Early proliferation of CCR5+ CD38+++ antigen-specific CD4+ Th1 effector cells during primary HIV-1 infection

doi:10.1182/blood-2005-01-0206Prepublished online May 19, 2005;

Cooper and Anthony D KelleherRamacciotti, Dick Quan, Robert Finlayson, John Kaldor, Eric S Rosenberg, Bruce D Walker, David A John J Zaunders, Mee Ling Munier, Daniel E Kaufmann, Susanna Ip, Pat Grey, Don Smith, Tim cells during primary HIV-1 infectionEarly proliferation of CCR5+ CD38+++ antigen-specific CD4+ Th1 effector

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1

TITLE: Early proliferation of CCR5+ CD38+++ antigen-specific CD4+ Th1

effector cells during primary HIV-1 infection.

AUTHORS: John J. Zaunders1, Mee Ling Munier1,3, Daniel E. Kaufmann2, Susanna

Ip1,3, Pat Grey3, Don Smith3, Tim Ramacciotti3, Dick Quan4, Robert

Finlayson5, John Kaldor3, Eric S. Rosenberg2, Bruce D. Walker2, David

A. Cooper1,3 and Anthony D. Kelleher1,3, on behalf of the PHAEDRA

Study Team 6

RUNNING TITLE: HIV-specific CD4+ T cells in acute infection

AFFILIATIONS: 1 Centre for Immunology, St. Vincent’s Hospital, Sydney, NSW, Australia2 Partners AIDS Research Center, Massachusetts General Hospital, Boston, MA, USA3 National Centre in HIV Epidemiology and Clinical Research, University of NSW, Sydney, NSW, Australia.4 Holdsworth House General Practice, Sydney, NSW Australia5 Taylor Square Private Clinic, Sydney, NSW, Australia

Email: [email protected] for correspondence: John Zaunders,

Centre for Immunology,St Vincent’s Hospital,Victoria St, Darlinghurst, NSW 2010 Australia

Phone: +61-2-8382-3700Fax: +61-2-8382-2391

Word count: Text: 4666 Abstract: 199

6 Members of the PHAEDRA Study Team are: P.Grey, J.Kaldor, D.A.Cooper, T.Ramacciotti, K.Petoumenos, D.Smith, M.Bloch, N.Medland, R.Finlayson, A.McFarlane, N.J.Roth, C.Workman, A.Carr, A.D.Kelleher, J.Zaunders and P.Cunningham.

Abbreviations: PHI (primary HIV-1 infection); ICC (intracellular cytokine assay).

Keywords: HIV-1, CD4+ T cells, CCR5, Antigen-specific, Cytotoxic T Lymphocytes

Blood First Edition Paper, prepublished online May 19, 2005; DOI 10.1182/blood-2005-01-0206

Copyright © 2005 American Society of Hematology




2

Financial support: The National Centre in HIV Epidemiology and Clinical Research is supported by the Commonwealth Department of Health and Ageing through the Australian National Council on AIDS, Hepatitis C and Related Diseases. This project was funded by an AIEDRP grant through the NIH Division of AIDS, and a program grant from the Australian National Health and Medical Research Council. The authors have no conflicting financial interests.




3

Abstract

We investigated whether HIV-1 antigen-specific CD4+ T cells expressed the viral

co-receptor, CCR5, during primary HIV-1 infection (PHI). In the peripheral blood of

subjects with very early PHI (<22 days post-onset of symptoms), there was a 10 to 20-

fold increase in the proportion of highly activated (CD38+++) and proliferating (Ki-67+)

CD4+ T cells which expressed CCR5+, and were mostly TIA-1+perforin+granzymeB+.

In the same patient samples, CD4+ T cells producing IFN-γ in response to HIV Gag

peptides were readily detected (median 0.58%) by intracellular cytokine assay – these

cells were again predominantly CD38+++, Ki-67+ and TIA-1+, as well as Bcl-2low. On

average, 20% of the Gag-specific CD4+ T cells also expressed IL-2 and were CD127(IL-

7R)+. Taken together, these results suggest that Gag-specific Th1 effector cells express

CCR5+ during the primary response and may include precursors of long-term self-

renewing memory cells. However, in PHI subjects with later presentation, antigen-

specific CD4+ T cells could not be readily detected (median 0.08%), coinciding with a 5-

fold lower level of the CCR5+CD38+++ CD4+ T cells. These results suggest that the

anti-viral response to HIV-1 infection includes highly activated CCR5+CD4+ cytotoxic

effector cells, which are susceptible to both apoptosis and cytopathic infection with HIV-

1, and rapidly decline.




4

Introduction

Antigen-specific memory CD4+ T cells are not often found in untreated chronic

HIV-1 infection, using the standard in vitro proliferation assay 1. It remains unknown

whether the scarcity of proliferative HIV-specific CD4+ T cells is due to dysfunction 2,3,

inappropriate apoptosis 4 or is a result of cytopathic infection of these cells 5. This deficit

of antigen-specific CD4+ T cells may represent a major impediment to immune control

of HIV-1 infection. In most, but not all, animal models of adaptive immune responses to

viral infection, optimal clearance of virus depends on synergistic interactions between

antigen-specific populations of helper CD4+ T cells, antibody-producing B cells and

cytotoxic CD8+ T cells 6,7. In particular, it is believed that effective CD8+ T cell function

in HIV-1 infection is reliant on CD4+ T cell function 8.

Previous studies of primary immune responses to viral infection in mice have

shown that antigen-specific Th1 CD4 responses can be readily detected in the early

stages of the infection, but rapidly decline as antigen is cleared 9,10. Similarly, human

CD4+ T cell immune responses to primary herpesvirus infections exhibit a peak response

in the first few weeks 11,12 with markedly reduced responses at follow-up. These results

suggest that antigen-specific CD4+ T cells should be generated at a relatively high level

during primary HIV-1 infection.

IFN-γ producing antigen-specific CD4+ T cells have been demonstrated in

primary HIV-1 infection, despite high levels of viremia 13-17. Furthermore, proliferative

responses were maintained if antiretroviral therapy was instituted during acute HIV-1




5

infection 13,14,18. Further evidence for the presence of antigen-specific CD4 T cells is the

production of high affinity, isotype-switched antibodies to HIV-1, which presumably

requires the provision of help for B cell responses by CXCR5+ CD4+ follicular helper T

cells 19,20.

Although CD4+ T cells which proliferate in vitro in response to HIV-1 antigens

are mostly absent in untreated chronically infected subjects, an average of approximately

0.1% of peripheral blood CD4+ T cells capable of producing IFN-γ can be detected in

most HIV-infected individuals by ELISPOT or by intracellular cytokine assay 21,22. It is

an absence of those HIV-specific CD4+ T cells which synthesise IL-2 23-27 that probably

causes the proliferative defect in viremic patients 26,28. However, IL-2-producing CD4+

memory cells typically belong to the CCR7+, CCR5-negative central memory subset 29

and therefore are not directly susceptible to infection by CCR5-tropic HIV-1 strains in

early infection. Furthermore, two recent studies of HIV-specific IFN-γ producing CD4+

T cells during PHI has shown that the vast majority were CCR7-negative cells which did

not produce IL-2 16,30. In the latter study, IL-2-producing antigen-specific CD4+ T cells

were readily detected in control subjects with EBV or HSV infections 30.

