY FG-2.1 1/47

28 CASS LRDEQY FG-2.7 1/47

29.1 CSV EDSGNEQF FG-2.1 2/47

29.1 CSV EDRANEQY FG-2.7 1/47

29.1 CSV RAPPMTSPTDEQY FG-2.7 1/47

6.2 CASS LDGGETQY FG-2.5 1/47

6.2 CASS QDGGETQY FG-2.5 1/47

AC-02 7.2 CASS EPALAEQYYEQY FG-2.7 39/46

B8-FL8 7.2 CASS FTPGTGAPYSNQPQH FG-1.5 4/46

29.1 CSV EGGSAYEQY FG-2.7 3/46


7.3 CASS LDREVTGELF FG-2.2 1/45

20.1 CSA HLYRAYGYT FG-1.2 5/45

19 CASS MGQHSNQPQH FG-1.5 4/45

7.2 CASS LAWGRAESSYNEQF FG-2.1 2/45

29.1 CSV WGTGKTYEQY FG-2.7 1/45

AC-15 6.1 CAS KWDPGQGSHYSNQPQH FG-1.5 17/37 6.1 CAS KWDPGQGSHYSNQPQH FG-1.5 2/41

B8-FL8 29.1 CSV EPSGRARTYNEQF FG-2.1 10/37

30 CAW DVKDRRIGNEQF FG-2.1 5/37

20.1 CSA RDSGRGIENYEQY FG-2.7 2/37

20.1 CSA SSQRGGIYEQY FG-2.7 2/37

7.9 CASS LVGVGTSDEQF FG-2.1 1/37


27 CASS LGQGLANYGYT FG-2.1 11/41

27 CASS LSPGTSGSRANEQF FG-2.1 1/41

28 CASS LELAGEDYEQY FG-2.7 4/41

28 CASS LSSNEQF FG-2.1 2/41

9 CASS TLTSGGARDEQF FG-2.7 3/41

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AC-131 29.1 CSV RDGYEQY FG-2.7 23/46

B8-EI8 7.2 CASS PVRGRTEAF FG-1.1 4/46

7.2 CASS LVGPGGREKLF FG-1.4 3/46

9 CASS VLTGGRETQY FG-2.5 7/46

19 CASS IIKGNQPQH FG-1.5 4/46

19 CASS SGLPTNEKLF FG-1.4 3/46

6.1 CAS PYMDARSEAF FG-1.1 2/46

9 CASS VVGDFRETQY FG-2.5 48/48

AC-177 6.6 CASS YGGTEAF FG-1.1 23/49 6.6 CASS YGGTEAF FG-1.1 18/46

B8-EI8 TA C GG A GG C ACTGAAGC TT TC 14/23

TA C GG G GG C ACTGAAGC AT TC 1/23

TA C GG G GG C ACTGAAGC TC TC 1/23

TA C GG A GG G ACTGAAGC TT TC 4/23

TA C GG A GG A ACTGAAGC TT TC 1/23 TA C GG A GG A ACTGAAGC TT TC 10/18

TA T GG G GG G ACTGAAGC TT TC 2/23

TA C GG G GG C ACTGAAGC TT TC 5/18

TA C GG G GG G ACTGAAGC TT TC 3/18

7.2 CASS LPGQGRTPLH FG-1.6 4/49 7.2 CASS LPGQGRTPLH FG-1.6 7/46

TTACCTGGACAGGGAAGGAC A CCCCTCCAC 4/4 TTACCTGGACAGGGAAGGAC A CCCCTCCAC 6/7

TTACCTGGACAGGGAAGGAC G CCCCTCCAC 1/7

7.2 CASS LETRNSPLH FG-1.6 1/49

7.2 CASS FLPKNEQF FG-2.1 4/49

7.2 CASS LVRDDRIEQY FG-2.7 2/49

7.2 CASS FPGQGRTEAF FG-1.1 2/49

20.1 CSA RLSYEQY FG-2.7 7/49

20.1 CSA RDNYEQY FG-2.7 2/49

20.1 CSA RDSYEQY FG-2.7 1/49

20.1 CSA RLDYEQY FG-2.7 1/49

20.1 CSA RYDYEQY FG-2.7 1/49

7.3 CASS LIPSGGRNEQF FG-2.1 1/49

9 CASS AITSGGARDEQF FG-2.1 11/46

9 CASS VGGDHREEQH FG-2.7 3/46

9 CASS AGDIQY FG-2.4 2/46

9 CASS AGGIQY FG-2.4 1/46

10.3 CAT IRTGFSSYEQY FG-2.7 4/46

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AC-132 6.6 CASS YSRGSGNTIY FG-1.3 19/43 6.6 CASS YSRGSGNTIY FG-1.3 7/42

