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Viral Latency and
T-cell Responses
After HAART
Experimental Protocol: Ho et al.
Nature 1995
20 HIV-infected individuals with CD4+ T cell counts
ranging from 36 to 490 cells (mm-3)
Protease Inhibitor:ABT-538
Measure viral RNA in the plasma
Measure CD4+ T-cells in the blood
Plasma viral load before and after drug
treatment
RNA copies per ml (x 103)
1000
Slope: -0.21
t 1/2: 3.3 days
100
10
1
-10
-5
Ho et al. Nature 1995
0
5
10
15
Days
20
25
30
35
Viral Half-Life
•Extrapolating from the curves:
•Between 108 and 109 virions are cleared each day suggesting
that viral production is 108 to 109 virions per day
•The T ½ is ~2 days (current estimates place it as even shorter)
•Unfortunately, not all the virus is cleared
Viral Latency
•The latent viral pool persists in everyone following Highly Active
Anti-Retroviral Therapy (HAART)
•Is established soon after infection
•T1/2 of replication competent virus is ~44 months therefore
eradication could take up to 60 years.
Evidence of Viral
Reservoirs
Plasma Viral RNA
Primary Infection
Viral Rebound
Viral Setpoint
Cessation
Of HAART
HAART
50
copies
Infection
Forms of HIV-1 “Latency”
•Compartmentalization
•CD4 + cells
•Pre-integration complexes – labile vs. stable
•Post-integration proviral DNA
•Drug resistance:
While drug resistance does occur, there is evidence
of wild-type HIV-1 replication.
Model for establishment and
maintenance of HIV-1 reservoirs
Death
Activation:
antigen
Activated T-cell
Quiescent T-cell
Activated T-cell
and renewed
viral replication
What Researchers Know So
Far:
Infection of naïve cells occurs infrequently and tends
to result in more labile forms of virus.
Viral DNA is most abundant in CD45RA- T-cells:
Effector or memory? ~ 1 per 106 cells.
Viral RNA is more abundant in activated, HLA-DR+,
T-cells.
Macrophages are also a cellular reservoir for HIV-1,
but are short lived, therefore; their contribution
to the long-lived reservoir is unclear.
Increase in CD4+ cell counts after drug treatment
1000
CD4 cell count (per mm3)
Slope: 2.4
100
10
1
-15
-10
Ho et al. Nature 1995
-5
0
5
10
Days
15
20
25
30
35
Inverse correlation between baseline CD4 cell count
and CD4 cell recovery
0.1
Exponential slope of CD4 increase
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0
50
Ho et al. Nature 1995
100
150
200
250
Baseline CD4+ cell count
300
350
400
450
Autran et al.:Experimental
Protocol
8 HIV-infected, previously untreated individuals
with advanced HIV-infection.
Protease inhibitor; Ritonavir
AZT
Dideoxycytosine
Measured viral RNA in the plasma
Determined lymphocyte counts in the blood
Phenotyped the cells
Determined responses to recall antigens
Phenotype of CD4+ T-cells following drug therapy
300
250
Cells (per ml)
200
150
100
50
0
0
5
10
15
20
Weeks
Pakker et al. Nature Medicine 1998
25
30
35
40
Naïve CD4+ cells
Memory CD4+ cells
Total CD4+ cells
Phenotype of CD8+ T-cells following drug therapy
900
800
700
Cells per ml
600
500
400
300
200
100
0
0
5
10
15
20
Weeks
Pakker et al. Nature Medicine 1998
25
30
35
40
Naïve CD8+ cells
Memory CD8+ cells
Total CD8+ cells
Roederer Nature Medicine 1998
Changes in T-cell subsets with drug therapy
CD8+
T-cells
Memory
Naive
CD4+
T-cells
Memory
Naive
Years after infection
HAART
Re-expression of CD25 on CD4+ T-cells After Drug
CD4+ CD25+
Therapy
CD45RO+ CD25+
CD45RA+ CD25+
Total CD4+ T cells
30
% Positive Cells
25
20
15
10
5
0
0
2
4
6
8
Months
Autran et a. Science 1997
10
12
14
Decrease in HLA-DR expression in T-cells after
CD3+8+DR+
drug therapy
Infectious Cells
CD45RO+DR+
CD4+DR+
Total CD4+ cells
30
% Positive Cells
25
20
15
10
5
0
0
2
4
6
8
Months
Autran et al. Science 1997
10
12
14
Restoration of CD4+ T-cell responses to tuberculin
Stimulation Index
100
10
1
0.1
-1
0
1
2
3
Months
Autran et al. Science 1997
4
5
6
7
Response of CD8+CD38+
T-cells To HAART
CD38 molecules on CD8T cells
Median Number of Molecules/cell
10000
8000
6000
4000
2000
0
25
26
27
28
29
Visit
30
31
32
33
Summary
•With the addition of a protease inhibitor, plasma viral load
drops but is not completely eliminated = viral latency
•As viral load drops CD4+ T-cells increase
•CD8+ T-cells increase then decline slightly
•Activation antigens decrease on both subsets
•CD4+ T-cells re-express CD25 and regain proliferative
responses to recall antigens
•The greatest T-cell increases are in the memory
phenotype cells
•After about six weeks slow, but steady, increases are
observed in the naïve T-cell compartment
Where Do The T-cells Come
From?