A possible explanation may come from studies of murine models, which suggest

that resting memory CD4+ T cells arise directly from effector cells 31,32. Theoretically,

then, IL-2-producing HIV-specific memory cells may be derived from Th1 effector cells,

which reportedly express CCR5 33. We have previously found that approximately 50%,

on average, of CD4+ T cells proliferating in vivo during primary HIV-1 infection

express CCR5 34. If the proliferating CD4+ T cells included antigen-specific effector

cells, then it is likely that many of these activated effector cells were CCR5+. Recently




6

we confirmed the existence of CCR5+ antigen-specific CD4+ T cells, in a HIV+ long-

term non-progressor infected with an attenuated strain of HIV-1 35. These CCR5+ cells

produced IL-2 and proliferated strongly in response to HIV-1 p24. Furthermore, these

cells were also cytotoxic T lymphocytes (CTL), similar to cloned CD4+ T cells from

patients treated early in acute HIV-1 infection 36 and consistent with our previous finding

that perforin+ CD4+ T cells were elevated in HIV-1 infection 37. A similar population of

CMV-specific CCR5+ cytotoxic CD4+ T cells was also found in healthy adults, at

surprisingly high frequencies 35.

We hypothesized that there would be CCR5+ antigen-specific CD4+ T cells

produced at the earliest stages of primary HIV-1 infection. We first examined the

phenotype of activated, proliferating effector CD4+ T cells in PHI, and then used the

flow cytometric intracellular cytokine assay to determine the cell surface phenotype of

antigen-specific CD4+ T cells, which subsequently allowed us to further define these

cells in samples of fresh whole blood.




7

Materials and Methods

Subjects

A total of 33 subjects, diagnosed with primary HIV-1 infection 34, and who then

enrolled in the PHAEDRA observational cohort, were included in this study. All subjects

were males whose risk group was sex with males. Symptoms associated with primary

HIV-1 infection were recorded as previously described 38.

Subjects were subdivided into two groups, early PHI (n = 19) and late PHI (n =

14), based on serology at presentation and clinical history of onset of symptoms (Table

1). A Western Blot intensity score (Ramacciotti et al, manuscript in preparation) was

calculated as the sum of each band, multiplied by its intensity on a scale of 1+ to 3+ (for

example, a subject with no bands will have a score of 0, while a subject with three bands

of 1+ intensity and one band of 2+ intensity will have a score of 5). Early PHI was

defined as having a Western Blot intensity score ≤ 5, and in our subject group, this

corresponded to presentation within 22 days following onset of symptoms. Late PHI was

defined as an intensity score > 5, and all these subjects in our cohort had a score of 12 or

higher, and presented 28 days or more since onset of symptoms. Two subjects in the late

PHI group did not record any symptoms of PHI, and therefore the midpoint between their

last negative and first positive HIV antibody tests was used as an estimate of infection

date.

One subject, #9400101, was originally diagnosed as early PHI, but was

concurrently diagnosed as primary CMV infection, exhibiting a new CMV IgM+

reactivity, together with detectable CMV viral load, one week after PHI diagnosis.

Therefore this individual has been excluded from the cohort analyses, and has been




8

considered separately. CMV serology was performed by routine diagnostic assay (Vidas,

BioMerieux, Marcy-l'Etoile, France).

Healthy HIV-negative university and hospital staff members were recruited as

controls for this study. The PHAEDRA study was approved by the local institutional

ethics committee, and all subjects gave informed consent.

Cell surface phenotyping of peripheral blood CD4+ T cells

Staining of T lymphocyte subsets in fresh peripheral blood and 6-colour flow

cytometric analysis on a dual-laser LSR II flow cytometer (Becton-Dickinson), was

performed as previously described 35. Monoclonal antibodies used were CD3-PerCP-

Cy5.5, CD4-PE-Cy7, CD8-APC-Cy7, CD11a-FITC, CD27-FITC, CD28-PE, CD38-

APC, -PE and -FITC, HLA-DR-FITC, CD57-FITC, CD62L-FITC, CD95-PE,

CD154(CD40L)-PE, and IL-2Rα (CD25)-PE and –FITC (from Becton-Dickinson, San

Jose, CA); CXCR4-PE, CCR5-APC, -PE and –FITC, CCR7-PE, CD45RA-APC,

CD45RO-FITC, TNFR2 (CD120b)-PE, Ki-67-FITC, Bcl-2-PE and -FITC, IL-12Rβ1-PE,

Perforin-FITC and Granzyme A-FITC (Pharmingen); TIA-1/GMP-17-PE, IL-

2Rβ (CD122)-PE, and IL-7R (CD127)-PE (Beckman Coulter, Hialeah, FL); IL-18R-PE

(R&D Systems, Minneapolis, MN); and Granzyme B-APC (Caltag, Burlingame, CA).

For CCR5 analysis, whole blood was processed an average of 1.75 hr after

venepuncture, to minimize spontaneous loss of this marker, as previously described 35.

Intracellular staining was performed using FACSlyse and FACSPermeabilizing Reagents

(Becton-Dickinson) according to the manufacturer’s directions, and analysed as

previously described.34




9

Intracellular cytokine assay

A subset of 14 consecutive subjects (7 early PHI and 7 late PHI) were studied for

the presence of HIV Gag- and Nef-specific CD4+ T cells using a whole blood

intracellular cytokine (ICC) assay 35 using 6-colour flow cytometry. Overlapping HIV-1

Gag 15-mer peptides, from the sequence of strain HXB2, were obtained from the NIH

AIDS reference reagents program. Gag peptides were used as a pool of 122 peptides, at

an individual concentration of 2µg/ml each. For analysis, 300,000 events were collected,

T lymphocytes were first gated on CD3-PerCP-Cy5.5 versus side scatter, then on CD4-

PE-Cy7-positive/CD8-APC-Cy7-negative cells, and finally IFN-γ−APC+ cells were

analysed for the various FITC and PE antibodies. This method has a validated cut-off for

positive results of 0.08% of CD4+ T cells, based on background results plus 3 times the

standard deviation, from study of sixteen HIV-negative controls (Munier et al,

manuscript in preparation).

Statistics

Lymphocyte phenotyping results were expressed as a percentage of CD4+ T

lymphocytes. Results for each cohort were expressed as medians and interquartile ranges.

The Mann-Whitney U test was performed to compare early and late subgroups of primary

HIV-1 infection subjects with each other and with the HIV-negative controls, using

Statview v5.0 for Macintosh (Abacus Concepts, Berkeley, CA, USA). A two sided p

value <0.05 was considered statistically significant. The relationship between different

phenotypes was determined by Spearman Rank Correlation (Statview). Graphs of




10

longitudinal data were plotted with Lowess curve-fitting, with tension set to 66%

(Statview).




11

Results

Changes in CD4+ T cell subsets during early PHI

Several subsets of CD4+ T cells were present only as very minor subsets in HIV-

negative controls, but were found to be prominent in subjects with early PHI. In

particular, CCR5+CD38+++ CD4+ T cells (Figure 1a) were highly elevated during early

PHI, compared with late PHI and healthy adult controls (medians: 5.3% vs 1.0% vs 0.3%

of CD4+ T cells, respectively; Figure 1e).

CCR5+TIA+Ki-67+ (Figure 1b) and Perforin+GranzymeB+ (Figure 1c) CD4+ T

cells were also elevated during early PHI, compared with late PHI and healthy controls

(3.6% vs 1.0% vs 0.2% and 11.6% vs 5.% vs 1.6%, respectively; Figure 1e).

Similarly, there was an elevation in CD127 (IL-7R)-negative CD57-negative

(Figure 1d) CD4+ T cells during early PHI, compared with late PHI and healthy controls

(medians: 13.7% vs 6.9% vs 5.2%, respectively; Figure 1e). These CD127-negative

CD4+ T cells were also CD45RA-negative (not shown).