A3-QK10 TACTCTAGGGG A TCTGGAAACACCATATAT 6/19 TACTCTAGGGG A TCTGGAAACACCATATAT 5/7

TACTCTAGGGG C TCTGGAAACACCATATAT 13/19 TACTCTAGGGG C TCTGGAAACACCATATAT 2/7

6.6 CASS PYRGPNTEAF FG-1.1 1/43 6.6 CASS PYRGPNTEAF FG-1.1 1/42

10.3 CAI SAGASFVTRSTDTQY FG-2.1 6/43 10.3 CAI SAGASFVTRSTDTQY FG-2.1 1/42

10.3 CAI RSTDTQY FG-2.1 1/43

6.1 CAS RQQGFVFEAKNIQY FG-2.4 3/43

6.1 CASS EEVEAF FG-1.1 1/43

20.1 CSA PTSGSAAF FG-1.1 3/43

20.1 CSA RDSIQFSSNQPQH FG-1.5 1/43

6.2 CASS YSMTSGSFSDLGAKNIQY FG-2.4 1/43

6.2 CAS RPGPVKNTGELF FG-2.2 1/43 6.2 CAS RPGPVKNTGELF FG-2.2 7/42

9 CASS LYHNTGELF FG-2.2 1/43

9 CASS GGAHFSKIPLAGYNEQF FG-2.1 1/43 9 CASS GGAHFSKIPLAGYNEQF FG-2.1 3/42

5.4 CASS RTDFTAGELF FG-2.2 1/43

27 CASS LTGHPYEQY FG-2.7 1/43 27 CASS LTGHPYEQY FG-2.7 9/42

28 CASS PGEKYEQY FG-2.1 1/43

29.1 CSV EDRHYEQY FG-2.7 1/43 29.1 CSV EDRHYEQY FG-2.7 5/42

6.6 CASS YSRGAGNTIY FG-1.3 7/42

6.6 CAS TRSGGFRDEQY FG-2.7 1/42

7.9 CASS RRDHQETQY FG-2.5 1/42

AC-160 5.4 CASS LTAPDTEAF FG-1.1 30/45 5.4 CASS LTAPDTEAF FG-1.1 8/53

B27-KK10 5.4 CASS GTAPAAEAF FG-1.1 1/45

5.4 CASS STAPDTEAF FG-1.1 1/45

5.4 CASS TAPGTEAF FG-1.1 1/45

27 CASS RSTGELF FG-2.2 10/45 27 CASS RSTGELF FG-2.2 27/53

20.1 CSA RDQRDYQETQY FG-2.5 2/45 20.1 CSA RDQRDYQETQY FG-2.5 1/53

27 CASS VRTGELF FG-2.2 14/53

27 CASS PRTGELF FG-2.2 3/53

AC-121 19 CASS GQGYGYT FG-1.2 24/46 19 CASS GQGYGYT FG-1.2 25/45

B57-KF11 GG A CAGGG G TATGGCTACACC 23/24 GG A CAGGG G TATGGCTACACC 22/25

GG G CAGGG G TATGGCTACACC 1/24

GG A CAGGG A TATGGCTACACC 3/25

19 CAS TGGGYGYT FG-1.2 8/46

19 CASS GQDYGYT FG-1.2 5/46 19 CASS GQDYGYT FG-1.2 10/45

19 CASS GQGYGYA FG-1.2 1/46

19 CAS TGSGYGYT FG-1.2 1/46

19 CASS GQEYGYT FG-1.2 1/46 19 CASS GQEYGYT FG-1.2 1/45

6.1 CAS TDSYGYT FG-1.2 6/46 6.1 CAS TDSYGYT FG-1.2 5/45

ACTGACAGCTATGGCTA C ACC 6/6 ACTGACAGCTATGGCTA C ACC 4/5

ACTGACAGCTATGGCTA T ACC 1/5

19 CASS GGSYGYT FG-1.2 4/45

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Table 3: Sequences of the CD8+ T cell target epitopes in the autologous virus of the study patients.

