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JVI Accepts, published online ahead of print on 18 May 2011 J. Virol. doi:10.1128/JVI.

00738-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Reduction of CD4+ T cells in vivo does not affect virus load in macaque elite controllers

Running Title: CD4 T cells in elite control

Philip A. Mudd1,2, Adam J. Ericsen1, Andrew A. Price3, Nancy A. Wilson1,4, Keith A. Reimann3 and David I. Watkins1,4*

Department of Pathology and Laboratory Medicine, University of Wisconsin-

Madison, Madison, WI 53711, 2Medical Scientist Training Program, University of Wisconsin-Madison, Madison, WI 53711, 3Nonhuman Primate Reagent Resource, Beth Israel Deaconess Medical Center, Boston, MA 02215,
4

Wisconsin National Primate Research Center, University of Wisconsin-Madison,

Madison, WI 53711

*Corresponding Author: David I. Watkins Department of Pathology and Laboratory Medicine, University of WisconsinMadison, 555 Science Dr., Madison, WI 53711 Phone: (608)265-3380, Fax: (608)265-8084 Email: watkins@primate.wisc.edu

Abstract word count: 89 / Main text word count: 2004

Abstract:

A small percentage of HIV and SIV-infected individuals spontaneously control virus replication. The majority of these elite controllers mount high frequency virus-specific CD4+ T cell responses. To evaluate the role these responses might play in viral control, we depleted CD4+ cells from two elite controller macaques. SIV-specific CD4+ T cell responses did not return to baseline until 8 weeks post-depletion. Viral loads remained stable throughout the experiment, suggesting that SIV-specific CD4+ T cell responses may not play a direct role in controlling chronic viral replication in these elite controllers.

Text:

Rhesus macaque models of immunodeficiency virus replication have proven invaluable in delineating cellular determinants of viral control. The importance of CD8+ T cells in control of viral replication was initially demonstrated in vitro [7, 26], but was shown to be critical to the control of acute [14, 22, 23] and chronic [2, 6, 23] immunodeficiency virus replication in vivo using the rhesus macaque model. In these studies, depletion of CD8+ lymphocytes by the application of depleting antibody resulted in elevations in viral replication that coincide with the loss of CD8+ T cells, or in the case of acute viral replication, resulted in failure of post-peak viral containment, which normally precedes the establishment of chronic phase set point viral load. Elite controllers (ECs) maintain low or undetectable chronic phase set point viral loads. Elite control is associated with certain MHC class I alleles, including HLA-B*5701 and HLA-B*2705, implying a key role for the immune system and more specifically CD8+ T lymphocytes in establishing control of viral replication [5]. Our group has described an animal model of MHC class Iassociated elite control involving the rhesus macaque alleles Mamu-B*08 and Mamu-B*17 [13, 27]. We have further shown the importance of CD8+ lymphocytes in the maintenance of viral control in this model by demonstrating increased viral replication after the application of a CD8-depleting antibody [2]. This recrudescence was followed by the re-establishment of viral control

concomitant with the emergence of CD8+ T lymphocytes restricted by the elite control-associated MHC class I alleles [2, 12]. HIV-infected ECs have robust HIV-specific CD4+ T cell responses compared with individuals who do not control viral replication [20]. These responses are largely focused on epitopes within Gag and Nef [8, 20]. We have also observed similar robust SIV-specific CD4+ T cell responses in rhesus macaque ECs [4, 3, 21, 2]. The precise role of these virus-specific CD4+ T cells in the effective immune response that limits retroviral replication in ECs in vivo is unknown. In a recent study, we demonstrated that SIV-specific CD4+ T cell clones from ECs responded to infected macrophages by directly eliminating SIVinfected macrophages in vitro [21]. This finding suggests that SIV-specific CD4+ T cells may play a direct role in controlling viral replication. To determine whether or not SIV-specific CD4+ T cells play a direct role in the control of chronic SIV replication in vivo, we depleted CD4+ lymphocytes from two Mamu-B*08+ EC animals. We utilized a new rhesus/mouse recombinant antibody based on the mouse anti-human CD4 monoclonal antibody, OKT4A [19]. Complementarity determining regions of OKT4A were grafted into rhesus variable region frameworks and combined with rhesus IgG1 constant regions. The recombinant antibody was expressed in CHO cells. We tested this antibody for in vivo CD4-depletion activity by administering a single 50 mg/kg dose intravenously to four SIV-negative Indian rhesus macaques. All four animals experienced profound depletion of CD4+ T lymphocytes from their peripheral blood lymphocyte population within 24 hours of antibody administration, with