All the above subset changes in early PHI were statistically significant when

compared to healthy adult controls (p<0.001), and also when compared with late PHI

(p<0.01). Differences between late PHI subjects and HIV-negative controls were also

statistically significant (p<0.01; except for CD127(IL-7R)-negative CD57-negative

CD4+ T cells, p=0.05).

Intracellular cytokine assay of HIV-1 Gag-specific CD4+ T cells during PHI

In six out of seven subjects studied during early PHI, Gag-specific CD4+ T cells

were detected by IFN-γ production in the ICC assay, ranging from 0.3% to 1.6% of




12

CD4+ T cells. Furthermore, HIV-1 Gag-specific IFN-γ+ CD4+ T cells were consistently

CD38+++ and Bcl-2 dim (Figure 2a), mostly Ki-67+ and TIA-1/GMP-17+ (Figure 2b),

CD40L+ (Figure 2c) and CD57-negative (Figure 2d). A small, but consistent, proportion

of IFN-γ+ Gag-specific CD4+ T cells also produced IL-2 (Fig. 2c) and expressed cell

surface CD127 (Fig. 2d).

Gag-specific IFN-γ+ CD4+ T cells were found in only one in subject with late

PHI and were too low to obtain reliable phenotype data (not shown).

In contrast, CMV-specific IFN-γ+ CD4+ T cells in five of the same early PHI

individuals who were also CMV IgG seropositive, at the same time points, were

predominantly CD38low and Bcl-2-high (Figure 3a), Ki-67-negative (Figure 3b) and

CD57-positive (Figure 3c). CMV-specific CD4+ T cells were similar to Gag-specific

CD4+ T cells in their expression of TIA-1/GMP-17 (Figure 3b), CD127 (Figure 3c),

CD40L and IL-2 (not shown). The phenotyping results for HIV-specific and CMV-

specific CD4+ T cells from all early PHI subjects studied are summarized in Figures 3d

and 3e, respectively.

Of interest, subject #90400101, undergoing concurrent primary HIV-1 and CMV

infections, had 4.0 % CMV-specific CD4+ T cells which were phenotypically very

similar to his HIV-specific CD4+ T cells, being CD38+++ and Bcl-2 dim, CD57-

negative, Ki-67+, TIA-1/GMP-17+ and CD40L+ (data not shown).

In subjects with late PHI and in HIV-negative adults (who were CMV-

seropositive), CMV-specific IFN-γ+ CD4+ T cells were also CD38low and Bcl-2-high,

Ki-67-negative and CD57-positive, TIA-1/GMP-17+, and CD40L+ (summarized in

Figure 3e). However, in HIV-negative adult controls, there was an increased proportion




13

of CMV-specific IFN-γ+ CD4+ T cells which produced IL-2 and were CD127+ and

CD57-negative, compared with both early (p<0.01) and late PHI subjects (p<0.02; Figure

3e). It is important to note that the low expression of CD38 and Ki-67 by CMV-specific

CD4+ T cells suggests that their expression is not acutely up-regulated by the 6 hr

exposure to antigen in vitro.

Close relationship of circulating CCR5+CD38+++ and Gag-specific CD4+ T cells

When we compared the CD38+++ phenotype of Gag-specific CD4+ T cells,

obtained from the ICC assays (Fig. 2a), with the co-expression of CCR5 on CD38+++

CD4+ T cells in whole blood samples from the same subjects at the same time points

(Fig. 1a), the results infer that antigen-specific CD4+ T cells were CCR5+ immediately

ex vivo. Direct examination of CCR5 expression in the ICC assay was precluded by

down-regulation of CCR5 both spontaneously and in response to antigen 35.

Overall, Gag-specific CD4+ T cells detected by IFN-γ production, in subjects

studied during early PHI, represented a median of 0.58% of CD4+ T cells, whereas

subjects with late PHI had a significantly lower median, 0.08% (p<0.01; Figure 4a), very

close to the detection limit of the ICC. Again, this is consistent with the relative levels of

CCR5+CD38+++ CD4+ T cells in these two subject subgroups (Fig. 1e).

Moreover, we observed a very close correlation between the proportion of

CCR5+CD38+++ CD4+ T cells and the proportion of IFN-γ+ Gag-specific CD4+ T cells

(rho = 0.76; p = 0.004; Figure 4b). The results show, however, that the proportion of

Gag-specific CD4+ T cells consistently represented only one-tenth of the proportion of

CCR5+CD38+++ CD4+ T cells (Figure 4b).




14

Subject #90400101, who was undergoing both primary HIV-1 and CMV

infection, had 55% of CD4+ T cells with a CCR5+CD38+++ phenotype, and had 1.1%

HIV-specific and 4.0% CMV-specific CD4+ T cells. Therefore, in this individual, the

combined proportions of HIV- and CMV-specific CD4+ T cells were also close to one-

tenth of the CCR5+CD38+++ CD4+ T cells. The much larger primary CD4+ T cell

response to CMV, compared with the response to HIV-1 Gag, has previously been

reported in four similarly co-infected subjects 16.

In this cross-sectional study of PHI subjects at presentation, the level of

CCR5+CD38+++ CD4+ T cells peaked within the first 22 days following onset of

symptoms, and appeared to decline rapidly after that time (Figure 4c). A similar peak was

also observed when plotted against WB intensity score (not shown). The level of

CCR5+CD38+++ CD4+ T cells was highly variable and not all early PHI subjects

exhibited a large population of CCR5+CD38+++ CD4+ T cells. Nearly all of the early

PHI subjects in the current study commenced treatment, so it has not been possible so far

to conclusively define the natural history of the CCR5+CD38+++ CD4+ T cells in

individual subjects.

The level of CCR5+CD38+++ CD4+ T cells appeared to have a complex

relationship with viral load, but there was an association between the two variables (rho =

0.63; p = 0.003; Figure 4d).




15

Further phenotyping of CCR5+CD38+++ CD4+ T cells in fresh whole blood

We investigated the expression of trafficking markers, adhesion molecules, co-

stimulatory molecules and cytokine receptors on the CCR5+CD38+++ CD4+ T cells in

samples of fresh whole blood, from 4 subjects with early PHI. Representative results are

shown in Figure 5.

The CCR5+CD38+++ CD4+ T cells exhibited relative upregulation of the

chemokine receptor CXCR3 and the cytokine receptor IL-12Rß1 (Figure 5a), consistent

with the phenotype of IFN-γ and IL-2 producing Th1 CD4+ T cells 33.

The CCR5+CD38+++ CD4+ T cells slightly up-regulated CD122 (Figure 5b) and

CD132 (not shown) but not CD25 (not shown), representing IL-2R ß-, γ-, and α-chains,

respectively. Expression of TNFR2 (CD120b) was also increased (Figure 5b).

The cell surface expression of IL-7R (CD127) was mostly down-regulated,

although one-third of cells retained this receptor (Figure 5c). Also, there was a

consistently observed minority of CCR5+CD38+++ which co-expressed CCR7 (Figure

5c), but these cells had completely down-regulated CD45RA (not shown). These cells

also maintained expression of CD62L (not shown) and had increased expression of

CD49d, integrin ß7 and LFA-1 (not shown), relative to other CD4+ T cells.

The CCR5+CD38+++ CD4+ T cells also maintained expression of the co-

stimulation molecules, CD28 and CD27 (not shown).




16

Discussion

The current study of the phenotype of HIV-specific CD4+ T cells, in fresh whole

blood samples obtained during primary HIV-1 infection, demonstrated that these cells

were highly activated CD38+++ cells. Parallel analysis of the same blood samples

showed that the CD38+++ CD4+ T cells co-expressed the HIV-1 co-receptor, CCR5.

