Transcript IMPORTANCE OF HIV SPECIFIC CTL

Immune Response to HIV
• Assays to measure immune responses
• CD4: Helper T-cell responses
• CTL: Cytotoxic T-cell responses
• B-cell: Antibody responses
• APC: Antigen Presenting Cells
Acute
Asymptomatic
Infection
AIDS and
Death
Levels (Separate Scales)
CD8+
T-cell
HIV
Antibodies
4–8
weeks
Years
CD4+
T-cell
Flow Cytometry and Cell
Sorting
CD62L
Anti-CD62L
Anti-CD45RA
Anti-CD8
Y
CD45RA
Side Scatter
(How complex the cell is)
Lymphocyte Gating
R1
R1
Forward Scatter
(How large the cell is)
Flow Cytometry
Gated on CD4
30%
CD62L
49%
(Dimmer)
5.2%
CD45RA
(Brighter)
T-Cell Phenotypes
Immunophenotyping
CD3+CD4+CD45+ = CD4+ T-cell
CD3+CD8+CD45+ = CD8+ T-cell
CD3-CD19+CD45+ = B-cells
CD3+CD4+CD28+CD45RA+ = naïve CD4+ T-cell
CD3+CD4+CD28+/-CD45RA- (or RO+) = effector/memory
CD4+ T-cell
CD4+ T-cell Responses To HIV
CD4+ T-cell responses to antigens are usually indirectly
measured by proliferation (cell division).
3H-Thy uptake
•
•
CFSE
•Cytokine production is another measure of activation
•Eliza
•ELISpot
•Flow cytometry: Polychromatic flow
3H-thymidine
uptake for measuring CD4+ T-cell proliferation.
3H
Thymidine
Pros:
• Relatively easy
• Well accepted method
Cons:
• Measures relative amounts of proliferation
• Doesn’t tell you how many cells
• Doesn’t identify which cells
Analysis of lymphocyte proliferation by CFSE
(Carboxyfluorescein diacetate succinimidyl ester)
2nd division
1st division
CFSE
Undivided
Proliferative response of peripheral CD4+
T-cells to tetanus.
Healthy Control Day 7
Medium/IL-2
Media
1 04
10 4
4%
38%
1 03
10 3
1 02
10 2
1 01
10 1
CD4
CD4
Tetanus/IL-2
Tetanus
1 00
10 0
101
1 02
CF SE
103
1 04
10 0
1 00
CFSE
10 1
1 02
CF SE
10 3
1 04
Proliferative response of peripheral CD4+
T-cells to tetanus.
LTNP #1 Day 7
Tetanus
Medium
4%
CD4
<1%
CFSE
Proliferative response of peripheral CD4+
T-cells to p24
LTNP #1
Medium
p24
3%
CD4
<1%
CFSE
CFSE ASSAY
Pros:
• Gives percentage of cells that proliferated
• Allows identification of cells that proliferated
• Detects the number of times cells proliferated
• Doesn’t use radioactive isotopes
Cons:
• A little more complex to perform
• CFSE can have some low level toxicity to
cells
• Requires more skill to interpret
ELISPOT Analysis For
Cytokine Production
Y Y Y Y
Y
1. Coat a well with antibody
to cytokine to be measured.
(IL-2, IL-5, IFN-g etc.)
Y Y Y Y
2. Add cells and antigen.
Incubate overnight.
3. Cells that recognize the antigen
Y Y Y Y
will become activated and
secrete cytokines
which will be captured by the antibody.
ELISPOT Analysis Cont.
Y Y Y
4. Wash away cells.
Y
Y Y Y Y
Y Y Y Y
5. Add a second antibody to the
cytokine, linked to an enzyme.
6. Develop the plate by adding a
colored substrate to react with
the enzyme. Read the plate with
computerized ELISpot reader.
ELISpot ASSAY
Pros:
• Relatively easy
• Highly quantitative. Allows determination of
the
number of responding cells.