maximum depletion reached 7 days post-administration. Average CD4+ T cell counts in these four animals were reduced from 2,247 per L of blood to 61 per L of blood, representing an average 97.3% depletion of circulating peripheral blood CD4+ T cells. To test the hypothesis that broad, high frequency SIV-specific CD4+ T cell responses contribute to the maintenance of control of chronic viral replication in rhesus macaque ECs, we used this new antibody to deplete CD4+ cells from two Mamu-B*08+ elite controller macaques. Animals r96067 and r02019 [10, 24] had been infected for more than 100 weeks with the pathogenic molecular clone SIVmac239 and met the criteria for EC in our model by maintaining chronic phase set point viral loads below 103 viral copies per mL of plasma (Figure 1). Both animals demonstrated broad, high frequency SIV-specific CD4+ T cell responses, many of which recognized epitopes in Gag (Figure 1). We gave both animals a single 50 mg/kg intravenous dose of the rhesus recombinant anti-CD4 antibody. CD4+ T cell counts in peripheral blood were reduced as early as 3 days post-depletion with maximal depletion achieved 1 week post-depletion (Figure 2A), as previously seen in our four SIV-nave animals. Average CD4+ T cell counts in peripheral blood were reduced from 1,072 per L of blood to 116 per L of blood. Peripheral blood B cell (CD20+ lymphocytes) and CD8+ T cell populations were unaffected by the CD4-depletion antibody (Figure 2A). Therefore, the rhesus recombinant anti-CD4 antibody selectively reduced peripheral blood CD4+ T cell counts by approximately 90% in

these two Mamu-B*08+ elite controllers without affecting other circulating lymphocyte populations. We also monitored the absolute number of peripheral blood memory and nave CD4+ T cell subpopulations prior to and after depletion (Figure 2B). We classified these populations by expression of the surface markers CD28 and CD95 (nave cells = CD28+CD95-; central memory cells = CD28+CD95+; effector memory cells = CD28-CD95+) [18]. The majority of CD28+CD95- CD4+ T cells were eliminated by the CD4-depleting antibody with very little recovery during follow-up. This apparent intrinsic susceptibility of CD28+CD95- CD4+ T cells to depletion with the OKT4A antibody is in agreement with a recent study [1]. Interestingly, the CD28+CD95+ CD4+ T cell population was relatively resistant to depletion, with frequencies of these cells never dropping below 67 cells per L of blood in either of the depleted animals. In spite of this relative protection from depletion, the absolute number of CD28+CD95+ CD4+ T cells remained low and did not completely recover during the 16 weeks of the experiment. CD28-CD95+ CD4+ T cells in both animals were essentially eliminated from circulating peripheral blood lymphocytes by 1 week post-CD4 depletion, however these cells rapidly recovered with increasing counts by 3 weeks post-depletion and complete recovery between 6 and 8 weeks post-depletion. In summary, the total absolute number of peripheral blood CD4+ T cells decreased substantially post-depletion with most residual peripheral blood CD4+ T cells at 1 week post-depletion residing within the CD28+CD95+ subset. Absolute numbers of all peripheral

blood CD4+ T cell populations analyzed except CD28-CD95+ T cells remained diminished for at least 16 weeks post-depletion. We further examined various lymphocyte subsets in three immunologically important tissues: inguinal lymph nodes, bronchoalveolar lavage fluid (BAL) and sigmoid colon biopsy specimens. We obtained samples four weeks prior to and one week after antibody administration. Multiple pinch biopsies from the sigmoid colon were obtained and prepared according to a previously published protocol [25]. The collagenase type II used in the preparation of the colon biopsy specimens can occasionally cleave the CD4 molecule from the cell surface without affecting expression of CD3 or CD8, therefore we classified CD4+ T cells in these samples as CD3+CD8- lymphocytes. Results from FACS analysis of these tissue specimens are summarized in Figure 2C. The largest reduction in the percentage of live lymphocytes classified as CD4+ T lymphocytes occurred in PBMC. We observed smaller reductions in the other three tissues analyzed. This suggests that either resistance to depletion in these tissues or incomplete distribution of the CD4-depleting antibody occurred in the sampled tissues at one week post-antibody administration. Interestingly, the percentage of CD28+CD95+ CD4+ T cells found within the lymph nodes did not change after depleting antibody administration. The lymph node may be a protected reservoir for this subset of CD4+ T cells, which were not completely eliminated from peripheral blood circulation. All other tissue compartments sampled demonstrated a reduction in CD4+ T cell frequency post-CD4-depletion. We also noted a reduction in CD8+ T cells in sigmoid colon biopsy specimens, indicating a global