Patient ID Timepoint of viral sequencing

Epitope sequence in autologous virus

AC-15 Day 43 p. p.

Day 1332 p. p.

Day 1823 p. p.

FLKEKGGL

FLKEKGGL

FLKEKGGL

AC-14 Day 81 p. p.

Day 672 p. p.

FLKEKGGL

FLKEKGGL

AC-46 Day 76 p. p.

Day 1040 p. p.

FLKEKGGL

FLKEKGGL

AC-02 Day 28 p. p.

Day 934 p. p.

FLKEKGGL

FLKEKGGL

AC-09 Day 1 p. p.

Day 904 p. p.

ILKEPVHGV

ILKEPVHGV

AC-131 Day 94 p. p.

Day 477 p. p.

EIYKRWII

EIYKRWII

AC-177 Day 27 p. p.

Day 189 p. p

EIYKRWII

EIYKRWII

AC-132 Day 6 p. p.

Day 27 p. p.

QVPLRPMTYK

QVPLRPMTYK

AC-160 Day 33 p. p.

Day 205 p. p.

KRWIILGLNK

KRWIILGLNK

AC-121 Day 63 p. p.

Day 476 p. p.

Day 562 p. p.

KAFSPEVIPMF

KAFSPEVIPMF

KAFSPEVIPMF

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0

1

2

3

5

MIP-1b IFN-g TNF-a CD107a/b

CD

8+

T c

ells

(%

)

p=0.01p<0.01 p=0.14 p=0.02

Figure 1

chronic infection primary infection

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10 -4 10 -3 10 -2 10 -1 100 1010.0

0.4

0.8

1.2

peptide concentration (µg/ml)

10 -4 10 -3 10 -2 10 -1 1000.0

0.4

0.8

1.2


10 -4 10-3 10-2 10-1 100 1010.0

0.4

0.8

1.2


norm

alize

d p

roport

ion o

fC

D8+

T c

ells

10 -4 10-3 10-2 10-1 100 1010.0

0.4

0.8

1.2


norm

alize

d p

roport

ion o

fC

D8+

T c

ells

EC50

=0.0006

EC50

=0.032

10 -4 10-3 10-2 10-1 100 1010.0

0.4

0.8

1.2


norm

alize

d p

roport

ion o

fC

D8+

T c

ells

10 -4 10-3 10-2 10-1 100 1010.0

0.4

0.8

1.2


norm

alize

d p

roport

ion o

fC

D8+

T c

ells

A

CD

8primary HIV-1

infection

chronic HIV-1

infection

CD

8

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

interferon-γ

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

interferon-γ

0.001µg/ml 0.01µg/ml 0.1µg/ml 1µg/ml 10µg/ml

0.03% 0.21% 0.27% 0.28% 0.26%

0.03% 0.06% 0.14% 0.15% 0.22%

AC-46

AC-02

AC-131

AC-160 AC-121

B

C

D

E

10 -4 10 -3 10 -2 10 -1 1000.0

0.4

0.8

1.2


AC-14

10 -4 10 -3 10 -2 10 -1 100 1010.0

0.4

0.8

1.2


AC-15

10 -4 10 -3 10 -2 10 -1 100 1010.0

0.4

0.8

1.2


AC-177

10 -4 10 -3 10 -2 10 -1 100 1010.0

0.4

0.8

1.2


AC-132

AC-09

EC50

=0.005

EC50

=0.012

EC50

=0.0006

EC50

=0.009

EC50

=0.0006

EC50

=0.0028

EC50

=0.0042

EC50

=0.026

EC50

=0.004

EC50

=0.029

EC50

=0.002

EC50

=0.01

EC50

=0.004

EC50

=0.11

EC50

=0.006

EC50

=0.0085

EC50

=0.0008

EC50

=0.001

Interferon-γ CD107a/b MIP-1β

primary

infection

primary

infection

chronic

infection

chronic

infection

p=0.05 p=0.04 p=0.02

0.000

0.025

0.050.05

0.15

EC

50

(µg/m

l)

primary

infection

chronic

infection

0.000

0.005

0.010

EC

50 (

µg/m

l)

primary

infection

chronic

infection

TNF-α

p=0.16

Figure 2

0.000

0.005

0.010

EC

50 (

µg/m

l)