• Dot size can indirectly allow relative
assessment of cytokine amounts (-, +, ++,
etc.)
Cons:
• Currently doesn’t allow phenotyping of
CD4+ T-cell Response To HIV
cont.
CD4+ T-cell responses are predictive of disease progression.
In most individuals, the following pattern is observed:
CD4+ T-cell responses decline at various stages:
response to HIV and recall antigens
(early)
response to alloantigens
(mid)
response to mitogens
(late)
expression of IL-2 receptor (CD25)
In addition, there is aberrant cytokine production
production of IFN-g, IL-2
production of IL-4, IL-10
Mandell & Mildvan I AIDS
CD8+ T-cells: Two potential
mechanisms for viral control.
CTL Responses To HIV
CTL responses are measured by
51Cr release assay: The “gold standard” for killing.
•
While it gives relative levels of CTL activity, it
doesn’t quantify the number of epitope specific cells.
•
ELISpot: Does not directly demonstrate killing, but
does allow quantification of epitope specific cells.
•
Tetramer staining: Quantifies epitope specific CD8+
T-cells and allows for phenotypic analysis.
Restricted to one epitope and requires knowledge of
person’s HLA type.
51Cr
Release Assay For CTL Activity
51Cr
Release Assay For CTL Activity
51Cr
Release Assay
Pros:
• The only assay that measures killing with the
caveat that flow-based assays are being
developed
•
•
•
•
•
Cons:
Measures relative amounts of killing
Doesn’t tell you how many cells did the killing
Doesn’t identify which cells did the killing
Uses a radioisotope
ELISPOT Analysis For CTL
Activity
Y Y Y Y
Y
1. Coat a well with antibody
to IFN-g or granzyme.
Y Y Y Y
2. Add cells and antigen.
Incubate overnight.
3. Cells that recognize the antigen
Y Y Y Y
will become activated and
secrete INF-g and granzyme
which will be captured by the antibody.
ELISPOT Analysis For CTL
Activity
Y Y Y
4. Wash away cells.
Y
Y Y Y Y
Y Y Y Y
5. Add a second antibody to IFN-g
or granzyme, linked to an enzyme.
6. Develop the plate by adding a
colored substrate to react with
the enzyme. Read the plate with
computerized ELISpot reader.
Black Bars = Freshly obtained peripheral blood CD8+ T-cells
Striped Bars = Expanded peripheral blood CD8+ T-cells
Vpu-B
Vpr-B
Vpu-A
Vpr-A
Vif-C
Vif-A
Vif-B
Tat-B
Tat-A
Rev-A
Rev-B
Pol-P
Pol-O
Pol-M
Pol-N
Pol-L
Pol-K
Pol-I
Pol-J
Pol-H
Pol-G
Pol-E
Pol-F
Pol-D
Pol-C
Pol-A
Pol-B
Nef-D
Nef-C
Nef-B
Gag-H
Nef-A
Gag-G
Gag-F
Gag-D
Gag-E
Gag-C
Gag-B
Env-N
Gag-A
Env-M
Env-L
Env-J
Env-K
Env-I
Env-H
Env-F
Env-G
Env-E
Env-D
Env-B
Env-C
Env-A
Mean SFC/million cells
ELISpot Analysis of CD8+ T-cell responses to HIV During
Chronic Infection
600
500
400
300
200
100
0
Targeting of HIV proteins by CD8+ T-cells during Chronic
HIV infection
SFC/Million CD8+/AA
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Env
Gag
Nef
Pol
Rev
Nef>Gag>Pol
Tat
Vif
Vpr
Vpu
Tetramer staining to
quantify antigen specific CD8+
T-cells
1. Construct HLA class of the type present in the person
you are studying. Link them into tetramers folded
around the peptide of interest.