reduction in T cell populations within the gut-associated lymphoid tissue postCD4-depletion. We continually monitored the individual SIV-specific CD4+ T cell responses found in the peripheral blood of each animal (Figure 1) using CD8depleted PBMC ELISPOT. SIV-specific CD4+ T cell responses were eliminated from PBMC following CD4-depletion and did not return to baseline values until at least 8 weeks post-depletion (Figure 3A). Viral loads throughout the entire experiment remained at or below approximately 102 viral copies per mL of plasma (Figure 3B), the baseline value that these two animals had maintained for several weeks prior to CD4-depletion (Figure 1). The frequency of MamuB*08-restricted SIV-specific CD8+ T cell responses did not change over the course of the experiment (data not shown). The OKT4A antibody can block receptor binding and viral entry and can, therefore interfere with viral replication [15, 16]. To determine if the depleting antibody inhibited viral replication and consequently prevented us from observing a possible increase in viral replication post-CD4-depletion, we used a FACS assay to ascertain when the antibody was cleared from circulating plasma. Briefly, we stained total PBMC from undepleted SIV-negative animals with 10 L of plasma obtained from the CD4-depleted animals at separate time points for 45 minutes in 100 L FACS buffer (1xPBS with 10% FCS) at room temperature. Then, we washed the cells twice with FACS buffer and added surface stains for CD3 and CD8, along with anti-rhesus IgG and stained for an additional 30 minutes at room temperature. Finally, we washed and fixed the cells and

analyzed the CD3+CD8- population for anti-rhesus IgG antibody binding using flow cytometry. We verified our ability to reliably detect the depleting antibody to concentrations less than 0.5 g/mL by concurrent experiments diluting the stock of depleting antibody into fresh plasma. The staining scheme and results are summarized in Figure 3C. We detected the depleting antibody at concentrations above the limit of detection (0.5 g/mL) in the plasma of r02019 for 4 weeks and in r96067 for 5 weeks after antibody administration. In summary, we successfully depleted CD4+ T lymphocytes and essentially eliminated all SIV-specific CD4+ T cell responses from the peripheral blood of two Mamu-B*08+ EC macaques using a rhesus recombinant antibody. We created a window of approximately two weeks when these animals did not have broad, high frequency SIV-specific CD4+ T cell responses detectable in their peripheral blood or depleting antibody present in their plasma. Viral replication did not increase above baseline during this two week window. The CD4-depleting antibody also selectively eliminates the principle target of viral replication in SIV-infected primates. A previous study evaluating CD4depleted SIV-infected sooty mangabeys demonstrated a reduction in cell associated SIV-RNA following depletion, concurrent with a reduction in plasma viral load. The viral load reduction observed may, therefore, have been dependent upon the elimination of virus-infected target cells [9]. This kind of reduction in infected target cells may also be occurring after CD4-depletion in our EC macaques. However, it is possible that sufficient viral replication targets were present to observe any potential recrudescent viral replication during the two

week window for two principle reasons. CD28-CD95+ CD4+ T cell counts were normal and CD28+CD95+ CD4+ T cell counts were diminished yet still present in peripheral blood. These T cell subpopulations are believed to be the principle targets of viral replication in vivo. We also observed continued detectable low levels of plasma viral replication consistent with baseline viral replication prior to CD4-depletion during the two week window, suggesting that sufficient targets for viral production and replication were available despite the diminished overall CD4+ T cell count. For these reasons, we conclude that during this very brief 2 week window, SIV-specific CD4+ T cell responses were not necessary for the maintenance of viral containment. By contrast, in vivo depletion of CD8+ cells for only 2-3 weeks in macaque ECs resulted in increased viral replication of at least 1.5 log10 in 6/6 animals [2]. It should be emphasized that SIV-specific CD4+ T cell responses were abolished in these macaques for only a limited period. Our results do not, therefore, eliminate the possibility that SIV-specific CD4+ T cell responses provide long-term help for the maintenance of CD8+ T cell responses in ECs during chronic infection. Our results more narrowly suggest that SIV-specific CD4+ T cell responses do not directly suppress viral replication during chronic infection in ECs in the same way that SIV-specific CD8+ T cell responses appear to. Furthermore, it remains to be seen what role, if any, SIV-specific CD4+ T cell responses play in the establishment of elite control during acute infection.