•Homeostasis mechanisms trigger proliferation of surviving cells.
•Redistribution of cells from inflammed tissues, back into blood
causes an observed increase in cells.
•Naïve cells: Thymus vs. gut vs. proliferation?
Can The Thymus of
Adults Contribute To
T-cell Reconstitution?
Phenotypic markers of T-cell
development in the Thymus
BM
CD34+
CD1CD3CD4CD5+/CD8-
Thymus
CD34+/CD1+
CD3CD4+
CD5+
CD8-
CD34CD1++
CD3CD4+
CD5+
CD8+
CD34CD1++
CD3+
CD4+
CD5+
CD8+
CD69+
PB
CD1+
CD3+
CD4+
CD8CD69+
CD1CD3+
CD4+
CD8CD69+
CD1CD3+
CD4+
CD8CD69-
CD1+
CD3+
CD4CD8+
CD69+
CD1CD3+
CD4CD8+
CD69+
CD1CD3+
CD4CD8+
CD69-
Summary of Adult Thymus
Specimens
Thymus#
Fetal
Fetal
1*
2*
3
4
5
6
7
8
9
10
11
12*
Age
0
0
23
23
23
33
33
36
36
37
48
52
54
56
CD4+CD8+
85
82
86
70
65
35
88
76
15
2
48
79
22
69
*Corrective Cardiac Surgery
Human Thymocyte
Differentiation
CD4+
TCR+
CD4+/-
CD4+
CD4+
CD8+
TCR+
CD8+
TCR+
CD34
CD5
CD1a
CD69
CD3
CD62L
Adult And Fetal Thymocytes Demonstrate
Similar Subset Distribution
Age
11%
85%
4%
88%
Fetal
10%
86%
6%
CD1a-PE
23
yrs.
CD4-TC
4%
2%
3%
13%
79%
89%
8%
2%
83%
52 yrs.
6%
CD8-FITC
14%
CD5-FITC
Adult And Fetal Thymocytes Demonstrate
Similar Subset Distribution
Age
8%
15%
80%
Fetal
11%
8%
16%
6%
CD3-PE
23
yrs.
CD69-PE
72%
14%
6%
72%
12%
22%
77%
14%
69%
52 yrs.
62%
CD8-FITC
14%
CD8-FITC
Phenotypic markers of T-cell
development in the Thymus
BM
CD34+
CD1CD3CD4CD5+/CD8-
Thymus
CD34+/CD1+
CD3CD4+
CD5+
CD8-
CD34CD1++
CD3CD4+
CD5+
CD8+
CD34CD1++
CD3+
CD4+
CD5+
CD8+
CD69+
PB
CD1+
CD3+
CD4+
CD8CD69+
CD1CD3+
CD4+
CD8CD69+
CD1CD3+
CD4+
CD8CD69-
CD1+
CD3+
CD4CD8+
CD69+
CD1CD3+
CD4CD8+
CD69+
CD1CD3+
CD4CD8+
CD69-
Co-stimulation of Adult Thymocytes
Unstimulated
Medium
Stimulated
Medium
Anti-CD28
Anti-CD3
3 Days
3 Days
Harvest
Stain for activation markers
Adult Thymocytes
Respond To Costimulatory Signals
CD25
Thymus #
Fetal
SCID-hu
1
3
4
5
8
11
Age
0
0
23
23
33
33
37
54
Unstim
8
1
2
1
9
2
<1
ND
CD69
Stim
74
47
80
49
96
46
62
ND
Unstim
10
18
22
20
49
17
7
29
Stim
86
93
67
57
96
80
75
67
10%
CD8+
8%
6%
4%
2%
Adult
0%
Thymocytes
8%
6%
Display A
Diverse Vb
Repertoire
4%
2%
0%
8%
6%
4%
2%
0%
Adult
Fetal
Can Adult
Thymocytes Migrate
To The Periphery?