Together, these results establish that circulating HIV-1 antigen-specific CD4+ T cells

were CCR5+ during primary HIV-1 infection. Expression of CCR5 on these highly

activated antigen-specific CD4+ T cells suggests that they are likely to be particularly

susceptible to HIV-1 infection, and possibly lost as a result of either direct cytopathic

effect by HIV-1, or lysis by CD8+ CTL.

Consistent with this possibility, our cross-sectional study of patients at

presentation found that there was an apparent rapid decline in antigen-specific CD4 T

cells over time, as defined both functionally (by ICC assay) and phenotypically

(CCR5+CD38+++). In order to establish whether loss of antigen-specific CD4+ T cells is

due to HIV-1 infection, it will be important to confirm the kinetics in more detailed

longitudinal studies of individual subjects. It will also be necessary to perform cell

sorting of CD38+++ CD4+ T cells during early primary infection and determine whether

they preferentially contain HIV-1 DNA. Previous studies of CD4+ T cells subsets

containing HIV-1 DNA have shown relatively increased infection of antigen-specific

CD4+ T cells 5 but the cell surface expression of CCR5 in that analysis was not

examined.

In the present study, HIV-1 Gag-specific CD4+ T cells were also predominantly

Ki-67+, indicating proliferative expansion. It has been shown during acute SIV infection




17

that Ki-67+ CD4+ T cells in lymphoid tissue increase dramatically about 10 days

following inoculation, and are the main producers of SIV during acute infection 39

although again in that study expression of CCR5 was not reported. Our results would

suggest that proliferation of antigen-specific CD4+ T cells contributes to an increase in

target cells in lymphoid tissue. However, cell-cell transmission of SIV to adjacent Ki-67-

negative cells in lymphoid tissue was also observed during acute infection 39, suggesting

that highly productively infected Ki-67+ CD4+ T cells may act as a focus of spreading

infection.

An important question is whether all the observed CCR5+ CD38+++ CD4+ T

cells were HIV-specific. Our findings agree with earlier descriptions of CD38+, Ki-67+

CMV- and EBV-specific CD4+ T cells during primary infection with these respective

viruses 11,12. In the present study, one early PHI subject concurrently underwent primary

CMV infection and had high levels of both CMV- and HIV-specific CD4+ T cells which

were similarly CCR5+CD38+++ and Ki-67+. Conversely, in the other PHI subjects

(without evidence of primary CMV infection), our results showed that “bystander” CMV-

specific effector memory CD4+ T cells, at the same time points, were neither activated,

nor proliferating. It should be noted, however, that a large fraction of these resting CMV-

specific memory CD4+ T cells were CD57+ and did not produce IL-2, suggesting that

they were terminally differentiated effector cells, which previous in vitro studies would

predict were unlikely to proliferate 40.

There are several possible reasons why the level of HIV-specific CD4+ T cells

measured in our assay may not match the level of CD38+++ CD4+ T cells in the blood

samples, even if these cells were largely antigen-specific. We have not completely




18

investigated the extent of CD4+ T cell responses to all HIV-1 proteins. Most studies do

suggest that CD4+ T cell responses to HIV-1 Gag are highly immunodominant 1,

particularly in subjects during acute infection 16 as well as subjects treated during acute

infection who underwent treatment interruption 41,42. However, gp160-specific CD4+ T

cell responses were also commonly found in very early PHI in one report 41. Additional

responses might also be detected to variant autologous sequences 43, to frame-shifted

peptides 44, or to peptides from recombinant variants 45, but these were not tested in the

present study. Moreover, there may be a sub-lineage of antigen-specific Th1 effector cells

which do not express IFN-γ, and may be precursors of memory cells 46. It has also been

reported that MHC class I tetramer+ HIV antigen-specific CD8+ T cells do not uniformly

produce IFN-γ in response to their cognate antigen during periods of high viremia

following treatment interruptions 47.

Increased activation of CD4+ T cells is prominent in PHI 48 and this activation is

dramatically reduced following initiation of anti-retroviral therapy 34,49. Increased

expression of CD38 on T cells, particularly on CD8+ T cells, is highly correlated with

disease progression during chronic HIV-1 infection 50. Elevated CD38 expression has

also been reported on HIV-specific CD8+ T cells during acute HIV-1, although at the

same time it was observed that CMV- and EBV-specific CD8+ T cells similarly

expressed relatively high levels of CD38 51,52. Therefore the question of whether elevated

activation is limited to HIV-specific T cells remains unclear, but overall the present

results indicate that CD38 up-regulation on CD4+ T cells, early in infection, is related to

antigen-specific activation. It will also be important to determine whether CCR5

expression is coupled to elevated CD38 expression throughout HIV-1 infection.




19

However, HIV-1 infection of CCR5+CD38+++ CD4+ T cells is probably not the

main reason why these cells decline in the circulation as the primary infection resolves.

Studies in murine models have shown that CD4+ T cell responses to viral infection show

a peak response in the range of 0.5 – 10% of CD4+ T cells, followed by a decrease of 10-

to 20-fold 9,10. Also, a similar peak and rapid decline in CMV-specific CD4+ T cells has

been observed in primary CMV infection 11. Possible explanations for such a normal

decline in viral antigen-specific CD4+ T cells include apoptosis and trafficking. Firstly,

we have now directly shown that the antigen-specific CD38+++ CD4+ T cells also

contained decreased levels of Bcl-2. We had previously shown that CCR5+ and CD38+ T

cells during acute HIV-1 infection contained low levels of Bcl-2 and spontaneously

underwent apoptosis in vitro 53, but these cells could be rescued from apoptosis by

incubation with IL-15 or IL-2, which increased intracellular levels of Bcl-2 53. Th1

effector cells are particularly susceptible to apoptosis 54, and interestingly, the products of

these cells, including IL-2, IFN-γ and perforin, have all been shown to be involved in

feedback regulation of T cell responses in vivo 55-58. Comparison of primary HIV-

1infection with acute EBV infection, where we have also shown an elevation of CD38++

CD4+ T cells, as well as proliferating CCR5+ CD4+ T cells with low Bcl-2 and increased

spontaneous apoptosis 34,53, may allow study of a normal contraction of these CD4+

effector cells.

Another possible reason for the observed decline of CCR5+CD38+++ CD4+ T

cells may be trafficking out of the circulation to sites of inflammation, consistent with the

cell surface phenotype of CXCR3+ and CCR5+ 59, as well as high expression of LFA-1

and CD62L 60. Anti-viral CD8+ effector T cells proliferate in lymph nodes and the




20

spleen, exit into the circulation, and then approximately half of these cells traffic to

organs such as the bone marrow, lung, liver and gut (reviewed in 61). In particular, it is

believed that CCR5+ CD4+ T cells in gut-associated lymphoid tissue represent a main

target of HIV-1 in acute infection 62,63. We found that the majority of CCR5+ CD38+++

CD4+ T cells expressed the gut-homing marker, integrin α4β7. Interestingly, a previous

study showed that there was a selective loss of the integrin α4β7+ subset of CCR5+

CD4+ T cells from the circulation during acute HIV-1 infection 64.

However, the apparent decline in antigen-specific T cells may alternatively

simply reflect anergy of these cells, resulting in an inability to detect them by functional

assays in vitro. Exhaustion of antigen-specific CD4+ T cells has been observed in

persistent viral infections in mice 65. Similarly, exposure to persistent antigen in vivo led

to a dramatic reduction in cytokine production by recently proliferated CD4+ T cells in a

mouse model of anergy 66. It is important to note that a histologic feature of acute HIV-

1infection is the accumulation of large numbers of virions retained by the follicular

dendritic cell network within lymphoid tissue 67. Constant exposure to HIV-1 antigens,

therefore, may prevent the transition to resting memory cells, or induce exhaustion. One

study has already demonstrated that CD4+ T cells specific for HIV-1 envelope, which

had been present at a detectable level during primary HIV-1 infection, only persisted at a

greatly reduced, functionally undetectable level following resolution of the acute phase of

infection 41 and required prolonged incubation in vitro in IL-2 containing medium for

detection.