The authors would like to thank Jonah Sacha and Matt Reynolds for helpful discussions. This work was supported by National Institutes of Health (NIH) grants R37 AI052056, R01 AI049120, R01 AI076114, R24 RR015371, R24 RR016038, and R21 PRJ27JP to DIW, and by R24 RR016001 and NIAID contract HHSN272200900037C to KAR. This publication was made possible in part by Grant Number P51 RR000167 from the National Center for Research Resources (NCRR), a component of the NIH, to the Wisconsin National Primate Research Center, University of Wisconsin-Madison. This publications contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or NIH.

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Figure Legends:

Figure 1. Viral load profiles and complete SIV-specific CD4+ T cell response mapping in study animals prior to CD4-depletion. We measured viral loads using a previously published protocol [11]. Briefly, viral RNA was isolated from plasma and detected using a one-step quantitative RT-PCR kit (Invitrogen, Carlsbad, CA, USA). The forward primer sequence was 5-GTCTGCGTCATCTGGTGCATTC-

3. The reverse primer sequence was 5CACTAGCTGTCTCTGCACTATGTGTTTTG-3. The detection probe sequence was 5-6-carboxyfluorescein-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-6carboxytetramethylrhodamine-3. We ran internal standards of synthetic SIV-gag RNA transcript concurrently with each individual viral load assay performed. The threshold for reproducible quantification in this assay is 30 viral RNA copy equivalents per mL plasma (V/mL; indicated with dashed line), although lower viral loads can be detected, but not reliably quantified. To map SIV-specific CD4+ T cell responses, PBMC were obtained from both animals by Ficoll density centrifugation and were depleted of CD8+ cells using a non-human primate magnetic bead separation protocol (Miltenyi Biotec, Auburn, CA, USA). CD8depleted PBMC were then evaluated for reactivity against the entire proteome of SIVmac239 using a set of 15-mer peptides that overlap by 11 (NIH AIDS Research and Reference Reagent Program, Germantown, MD, USA) in interferon gamma ELISPOT assays (methods described in [11] with the exception that results were calculated as described in [17]). Positive response magnitude is reported in spot forming units (SFU) per 106 CD8-depleted PBMC. The minimum detection threshold for a positive response was set at 50 SFU/106 (dashed line). Both animals made 9 separate CD8-depleted PBMC responses against SIVmac239. Responses in both animals are principally directed at Gag, similar to previous findings in both HIV- and SIV-infected elite controllers.

Figure 2. Lymphocyte counts following CD4-depletion. A) CD4+ T cell counts were greatly reduced following a single intravenous 50 mg/kg dose of rhesus recombinant depleting antibody. CD8+ T cell and CD20+ B cell counts were unaffected by antibody administration. B) CD28+CD95- and CD28-CD95+ CD4+ T cells were nearly eliminated by the depleting antibody, whereas a small number of CD28+CD95+ CD4+ T cells were preserved. CD28-CD95+ CD4+ T cells demonstrated brisk recovery from depletion within 7 weeks. CD28+CD95- and CD28+CD95+ CD4+ T cells did not regain their pre-depletion levels during the 16 weeks of follow-up. C) The frequency of live lymphocytes in PBMC, lymph nodes, bronchoalveolar lavage fluid (BAL) and sigmoid colon which express the indicated surface phenotype were measured 4 weeks before CD4-depletion and 1 week after CD4-depletion. Graphs labeled as T cell populations are gated on CD3+ events. CD4+ T cells = CD3+CD4+ live lymphocytes, except for colon biopsy samples where CD4+ T cells = CD3+CD8- live lymphocytes. Lymph node specimens did not contain significant numbers of CD28-CD95+ CD4+ T cells, whereas BAL fluid did not contain significant numbers of CD28+CD95- CD4+ T cells.

Figure 3. Kinetics of SIV-specific CD4+ T cell responses, viral load and circulating CD4-depletion antibody post-CD4-depletion. A) Total SIV-specific CD4+ T cell responses were greatly diminished and did not re-establish predepletion frequencies until 8 weeks post-depletion. B) SIVmac239 viral loads in both animals remained at or below 102 viral copies per mL of plasma throughout

the experiment as determined by quantitative PCR. C) Circulating rhesus recombinant anti-CD4 depleting antibody was measured in the plasma of the depleted animals using FACS. Plasma samples were stored at -80C throughout the experiment and then thawed once to evaluate the presence of rhesus recombinant CD4 depleting antibody. 10 L of plasma was used to stain one million PBMC from a SIV-negative rhesus macaque. Secondary antibodies (antiCD3 [SP34-2], anti-CD8 [RPA-T8] and anti-rhesus IgG [1B3]) were then added. The gating strategy used to evaluate expression of CD4 on the SIV-negative animals PBMC is outlined in the first panel using data from r02019. The increased geometric mean fluorescence intensity (MFI) represents an almost complete (>90%) staining of all CD3+CD8- lymphocytes with both the primary anti-CD4 depleting antibody and the secondary anti-rhesus IgG antibody. The sensitivity for the detection of depleting antibody in plasma was less than 0.5 g/mL final rhesus recombinant anti-CD4 antibody concentration. The plasma concentration of the depleting antibody in r02019 was < 0.5 g/mL before 5 weeks post-depletion and was < 0.5 g/mL in r96067 before 6 weeks postdepletion, indicating removal of the antibody from the circulating plasma of both animals within 6 weeks of administration. The assay was performed twice using PBMC from two separate SIV-negative animals each time (four replicates total).