0.000

0.005

0.010

EC

50 (

µg/m

l)

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0.00

0.01

0.02

Kof

f (m

in-1

)

0 30 60 90 12032

64

128

time (minutes)

rela

tive

tota

l fluore

scence

(log

2)

Koff=0.0006Koff=0.0023

AC-46

0 30 60 90 12032

64

128

time (minutes)

rela

tive

tota

l fluore

scence

(log 2

)

0 30 60 90 12032

64

128

time (minutes)

rela

tive

tota

l fluore

scence

(log 2

)

Koff=0.0016

Koff=0.003

AC-02

0 30 60 90 12032

64

128

time (minutes)

AC-14

Koff=0.002Koff=0.0057

0 30 60 90 12032

64

128

time (minutes)

AC-15

Koff=0.003Koff=0.0053

0 30 60 90 12032

64

128

time (minutes)

AC-09

Koff=0.0018Koff=0.0066

0 30 60 90 12032

64

128

time (minutes)

rela

tive

tota

l fluore

scence

(log

2)

AC-131

Koff=0.004

Koff=0.0072

0 30 60 90 12032

64

128

time (minutes)

AC-177

Koff=0.0038

Koff=0.0062

0 30 60 90 12016

32

64

128

time (minutes)

AC-132

Koff=0.009

Koff=0.015

A

B

C

D

AC-160

Koff=0.0049Koff=0.0062

0 30 60 90 12032

64

128

time (minutes)

AC-121

Koff=0.0042Koff=0.0049

primary

infection

chronic

infection

p<0.01

Figure 3

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early HIV-1

infection

chronic HIV-1

infectionA

AC-02

B8-FL8

AC-14

B8-FL8

AC-09

A2-IV9

AC-15

B8-FL8

AC-46

B8-FL8

Bearly HIV-1

infection

chronic HIV-1

infection

AC-131

B8-EI8

AC-177

B8-EI8

AC-132

A3-QK10

AC-160

B27-KK10

AC-121

B57-KF11

C

Figure 4

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0 30 60 90 12032

64

128

time (minutes)

rela

tive tota

lflu

ore

scen

ce

(lo

g2)

0 30 60 90 12032

64

128

time (minutes)

rela

tive tota

lflu

ore

scen

ce

(lo

g2)

Vβ27

A

B

Figure 5

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

101

102

103

104

101 102 103 1040

B8

-FL

8 t

et

A2

-IV

9 t

et

Vβ20Vβ9

Vβ20 Vβ10.3

9% 33%

30% 7%

B8

-FL

8 t

et

101

102

103

104

101 102 103 1040

29%8%

Vβ28

101

102

103

104

101 102 103 1040

43%

AC-46

AC-09

AC-14

0 30 60 90 12016

32

64

128

time (minutes)

rela

tive tota

lflu

ore

scen

ce

(lo

g2)

Vβ9 (Koff=0.0006)

Vβ20.1 (Koff=0.006)

AC-46

AC-09

AC-14

Vβ20.1 (Koff=0.0017)

Vβ10.3 (Koff=0.012)

Vβ27 (Koff=0.002)

Vβ28 (Koff=0.0048)

101 102 103 1040

20

10

30

40

101 102 103 1040

20

10

30

40

101 102 103 1040

20

10

30

40

Ki-67CD127

(IL-7R α chain)CD38

101 102 103 1040

20

10

30

40

101 102 103 1040

20

10

30

40

101 102 103 1040

20

10

30

40

C

101 102 103 1040

20

10

30

40

101 102 103 1040

20

10

30

40

101 102 103 1040

20

10

30

40

AC-46

AC-09

AC-14

MFI=89MFI=22

MFI=68MFI=27

MFI=134MFI=51

MFI=71MFI=256

MFI=313MFI=632

MFI=401MFI=541

MFI=12MFI=18

MFI=21MFI=36

MFI=16MFI=67

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selective depletion of high-avidity hiv-1-specific cd8+ t...

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