Finally, it is possible that the antigen-specific CD4+ T cells switch to production

of alternative cytokines not measured in the current study, such as IL-10, rather than IFN-




21

γ 68. A more comprehensive analysis of cytokines and effector molecules produced by the

Gag-specific CD4+ T cells in PHI is now feasible and warranted. Similarly, development

of CD25+ suppressor cells 69 may occur after resolution of the primary infection,

blocking in vitro responsiveness and studies of the effect of depletion of such CD25+

CD4+ T cells may be informative.

Approximately half of the activated, proliferating CD4+ T cells also expressed

markers of cytotoxic T lymphocytes, including TIA-1/GMP-17/NKG-7, as well as

granzyme B and perforin. The results of the current study are in agreement with previous

findings that immune responses to HIV-1 and CMV include CD4+ T cells with a CTL

phenotype and function 35-37,70. These consistent findings suggest that the normal role of

CD4+ T cells is not just to help B cells and CD8+ T cells, but also indicates a direct

antiviral effect. It had been reported that MHC Class II+ cells in vivo can present

endogenously produced viral antigenic peptides to CD4+ T cells 71 and a recent paper has

now shown that CD4+ CTL specifically clear viral peptide loaded target cells in vivo 72.

Most CCR5+CD38+++ CD4+ T cells exhibited an upregulation of the IL-2R ß

chain, but not the IL-2Rα chain, consistent with a role for IL-15 in survival of these cells

73. We also found an upregulation of the IL-12Rß1 chain on these cells, at least during the

acute phase. Previous studies have shown that incubation with IL-15 74,75 or IL-12 76

increased antigen-specific responses in vitro. The presence of these receptors is consistent

with the ability of IL-15 to induce CCR5 expression on resting T cells 77 and IL-12 to

regulate CCR5 expression following T cell activation 78. We also found an upregulation

of the TNFR2 on the CCR5+ CD38+++ CD4+ T cells, which may contribute to apoptosis




22

in combination with low Bcl-2 79, but may also be involved in enhancement of HIV-1

replication 80.

Studies of effector CD4+ cells in murine models suggest that as viral antigen is

cleared, a subset of effector cells will convert to resting memory cells 31,32. In particular,

those antigen-specific CD4+T cells which retain expression of the IL-7R are precursors

of memory cells 81. In our earlier study 35, IL-7R-negative CD38++ T cells from subjects

during primary HIV-1 infection underwent spontaneous apoptosis in vitro, but we did not

study whether there was a subset of activated T cells which retained IL-7R and could be

induced to become resting memory cells by incubation in vitro with IL-7, as suggested in

the murine studies 81.

We had also previously studied the TCR V beta repertoire of Ki-67+, CD38++

and CCR5+ CD4+ T cells and found that it was surprisingly broad 53. Taken together

with the current results, it is likely that many clones are involved in the initial antigen-

specific response to HIV. Patients treated during acute infection have the broadest

responses to HIV proteins 42, which also suggests a broad base of responses during acute

infection.

Our results showing expression of CCR5 on activated CD4+ T cells may help to

explain the effects of immunosuppressive treatment with hydroxyurea 82 and cyclosporine

A 83 in acute HIV-1 infection. The presence of CCR5 on antigen-specific CD4+ T cells

predicts that blockade of this receptor will be particularly beneficial, not only during

acute infection, but possibly also during therapeutic immunisation or treatment

interruptions. It will also be important to confirm that expression of IL-7R is an early




23

marker of long-term self-renewing memory cells, and may represent a useful guide to

vaccine efficacy.




24

Acknowledgements

The authors would like to thank the subjects and their physicians for their

participation, Kate McGhie, Ciara McGinley and Palanee Ammaranond for help with

specimen organization and logistics, as well as the NIH Reference Reagents Program for

provision of HIV-1 overlapping Gag peptides.




25

References

1. Picker LJ, Maino VC. The CD4(+) T cell response to HIV-1. Curr Opin Immunol.

2000;12:381-386.

2. Clerici M, Stocks NI, Zajac RA, et al. Detection of three distinct patterns of T

helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive

patients. Independence of CD4+ cell numbers and clinical staging. J Clin Invest.

1989;84:1892-1899.

3. McNeil AC, Shupert WL, Iyasere CA, et al. High-level HIV-1 viremia suppresses

viral antigen-specific CD4(+) T cell proliferation. Proc Natl Acad Sci U S A.

2001;98:13878-13883.

4. Badley AD, Pilon AA, Landay A, Lynch DH. Mechanisms of HIV-associated

lymphocyte apoptosis. Blood. 2000;96:2951-2964.

5. Douek DC, Brenchley JM, Betts MR, et al. HIV preferentially infects HIV-

specific CD4+ T cells. Nature. 2002;417:95-98.

6. Doherty PC, Topham DJ, Tripp RA, Cardin RD, Brooks JW, Stevenson PG.

Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus

infections. Immunol Rev. 1997;159:105-117.

7. Bachmann MF, Hunziker L, Zinkernagel RM, Storni T, Kopf M. Maintenance of

memory CTL responses by T helper cells and CD40-CD40 ligand: antibodies provide the

key. Eur J Immunol. 2004;34:317-326.

8. Kalams SA, Buchbinder SP, Rosenberg ES, et al. Association between virus-

specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus

type 1 infection. J Virol. 1999;73:6715-6720.

9. Topham DJ, Doherty PC. Longitudinal analysis of the acute Sendai virus-specific

CD4+ T cell response and memory. J Immunol. 1998;161:4530-4535.

10. Christensen JP, Doherty PC. Quantitative analysis of the acute and long-term

CD4(+) T-cell response to a persistent gammaherpesvirus. J Virol. 1999;73:4279-4283.

11. Rentenaar RJ, Gamadia LE, van DerHoek N, et al. Development of virus-specific

CD4(+) T cells during primary cytomegalovirus infection. J Clin Invest. 2000;105:541-

548.




26

12. Amyes E, Hatton C, Montamat-Sicotte D, et al. Characterization of the CD4+ T

cell response to Epstein-Barr virus during primary and persistent infection. J Exp Med.

2003;198:903-911.

13. Rosenberg ES, Billingsley JM, Caliendo AM, et al. Vigorous HIV-1-specific

CD4+ T cell responses associated with control of viremia. Science. 1997;278:1447-1450.

14. Rosenberg ES, Altfeld M, Poon SH, et al. Immune control of HIV-1 after early

treatment of acute infection. Nature. 2000;407:523-526.

15. Oxenius A, Price DA, Easterbrook PJ, et al. Early highly active antiretroviral

therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T

lymphocytes. Proc Natl Acad Sci U S A. 2000;97:3382-3387.

16. Harari A, Rizzardi GP, Ellefsen K, et al. Analysis of HIV-1- and CMV-specific

memory CD4 T-cell responses during primary and chronic infection. Blood.

2002;100:1381-1387.

17. Lichterfeld M, Kaufmann DE, Yu XG, et al. 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. 2004;200:701-712.

18. Malhotra U, Berrey MM, Huang Y, et al. Effect of combination antiretroviral

therapy on T-cell immunity in acute human immunodeficiency virus type 1 infection. J

Infect Dis. 2000;181:121-131.