Figure 1
750

108 107 Viral Load (V/mL) 106 105 104 103 102 101 100 0 10 20 20 105 190 190

r02019

SFU per 1x106 CD8-depleted PBMC

600

r02019

450

CD4-depletion

300

150

210

230

Weeks post-infection
750

Gag Pol
SFU per 1x106 CD8-depleted PBMC

Vpr Vif Rev Vpx Tat

Nef Env

108 107 Viral Load (V/mL) 106 105 104 103 102 101 100 0 10 20 20 60 100 100

r96067

600

r96067

450

CD4-depletion

300

150

120

140

Weeks post-infection

Gag Pol

Vpr Vif Rev Vpx Tat Env

Nef

Figure 2

# CD4+ T cells per uL blood

# CD8+ T cells per uL blood

# CD20+ cells per uL blood

A)

2000

r02019 r96067

3000

r02019 r96067

1000 800 600 400 200 0 -4

r02019 r96067

1500

2000

1000

1000

500

0 -4

12

16

0 -4

12

16

12

16

Weeks post-CD4 depletion


# CD28+CD95- CD4+ T cells per uL blood r02019 r96067

Weeks post-CD4 depletion

Weeks post-CD4 depletion

# CD28+CD95+ CD4+ T cells per uL blood

# CD28-CD95+ CD4+ T cells per uL blood

B)

1200

800

r02019 r96067

500 400 300 200 100 0 -4

r02019 r96067

900

600

600

400

300

200

0 -4

12

16

Weeks post-CD4 depletion

0 -4

12

16

12

16

Weeks post-CD4 depletion

Weeks post-CD4 depletion

C)
CD8+ T cells
50 40
20 30

CD20+ B cells
40

CD4+ T cells
20

CD28+CD95CD4+ T cells

15

CD28+CD95+ CD4+ T cells

15

CD28-CD95+ CD4+ T cells

30

15

10
20

10

PBMC

30

10

r02019 r96067

20
10

5
10

10 0
0 50 40

60

Pre

Post

Pre

Post

40

Pre

Post

30

Pre

Post

20

Pre

Post

5 4 3

Pre

Post

30

15

Lymph Node

40
30

20
20
20

10

2 10
10

20
10

1 0 5 4 3

0 60

Pre

Post

0 10 8

Pre

Post

0 50 40 30 20 10 0

Pre

Post

5 4 3

Pre

Post

40

Pre

Post

Pre

Post

30

40
6

BAL
4

20

2 1 0
10

2 1 0

20
2

0
10 8 6

Pre

Post

0 20

Pre

Post

Pre

Post

5 4 3

Pre

Post

0 5 4 3 2 1 0

Pre

Post

5 4 3 2 1 0

Pre

Post

15

Colon
4 2 0

10

2
5

1 0

Pre

Post

Pre

Post

Pre

Post

Pre

Post

Pre

Post

Pre

Post

Figure 3
Total SIV-Specific Responses (SFU per 1x106 CD8-depleted PBMC)

A)

8000

r02019 r96067

B)
Viral Load (V/mL)

108 107 106 105 104 103 102 101

r02019 r96067

6000

4000

2000

0 -4

12

16

100 -4

12

16

Weeks post-CD4 depletion

Weeks Post-CD4-depletion
5

C)

250K 200K 150K 100K

250K 200K

10

10 150K 10 100K

4000

r02019 r96067

FSC-H

SSC-A

50K 0 0 50K 100K 150K 200K 250K

50K 0 0 50K 100K 150K 200K 250K

10

Anti-Rhesus IgG gMFI

CD8

0 0 10
2

3000

10

10

10

FSC-A

FSC-A

CD3

2000

Week 0 day of depletion


1000

Week 1 post-depletion
500 400

1000

MFI = 293
800 600

MFI = 2094

300 400 200 0 0 102 103 104 105 200 100 0 0 102 103 104 105

0 -4

12

16

Weeks post-CD4 depletion

anti-rhesus IgG

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