Dale and Betty Bumpers

Vaccine Research CenterNational Institute of Allergy and Infectious DiseasesNational Institutes of Health

Richard A. KoupVaccine Research Center

Lymph node structure and HIV-1 infection:

T cell immunopathogenesis

Background

• HIV replication is active in lymph nodes throughout the course of HIV infection

• HIV leads to an initial hypertrophy, followed by involution of lymphoid tissues (Tim Schacker)

• SIV infection in rhesus macaques has a similar pathogenesis to HIV infection in humans (Guido Silvestri) and can be used to study the immuno-pathogenesis of HIV infection in lymph nodes

• In this talk I will concentrate on using acute and early SIV infection of rhesus macaques to model HIV pathogenesis in lymph nodes

Janeway’s Immunobiology (8th Edition)

The Lymph Node has a Complicated Structure

Simplified LN Structure

Paracortical Area(T cells)

Lymphoid Follicle(B cells)

Germinal Center(B and T cells)

Light Zone(T/B cell interaction)

Dark Zone(B cell proliferation)

Simplified LN Structure – HIV Infection

Uninfected Infected - Early

Germinal Center Hypertrophy

(increased germinal center T cells)

Loss of non-germinal

center T cells

Infected - Late

Outline

• Virus replication in LN during acute/early HIV/SIV infection

• Changes in CD4 T cell populations in LNs during acute/early HIV/SIV replication

• Underlying mechanisms• Non-T cell consequences

SIV as a model for HIV infection

Nature 434:1093-7, 2005

Pla

sma

Vira

l Loa

d (L

og10

)

Acute Infection: Plasma Viral Loads

CD

4 T

Cel

ls,

% o

f C

D3+

Total CD4 Cell Dynamics

0

20

40 InguinalLN

0

20

40

60Jejunum

0

20

40 MesentericLN

0

20

40PBMC

0 3 7 10 14 17 0 3 7 10 14 17

Days Post-infection

CD

4 M

emo

ry,

% o

f C

D3+

Memory CD4 T Cell Dynamics

Early expansion at d. 3

Loss of ~80% of cells by d. 17

Cell-Associated Viral Loads

0

1x105

2x105

0

1x105

2x105

0

1x105

2x105

0

1x105

2x105

Naive CD4 T Cells Memory CD4 T Cells

PBMC

Inguinal LN

Mesenteric LN

Jejunum

Days Post-infection3 7 10 14 17 3 7 10 14 17

Cel

l-A

sso

ciat

ed V

iral

Lo

ad (

gag

Co

pie

s / 1

05 ce

lls)

These data do not take into account structural localization within the LN

• Where is the virus replicating with respect to the paracortical T cell zone and the light zone of the germinal center?

• What is happening to CD4 T cell frequency in these areas during acute and early SIV infection?

Paracortical T cell zone

Light zone of the germinal center

Surface markers can distinguish CD4 T cells from these different areas

Paracortical T cell zone

Light zone of the germinal center

CD95

CD

28

CD8

CD

4

CD3

Aq

ua

FSC-A

FS

C-H

PD-1

CC

R7

0 103 10 4 105

0

10 2

10 3

10 4

10 5

70.8

19.5

Where does SIV replicate?

PD-1

CC

R7

0

0.5

1.0

1.5

Early SIV (>2 months)

0

1

2

3

4

5

Acute SIV (3-21 days)

SIV

Gag

DN

A (

cop

ies/

cell

)

SIV

Gag

DN

A (

cop

ies/

cell

)p=0.0078p=0.0156

0 103 104 105

0

102

103

104

105

ICO

S

CD150

Paracortical T cell zone

Light zone of the germinal center

Would expect to see depletion of CD4 T cells in

germinal centers

SIV: Relative accumulation of CD4 T cells in GCs

0 102

103

104

105

0

102

103

104

105

29.1

12.9

47.8

PD-1

CC

R7

CCR7high

PD-1low

CCR7high/low

PD-1dim

TFH

% o

f C

M C

D4

T c

ells

SIV acute SIV earlySIV-

CD4 PD1 Ki-67

Michael Gerner, Ron Germain

Light zone:T - B cell interaction

Dark zone:B cell proliferation

Accumulation of GC CD4 T cells during SIV infection: Abundant GCs with retained architecture

Mechanism?