Signal joint (sj) and coding joint (cj)
TREC production from the a/d locus
a locus
V
a
d locus
a locus
Vd dRec Dd Jd Cd yJa Ja Ca
sj PCR
cj PCR
Adapted from Martie C. M. Verschuren, Thesis
TREC:
-T-cell receptor Rearrangement Excision Circles
- are detected in phenotypically naive T-cells, but not
in memory T-cells or in B-cells.
- decline in the peripheral blood with age, after
thymectomy, and in HIV-infected individuals.
- increase in HIV-infected individuals undergoing
HAART.
Douek et al. Nature 1998
TREC Positive Cells In The Periphery of Adults
Peripheral CD4+ T-cells
Thymocytes
Sj TRECs per mg of DNA
100000
10000
1000
100
0
10
20
30
40
50
Age (years)
60
70
80
TREC changes during HAART
1 4 A -3 6
12
10
8
6
4
2
0
8
7
6
5
4
3
2
1
C -2 5
B -2 8
1 06
-
1 05
+
10
4
103
50
10 0
15 0
20 0
0
25 0
D - 29
50
1 00
150
20 0
25 0
0
50
10 0
2 00
2
250 1 0
6
10
F-3 6
E -2 9
1 50
+
1 05
10
-
4
103
0
50
100
150
2 00
2 50
0
8 G -2 7
7
6
5
4
3
2
1
0
1 00
150
20 0
25 0
H -3 2
0
50
100
15 0
200
I-2 2
25 0
102
1 06
1 05
104
10 3
50
10 0
15 0
20 0
8 J-6 3
7
6
5
4
3
2
1
0
50
250
1 06
1 05
10
4
103
50
10 0
15 0
20 0
250 1 0
0
50
1 00
150
20 0
25 0
0
50
100
15 0
200
25 0 1 0
2
Sj TREC x103 per mg naïve CD4 cell DNA
Naïve CD4 T cells x102 per ml blood
HIV RNA copies per ml of plasma
2
Douek et al. Nature 98
Conclusions
1. Thymopoiesis is ongoing in adults, resulting in
functional T-cells with a diverse TCR-Vb
repertoire.
2. CD4+, phenotypically naïve, TREC positive T-cells
increase following HAART suggesting that the adult
thymus contributes to T-cell reconstitution.
3. The thymic microenvironment remains functional
after exposure to HIV.
Douek et al. 1998 Nature 396: 690
Withers-Ward et al. 1997 Nature Med. 3: 1102
Jamieson et al. 1999 Immunity, 10: 569
Conclusions cont.
Taken together, these results suggest that therapeutic
strategies to enhance thymopoiesis in the adult may
increase T-cell reconstitution in HIV-infected
individuals treated with HAART, or in cancer patients
undergoing chemotherapy. In addition, these
strategies may improve T-cell responses in the elderly.
Strategies to Improve
T-cell reconstitution
Vishwa Deep Dixit
Current Opinion in Immunology
Volume 22, Issue 4, August 2010: 521-528
Therapeutic Strategies
1.
2.
3.
4.
5.
6.
7.
IL-7: Modulates thymic output, expands and supports
survival of naïve and memory T-cells. In HIV-1 infected
individuals see increases in functional T-cells(1).
IL-15: Supports survival of NK, NKT and CD8+ memory
T-cells(2)
Growth Hormone: acts as a pro-thymopoietic agent (3)
Ghrelin: Reduces pro-inflammatory cytokines and
reverses thymic involution (3)
Leptin: Increases peripheral IGF1, results in thymic
regeneration (3)
IGF1: expands progenitors in bone marrow, increases
thymic epithelial niches (3)
CR mimetics: Maintains TCR diversity and increases
thymopoiesis (3)
1.
2.
3.
Levy et al. Journal of Clinical Investigation 2009, 119:997
Van den Brink et al. Nature 2004, 4:856
Vishwa Deep Dixit, Current Opinion in Immunology, 2010:
521