19. Breitfeld D, Ohl L, Kremmer E, et al. Follicular B helper T cells express CXC

chemokine receptor 5, localize to B cell follicles, and support immunoglobulin

production. J Exp Med. 2000;192:1545-1552.

20. Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B. CXC

chemokine receptor 5 expression defines follicular homing T cells with B cell helper

function. J Exp Med. 2000;192:1553-1562.

21. Pitcher CJ, Quittner C, Peterson DM, et al. HIV-1-specific CD4+ T cells are

detectable in most individuals with active HIV-1 infection, but decline with prolonged

viral suppression. Nat Med. 1999;5:518-525.

22. Betts MR, Ambrozak DR, Douek DC, et al. Analysis of total human

immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses:

relationship to viral load in untreated HIV infection. J Virol. 2001;75:11983-11991.




27

23. Wilson JD, Imami N, Watkins A, et al. Loss of CD4+ T cell proliferative ability

but not loss of human immunodeficiency virus type 1 specificity equates with progression

to disease. J Infect Dis. 2000;182:792-798.

24. Boaz MJ, Waters A, Murad S, Easterbrook PJ, Vyakarnam A. Presence of HIV-1

Gag-specific IFN-gamma+IL-2+ and CD28+IL-2+ CD4 T cell responses is associated

with nonprogression in HIV-1 infection. J Immunol. 2002;169:6376-6385.

25. Iyasere C, Tilton JC, Johnson AJ, et al. Diminished proliferation of human

immunodeficiency virus-specific CD4+ T cells is associated with diminished interleukin-

2 (IL-2) production and is recovered by exogenous IL-2. J Virol. 2003;77:10900-10909.

26. Younes SA, Yassine-Diab B, Dumont AR, et al. HIV-1 viremia prevents the

establishment of interleukin 2-producing HIV-specific memory CD4+ T cells endowed

with proliferative capacity. J Exp Med. 2003;198:1909-1922.

27. Harari A, Petitpierre S, Vallelian F, Pantaleo G. Skewed representation of

functionally distinct populations of virus-specific CD4 T cells in HIV-1-infected subjects

with progressive disease: changes after antiretroviral therapy. Blood. 2004;103:966-972.

28. Palmer BE, Boritz E, Wilson CC. Effects of sustained HIV-1 plasma viremia on

HIV-1 Gag-specific CD4+ T cell maturation and function. J Immunol. 2004;172:3337-

3347.

29. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory

T lymphocytes with distinct homing potentials and effector functions. Nature.

1999;401:708-712.

30. Harari A, Vallelian F, Meylan P, Pantaleo G. Functional heterogeneity of memory

CD4 T cell responses in different conditions of antigen exposure and persistence. J

Immunol. 2005;174:1037-1045.

31. Hu H, Huston G, Duso D, Lepak N, Roman E, Swain SL. CD4(+) T cell effectors

can become memory cells with high efficiency and without further division. Nat

Immunol. 2001;2:705-710.

32. Harbertson J, Biederman E, Bennett KE, Kondrack RM, Bradley LM. Withdrawal

of stimulation may initiate the transition of effector to memory CD4 cells. J Immunol.

2002;168:1095-1102.




28

33. Bonecchi R, Bianchi G, Bordignon PP, et al. Differential expression of

chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and

Th2s. J Exp Med. 1998;187:129-134.

34. Zaunders JJ, Kaufmann GR, Cunningham PH, et al. Increased turnover of CCR5+

and redistribution of CCR5- CD4 T lymphocytes during primary human

immunodeficiency virus type 1 infection. J Infect Dis. 2001;183:736-743.

35. Zaunders JJ, Dyer WB, Wang B, et al. Identification of circulating antigen-

specific CD4+ T lymphocytes with a CCR5+, cytotoxic phenotype in an HIV-1 long-term

nonprogressor and in CMV infection. Blood. 2004;103:2238-2247.

36. Norris PJ, Sumaroka M, Brander C, et al. Multiple effector functions mediated by

human immunodeficiency virus-specific CD4(+) T-cell clones. J Virol. 2001;75:9771-

9779.

37. Appay V, Zaunders JJ, Papagno L, et al. Characterization of CD4(+) CTLs ex

vivo. J Immunol. 2002;168:5954-5958.

38. Vanhems P, Dassa C, Lambert J, et al. Comprehensive classification of symptoms

and signs reported among 218 patients with acute HIV-1 infection. J Acquir Immune

Defic Syndr. 1999;21:99-106.

39. Zhang ZQ, Wietgrefe SW, Li Q, et al. Roles of substrate availability and infection

of resting and activated CD4+ T cells in transmission and acute simian

immunodeficiency virus infection. Proc Natl Acad Sci U S A. 2004;101:5640-5645.

40. Brenchley JM, Karandikar NJ, Betts MR, et al. Expression of CD57 defines

replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood.

2003;101:2711-2720.

41. Malhotra U, Holte S, Zhu T, et al. Early induction and maintenance of Env-

specific T-helper cells following human immunodeficiency virus type 1 infection. J

Virol. 2003;77:2663-2674.

42. Kaufmann DE, Bailey PM, Sidney J, et al. Comprehensive analysis of human

immunodeficiency virus type 1-specific CD4 responses reveals marked

immunodominance of gag and nef and the presence of broadly recognized peptides. J

Virol. 2004;78:4463-4477.




29

43. Altfeld M, Addo MM, Shankarappa R, et al. Enhanced Detection of Human

Immunodeficiency Virus Type 1-Specific T-Cell Responses to Highly Variable Regions

by Using Peptides Based on Autologous Virus Sequences. J Virol. 2003;77:7330-7340.

44. Cardinaud S, Moris A, Fevrier M, et al. Identification of Cryptic MHC I-restricted

Epitopes Encoded by HIV-1 Alternative Reading Frames. J Exp Med. 2004;199:1053-

1063.

45. Shriner D, Rodrigo AG, Nickle DC, Mullins JI. Pervasive genomic recombination

of HIV-1 in vivo. Genetics. 2004;167:1573-1583.

46. Wu CY, Kirman JR, Rotte MJ, et al. Distinct lineages of T(H)1 cells have

differential capacities for memory cell generation in vivo. Nat Immunol. 2002;3:852-858.

47. Oxenius A, Sewell AK, Dawson SJ, et al. Functional discrepancies in HIV-

specific CD8+ T-lymphocyte populations are related to plasma virus load. J Clin

Immunol. 2002;22:363-374.

48. Zaunders J, Carr A, McNally L, Penny R, Cooper D. Effects of primary HIV-1

infection on subsets of CD4+ and CD8+ T lymphocytes. AIDS. 1995;9:561-566.

49. Tilling R, Kinloch S, Goh LE, et al. Parallel decline of CD8+/CD38++ T cells and

viraemia in response to quadruple highly active antiretroviral therapy in primary HIV

infection. Aids. 2002;16:589-596.

50. Liu Z, Cumberland WG, Hultin LE, Kaplan AH, Detels R, Giorgi JV. CD8+ T-

lymphocyte activation in HIV-1 disease reflects an aspect of pathogenesis distinct from

viral burden and immunodeficiency. J Acquir Immune Defic Syndr Hum Retrovirol.

1998;18:332-340.

51. Papagno L, Spina CA, Marchant A, et al. Immune Activation and CD8(+) T-Cell

Differentiation towards Senescence in HIV-1 Infection. PLoS Biol. 2004;2:E20.

52. Doisne JM, Urrutia A, Lacabaratz-Porret C, et al. CD8+ T cells specific for EBV,

cytomegalovirus, and influenza virus are activated during primary HIV infection. J

Immunol. 2004;173:2410-2418.