Uninfected Infected - Early

?

No correlation between VLs and percent GC T cells during SIV infection

Percent GC T cells in LN

Vira

l Loa

ds

GC T cells

% o

f C

M C

D4

T c

ells

Accumulation of GC T cells is associated with general immune activation (sCD14)

p=0.0013

p=0.0004

p=0.0164

sCD

14 (

x106

pg

/ml)

SIV acute SIV early (high % of TFH)SIV- SIV early (low % of TFH)

SIV: Relative accumulation of GC T cells

IL-6 signaling drives the up-regulation of Bcl-6 and enhanced T cell responses that are seen

during chronic LCMV infection in mice.

Does IL-6 production drive the accumulation of GC T cells during early SIV infection in monkeys?

SIV- SIV+ (early)

p=0.0215

Increase in the IL-6/IL-6R axis is associated with GC T cell accumulation during SIV infection

Pla

sm

a IL

-6 (

pg

/ml) p=0.0136

Percent GC T cells in LN

IL6R

a o

n G

C T

cel

ls (

MF

I)

Mechanism?

Uninfected Infected - Early

IL-62)

Altered phospho STAT (3>1)3)

GC T cell differentiation4)

Immune activation1)

Petrovas et al, J. Clin. Invest., 2012

SIV -

SIV chronic (low % GC T cells)

SIV chronic (high % GC T cells)

p=0.015

0 10 2 10 3 10 4 10 5

0

10 2

10 3

10 4

10 5

86.1

0 10 3 10 4 10 5

010 2

10 3

10 4

10 5

28.7

0 10 3 10 4 10 5

0

10 3

10 4

10 5

9.84

6.4922.3

61.3

0 10 3 10 4 10 5

0

10 3

10 4

10 5

15.8

0.721.87

81.5

Aq

ua

SSC CD20

CD

3

PNA

IgG

PBMC

LN

LN

PNAhighIgGlow

PNAhighIgGhigh

SIV- SIV+

low TSIV+

high T

GC T cells are TFH that influence B cells differentiation and antibody production

SIV chronic (low % GC T cells)

SIV chronic (high % GC T cells)

gp120

p=0.051

SIV

-sp

ecif

ic I

gG

(t

iter

, x1

04 )

0

2.5

5

7.5

10

Conclusions• HIV/SIV replication is profound during acute infection• This leads to a massive depletion of memory CD4 T cells

in the LN and gut• CD4 T cell depletion from the gut leads to microbial

translocation and general immune activation (discussed elsewhere)

• Immune activation promotes differentiation of T cells in the LN which move to and accumulate in the GCs (lymphoid hypertrophy)– Non-pathogenic SIV controls immune activation

• Continued immune activation ultimately leads to fibrosis of the lymph nodes

Simplified LN Structure – HIV Infection

Uninfected Infected - Early

Germinal Center Hypertrophy

(increased germinal center T cells)

Loss of non-germinal

center T cells

Infected - Late

Directinfection:

CD4 depletion

Immune activation:

GC hypertrophy followed by

fibrosis

Dale and Betty Bumpers

Vaccine Research CenterNational Institute of Allergy and Infectious DiseasesNational Institutes of Health

Costas PetrovasTakuya YamamotoKristin BoswellJoseph CasazzaRob ParisDavid Ambrozak

Immunology Laboratory NIAID/NCI Collaborators

Michael GernerRon Germain

Human Immunology Section

Netanya SandlerDaniel Douek

ImmunoTechnology Section

Mario Roederer

Lab Animal Medicine

John-Paul ToddSrinivas Rao