mudd 2011 reduction of cd4 t cells in vivo does not affect virus load in elite controllers

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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*

1Department 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,

4Wisconsin National Primate Research Center, University of Wisconsin-Madison,

Madison, WI 53711

*Corresponding Author:

David I. Watkins

Department of Pathology and Laboratory Medicine, University of Wisconsin-

Madison, 555 Science Dr., Madison, WI 53711

Phone: (608)265-3380, Fax: (608)265-8084

Email: [email protected]

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

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.00738-11JVI Accepts, published online ahead of print on 18 May 2011



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 I-

associated 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 SIV-

infected 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 IgG1κ 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 µL of blood to 61 per

µL 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-naïve

animals. Average CD4+ T cell counts in peripheral blood were reduced from

1,072 per µL of blood to 116 per µL 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

naïve CD4+ T cell subpopulations prior to and after depletion (Figure 2B). We

classified these populations by expression of the surface markers CD28 and

CD95 (naïve 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 µL 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



reduction in T cell populations within the gut-associated lymphoid tissue post-

CD4-depletion.

We continually monitored the individual SIV-specific CD4+ T cell

responses found in the peripheral blood of each animal (Figure 1) using CD8-

depleted 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 Mamu-

B*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 µL

of plasma obtained from the CD4-depleted animals at separate time points for 45

minutes in 100 µL 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 µg/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 µg/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 CD4-

depleted 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 publication’s 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 5’-

CACTAGCTGTCTCTGCACTATGTGTTTTG-3’. The detection probe sequence

was 5’-6-carboxyfluorescein-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-6-

carboxytetramethylrhodamine-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). CD8-

depleted 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 pre-

depletion 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 -80°C throughout

the experiment and then thawed once to evaluate the presence of rhesus

recombinant CD4 depleting antibody. 10 µL of plasma was used to stain one

million PBMC from a SIV-negative rhesus macaque. Secondary antibodies (anti-

CD3 [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

animal’s 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

µg/mL final rhesus recombinant anti-CD4 antibody concentration. The plasma

concentration of the depleting antibody in r02019 was < 0.5 µg/mL before 5

weeks post-depletion and was < 0.5 µg/mL in r96067 before 6 weeks post-

depletion, 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

0

150

300

450

600

750

S F U

p e r 1 x 1 0 6 C D 8 - d e p l e t e d P B M C

Gag

Pol

Vpr

Vpx

Vif

Tat

Rev

Env

Nef

r96067

0

150

300

450

600

750

S F U p e r 1 x 1 0 6 C D

8 - d e p l e t e d P B M C

Gag

Pol

Vpr

Vpx

Vif

Tat

Rev

Env

Nef

r02019

0 10 20 20 105 190 190 210 230100

101

102

103

104

105

106

107

108

r02019

Weeks post-infection

V i r a l L o a d

( V / m L )

0 10 20 20 60 100 100 120 140100

101

102

103

104

105

106

107

108

r96067

Weeks post-infection

V i r a l L o a d ( V / m L )

CD4-depletion

CD4-depletion



Figure 2

A)

B)

C)

-4 0 4 8 12 160

1000

2000

3000 r02019

r96067

Weeks post-CD4 depletion

# C D 8 + T

c e l l s p e r u L b l o o d

-4 0 4 8 12 160

200

400

600

800

1000 r02019

r96067


# C D 2 0 + c

e l l s p e r u L b l o o d

-4 0 4 8 12 160

500

1000

1500

2000 r02019

r96067


# C D 4 + T


-4 0 4 8 12 160

300

600

900

1200 r02019r96067

Weeks post-CD4 depletion # C D 2 8 + C D 9 5 - C D 4 + T


-4 0 4 8 12 160

200

400

600

800 r02019r96067

Weeks post-CD4 depletion # C D 2 8 + C D 9 5 + C

D 4 + T


-4 0 4 8 12 160

100

200

300

400

500 r02019r96067

Weeks post-CD4 depletion # C D 2 8 - C D 9 5 + C

D 4 + T


Pre Post0

10

20

30

40

50

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60

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CD8+ T cells CD20+ B cells CD4+ T cells CD28+CD95-

CD4+ T cells

CD28+CD95+

CD4+ T cells

CD28-CD95+

CD4+ T cells

PBMC

Lymph

Node

BAL

Colon

r02019r96067



A) B)

0 50K 100K 150K 200K 250K

0

50K

100K

150K

200K

250K

0 50K 100K 150K 200K 250K

0

50K

100K

150K

200K

250K

0 102

103

104

105

0

102

103

104

105

F S C - H

FSC-A FSC-A

S S C - A

C D 8

CD3

0 102

103

104

105

0

200

400

600

800

1000

MFI = 293

0 102

103

104

105

0

100

200

300

400

500MFI = 2094

anti-rhesus IgG

Week 0

day of depletion

Week 1

post-depletion

C)

Figure 3

-4 0 4 8 12 16100

101

102

103

104

105

106

107

108r02019r96067

Weeks Post-CD4-depletion

V i r a l L o a

d ( V / m L )

-4 0 4 8 12 160

2000

4000

6000

8000 r02019r96067


T o

t a l S I V - S p e c i f

i c R e s p o n s e s

( S F U p e r

1 x

1 0 6

C D 8

- d e p

l e t e d P B M C )

-4 0 4 8 12 160

1000

2000

3000

4000 r02019r96067


A n

t i - R h e s u s I g

G

g M F I

mudd 2011 reduction of cd4 t cells in vivo does not affect virus load in elite controllers

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