53. Zaunders JJ, Moutouh-de Parseval L, Kitada S, et al. Polyclonal proliferation and

apoptosis of CCR5+ T lymphocytes during primary human immunodeficiency virus type

1 infection: regulation by interleukin (IL)-2, IL-15, and Bcl-2. J Infect Dis.

2003;187:1735-1747.




30

54. Zhang X, Brunner T, Carter L, et al. Unequal death in T helper cell (Th)1 and Th2

effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J Exp

Med. 1997;185:1837-1849.

55. Lenardo M, Chan KM, Hornung F, et al. Mature T lymphocyte apoptosis--

immune regulation in a dynamic and unpredictable antigenic environment. Annu Rev

Immunol. 1999;17:221-253.

56. Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers. Nat

Rev Immunol. 2002;2:389-400.

57. Dalton DK, Haynes L, Chu CQ, Swain SL, Wittmer S. Interferon gamma

eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis

of activated CD4 T cells. J Exp Med. 2000;192:117-122.

58. Badovinac VP, Tvinnereim AR, Harty JT. Regulation of antigen-specific CD8+ T

cell homeostasis by perforin and interferon-gamma. Science. 2000;290:1354-1358.

59. Qin S, Rottman JB, Myers P, et al. The chemokine receptors CXCR3 and CCR5

mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest.

1998;101:746-754.

60. von Andrian UH, Mempel TR. Homing and cellular traffic in lymph nodes. Nat

Rev Immunol. 2003;3:867-878.

61. Masopust D, Lefrancois L. CD8 T-cell memory: the other half of the story.

Microbes Infect. 2003;5:221-226.

62. Brenchley JM, Schacker TW, Ruff LE, et al. CD4+ T cell depletion during all

stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med.

2004;200:749-759.

63. Mehandru S, Poles MA, Tenner-Racz K, et al. Primary HIV-1 infection is

associated with preferential depletion of CD4+ T lymphocytes from effector sites in the

gastrointestinal tract. J Exp Med. 2004;200:761-770.

64. Krzysiek R, Rudent A, Bouchet-Delbos L, et al. Preferential and persistent

depletion of CCR5+ T-helper lymphocytes with nonlymphoid homing potential despite

early treatment of primary HIV infection. Blood. 2001;98:3169-3171.




31

65. Oxenius A, Zinkernagel RM, Hengartner H. Comparison of activation versus

induction of unresponsiveness of virus-specific CD4+ and CD8+ T cells upon acute

versus persistent viral infection. Immunity. 1998;9:449-457.

66. Tanchot C, Barber DL, Chiodetti L, Schwartz RH. Adaptive tolerance of CD4+ T

cells in vivo: multiple thresholds in response to a constant level of antigen presentation. J

Immunol. 2001;167:2030-2039.

67. Schacker T, Little S, Connick E, et al. Rapid accumulation of human

immunodeficiency virus (HIV) in lymphatic tissue reservoirs during acute and early HIV

infection: implications for timing of antiretroviral therapy. J Infect Dis. 2000;181:354-

357.

68. Ostrowski MA, Gu JX, Kovacs C, Freedman J, Luscher MA, MacDonald KS.

Quantitative and qualitative assessment of human immunodeficiency virus type 1 (HIV-

1)-specific CD4+ T cell immunity to gag in HIV-1-infected individuals with differential

disease progression: reciprocal interferon-gamma and interleukin-10 responses. J Infect

Dis. 2001;184:1268-1278.

69. Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF. Human CD4+

CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and

cytomegalovirus antigens. J Virol. 2004;78:2454-2459.

70. Suni MA, Ghanekar SA, Houck DW, et al. CD4(+)CD8(dim) T lymphocytes

exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to

HCMV and HIV-1 antigens. Eur J Immunol. 2001;31:2512-2520.

71. Oxenius A, Bachmann MF, Mathis D, Benoist C, Zinkernagel RM, Hengartner H.

Functional in vivo MHC class II loading by endogenously synthesized glycoprotein

during viral infection. J Immunol. 1997;158:5717-5726.

72. Jellison ER, Kim SK, Welsh RM. Cutting Edge: MHC Class II-Restricted Killing

In Vivo during Viral Infection. J Immunol. 2005;174:614-618.

73. Marrack P, Kappler J. Control of T Cell Viability. Annu Rev Immunol.

2004;22:765-787.

74. Kanai T, Thomas EK, Yasutomi Y, Letvin NL. IL-15 stimulates the expansion of

AIDS virus-specific CTL. J Immunol. 1996;157:3681-3687.




32

75. Patki AH, Quinones-Mateu ME, Dorazio D, et al. Activation of antigen-induced

lymphocyte proliferation by interleukin-15 without the mitogenic effect of interleukin-2

that may induce human immunodeficiency virus-1 expression. J Clin Invest.

1996;98:616-621.

76. Clerici M, Lucey DR, Berzofsky JA, et al. Restoration of HIV-specific cell-

mediated immune responses by interleukin-12 in vitro. Science. 1993;262:1721-1724.

77. Perera LP, Goldman CK, Waldmann TA. IL-15 induces the expression of

chemokines and their receptors in T lymphocytes. J Immunol. 1999;162:2606-2612.

78. Iwasaki M, Mukai T, Gao P, et al. A critical role for IL-12 in CCR5 induction on

T cell receptor- triggered mouse CD4(+) and CD8(+) T cells. Eur J Immunol.

2001;31:2411-2420.

79. de Oliveira Pinto LM, Garcia S, Lecoeur H, Rapp C, Gougeon ML. Increased

sensitivity of T lymphocytes to tumor necrosis factor receptor 1 (TNFR1)- and TNFR2-

mediated apoptosis in HIV infection: relation to expression of Bcl-2 and active caspase-8

and caspase-3. Blood. 2002;99:1666-1675.

80. Kinter AL, Ostrowski M, Goletti D, et al. HIV replication in CD4+ T cells of

HIV-infected individuals is regulated by a balance between the viral suppressive effects

of endogenous beta-chemokines and the viral inductive effects of other endogenous

cytokines. Proc Natl Acad Sci U S A. 1996;93:14076-14081.

81. Li J, Huston G, Swain SL. IL-7 promotes the transition of CD4 effectors to

persistent memory cells. J Exp Med. 2003;198:1807-1815.

82. Lori F, Jessen H, Lieberman J, et al. Treatment of human immunodeficiency virus

infection with hydroxyurea, didanosine, and a protease inhibitor before seroconversion is

associated with normalized immune parameters and limited viral reservoir. J Infect Dis.

1999;180:1827-1832.

83. Rizzardi GP, Harari A, Capiluppi B, et al. Treatment of primary HIV-1 infection

with cyclosporin A coupled with highly active antiretroviral therapy. J Clin Invest.

2002;109:681-688.




33

Table 1 - Demographics of Primary HIV-1 Infection (PHI) Subjects

Early PHI

N = 19

Late PHI

N = 14

Age (years) 38 a

(33 – 43)b

35

(30 – 39)

Time since onset of

symptoms (days)

15

(11 - 16)

42

(32 - 76)

Symptom count 5

(3 – 7)

4

(1 – 7)

Western Blot intensity score 1

(0 - 3)

13

(12 - 20)

CD4 count

(cells / µl)

396

(239 - 554)

608

(566 -684)

Plasma HIV RNA

(log10 copies / ml)

5.9

(5.5 – 6.7)

4.5

(3.5 – 4.9)

Time from diagnostic visit

to study sample (days)

7

(0 – 11)

12

(8 – 25)

a Median

b Interquartile range




34

Figure Legends

Figure 1 – Changes in cell surface phenotypes of CD4+ T cells during primary HIV-1

infection

Flow cytometry histograms, gated on CD3+CD4+ T cells, show increases in (a)

CCR5+CD38+++; (b) CCR5+TIA-1+Ki-67+; (c) Perforin+Granzyme B+; and (d)

CD127-negative CD57-negative CD4+ T cells from representative early PHI subjects and

HIV-negative controls. Results from all patients and controls are summarized in (e). Box

plots show 10th, 25th, median, 75th and 90th percentiles for each marker for each cohort.

Figure 2 – Intracellular cytokine responses to HIV-1 Gag peptide pool

Flow cytometry histograms, gated on CD3+CD4+CD8-negative T cells, show the

phenotype of IFN-γ+ cells: (a) Bcl-2 and CD38; (b) Ki-67 and TIA-1; (c) IL-2 and

CD154 (CD40L); and (d) CD127 (IL-7R) and CD57. Histograms are representative of six

different subjects, all showing similar results.

Figure 3 - Intracellular cytokine responses to CMV lysate and comparison with responses

to HIV-1 Gag peptide pool

Flow cytometry histograms, gated on CD3+CD4+CD8-negative T cells, show the

phenotype of IFN-γ+ cells: (a) Bcl-2 and CD38; (b) Ki-67 and TIA-1; and (c) CD127 (IL-

7R) and CD57. Histograms shown are representative of results from six different

subjects, all showing similar results. Results from all patients and controls are

summarized in (d) and (e). Bars represent means ± SE for each group of patients.




35

Figure 4 – Relationship of Gag-specific CD4+ T cells to time since infection, subset

changes and viral load

(a) Comparison of levels of Gag-specific CD4+ T cells in early versus late PHI subjects.

Box plots show 10th, 25th, median, 75th and 90th percentiles for each cohort.

(b) Plot of CCR5+CD38+++ CD4+ T cells in all PHI subjects versus time since onset of

symptoms. The regression line is a Lowess curve.

(c) Correlation of Gag-specific CD4+ T cells with proportion of CCR5+CD38+++ CD4+

T cells in all PHI subjects.

(d) Plot of CCR5+CD38+++ CD4+ T cells in all PHI subjects versus plasma HIV-1 RNA

viral load. The linear regression curve is shown.

Figure 5 – Cell surface expression of chemokine and cytokine receptors on

CCR5+CD38+++ CD4+ T cells.

(a) Expression of IL-12Rß1 and CXCR3 on CCR5+CD38+++ CD4+ T cells (upper

histograms) versus expression on all other CD4+ T cells (lower histograms).

(b) Expression of IL-2Rß (CD122) and TNFR2 (CD120b) on CCR5+CD38+++ CD4+ T

cells (upper histograms) versus expression on all other CD4+ T cells (lower

histograms).

(c) Expression of CCR7 and IL-7R (CD127) on CCR5+CD38+++ CD4+ T cells (upper

histograms) versus expression on all other CD4+ T cells (lower histograms).




36

Histograms shown are representative of results from at least three different subjects, all

showing similar results.




37

Figure 1C

D38

-PE

CCR5-APCCCR5-APC

CONTROL CD3+CD4+ PHI CD3+CD4+

0.45% 16.9%

A

TIA

-1-P

E

CCR5-APC

CONTROL CD3+CD4+Ki-67+

CCR5-APC

PHI CD3+CD4+Ki-67+

61%11.2%

B

Perforin-FITC

Gra

nzy

me

B -

AP

C

Perforin-FITC

3.8% 10.1%

CONTROL CD3+CD4+ PHI CD3+CD4+C

CD57-FITC

IL-7

R (

CD

127)

- P

E

CD57-FITC

93.2% 81.8%

CONTROL CD3+CD4+ PHI CD3+CD4+

0.8%

4.5%

1.5%

0.4%

16.0%

0.4%

D

% o

f C

D4+

T c

ells

HIV-negative controls N = 13

Late PHI N = 14

Early PHI N = 16

CCR5+ CD38+++

CCR5+ TIA+ Ki-67+

Granzyme B+ Perforin+

CD127- CD57-

0

5

10

15

20E




38

Figure 2

IFN

-g -

AP

C

Bcl-2 - FITC CD38 - PE

PHI CD3+CD4+CD8- + Gag PHI CD3+CD4+CD8- + Gag

1.5% 0.5% 0.3% 1.7%

A

IFN

-g -

AP

C

Ki-67 - FITC TIA / GMP-17 - PE


0.4% 1.6% 0.7% 1.3%

B

PHI CD3+CD4+CD8- + Gag

IL-2 - FITC

1.3% 0.9%

PHI CD3+CD4+CD8- + Gag

CD40L - FITC

0.2% 0.4%

IFN

-g -

AP

C

C

IFN

-g -

AP

C

CD57-FITCIL-7R (CD127) - PE


1.2% 0.2% 1.4% 0.1%

D




39

Figure 3

IFN

-g-A

PC

Bcl-2 - FITC

PHI CD3+CD4+CD8- + CMV PHI CD3+CD4+CD8- + CMV

CD38 - PE

0.1% 1.3% 1.3% 0.1%

A

IFN

-g-A

PC

Ki-67 - FITC

PHI CD3+CD4+CD8- + CMV

1.4% 0%

TIA / GMP-17 - PE

PHI CD3+CD4+CD8- + CMV

0% 1.4%

B

IFN

-g-A

PC

CD57-FITCIL-7R (CD127) - PE

PHI CD3+CD4+CD8- + CMV PHI CD3+CD4+CD8- + CMV

1.1% 0.1% 0.1% 1.1%

C

0

20

40

60

80

100

CD38 high

Ki-67+ CD127+ CD57+IL-2+TIA-1+Bcl-2 low

CD40L+

%o

fCM

VIF

N+

CD

4+

HIV-negative controls N = 5

Late PHI N = 6

Early PHI N = 6

%of

GA

GIF

N+

CD

4+

0

20

40

60

80

100

CD38 high

Ki-67+ CD127+ CD57+IL-2+TIA-1+Bcl-2 low

CD40L+

D

E




40

Figure 4

0

.5

1

1.5

2

GA

G IF

N+

% o

f C

D4+

Late PHI N = 7

Early PHI N = 7

AL

og

Vir

al L

oad

CCR5+CD38+++% OF CD4

D

GA

G IF

N+

% o

f C

D4+

CCR5+CD38+++% OF CD4

1.2

1.6

0

.4

.8

2

0 5 10 15 20

rho = 0.76 p = 0.004

CC

R5+

CD

38++

+% O

F C

D4

Days since onset of symptoms

0

4

8

12

16

0 20 40 60 80 100 120 140

B C

2

3

4

5

6

7

0 4 8 12 16

rho = 0.63 p = 0.003




41

Figure 5a

Figure 5b

Figure 5c

IL-12Rß1 - PE

CCR5+CD38+++ CD4+

Total CD4+

CXCR3 - PE

CCR5+CD38+++ CD4+

Total CD4+

84%

30%

85%

35%

TNFR2 (CD120b) - PE

CCR5+CD38+++ CD4+

Total CD4+

IL-2Rß (CD122) - PE

CCR5+CD38+++ CD4+

Total CD4+

CCR7- PE

CCR5+CD38+++ CD4+

Total CD4+

IL-7R (CD127) - PE

CCR5+CD38+++ CD4+

Total CD4+

34%

87%




42




Early proliferation of CCR5+ CD38+++ antigen-specific CD4+ Th1 effector cells during primary HIV-1 infection

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