2. Bind these tetramers to a fluorescent tag.
3. React with lymphocytes.
4. Run through a flow cytometer.
Tetramer Staining
MHC molecule
Antigen
Fluorescently
Labeled
Strepavidin
Biotin
Y
Cytotoxic T-cell
Y
TCR
CD8 molecule
HIV Specific CTL are critical for
control of HIV Replication
•CTL responses are made to various epitopes on:
Gag, RT, Env, Pol, Nef, Vif, Vpr
•Inverse correlation between viral load and levels of circulating
HIV-specific CTL.
•Emergence of CTL escape mutants over time.
•Depletion of CD8+ T cells from macaques prior to infection
with SIV, leads to higher viral loads and more profound
immunosuppression.
•Absence of detectable HIV-specific CTL, or oligoclonal CTL
responses are associated with poor clinical outcome.
CTL fail to eliminate HIV-1
• Many chronically infected individuals have
vigorous HIV-1-specific CTL responses yet
they almost always fail to adequately suppress
the virus.
Why?
Epitope
CTL
escape?
Exhaustion?
Suboptimal
CTL?
Donor A: CD8 response to SL9
% Positive CD8 T cells
GAG tetramer
1
Gamma-INF
1.5
SLYNTVATL
1
0.5
0.5
0
01/85
0
01/87
01/89
01/91
Date
01/93
01/95
Proportion of SLYNTVATL
2
Emergence of epitope sequence mutations
ID
A
B
C
Years from
Seroconversion
0.26
"
1.52
"
1.74
"
3.08
"
"
"
5.51
"
8.00
8.98
9.98
Proportion
of clones
9/10
1/10
9/10
1/10
9/10
1/10
2/9
2/9
3/9
2/9
5/11
6/11
10/10
10/10
9/9
1.30
1.78
3.35
"
3.54
4.66
"
5.67
"
6.73
"
"
10/10
10/10
9/10
1/10
12/12
9/10
1/10
9/10
1/10
6/10
3/10
1/10
aa acid position within
Variant
Nomenclature S L Q T G
SL9
A - - - "
A - - - E
"
A - - - "
A - - - "
- - - - 79F
- - - - SL9
- - - - 83V
- - - - 79F
- - - - 79F-82I
- - - - 79F-84V
- - - - 82I-84V
- - - - "
- - - - "
- - - - "
- - - - SL9
"
"
"
84V
79H-84V
82I-84V
79F-84V
84V
"
"
79F-84V
A
A
A
-
-
-
-
-
gag p17: 77 78 79 80 81 82 83 84 85
S
-
E
-
E
-
L
-
R
K
K
K
K
K
S
-
L
-
Y
F
F
F
F
-
N
-
T
-
V
I
I
I
I
I
A
V
-
T
V
V
V
V
V
L
-
Y
-
C
-
V
-
H
-
Q
-
R
K
K
K
K
K
K
K
K
I
-
E
-
I
V
V
V
V
V
V
V
V
V
V
V
K
-
-
-
-
-
-
-
-
H
F
F
-
-
I
-
-
V
V
V
V
V
V
V
V
-
-
Y
Y
-
-
-
-
K
K
K
K
K
K
K
K
K
K
T
-
-
V
V
V
V
V
V
V
V
V
-
1.57
9/9
SL9
A - - - - - - - - - - - - - - - - - - - - - - - K - - V 3.35
7/8
"
- - - - - - - - - - - - - - - - - - - - - - - - K - - V "
1/8
"
- - - - - - K - - - - - - - - - - - - - - - - - K - - V 5.77
9/9
"
- - - - - - - - - - - - - - - - - - - - - - - - K - - - 6.48
2/9
"
- - - - - - - - - - - - - - - - - - - - - - - - K - - - "
5/9
"
A - - - - - - - - - - - - - - - - - - - - - - - K - - - "
1/9
"
A - - - - - - - - G - - - - - - - - - - - - - - K - - - "
1/9
84V
- - - - - - - - - - - - - - - - - V - - - - - - K - - - 8.24
13/17
"
- - - - - - - - - - - - - - - - - V - - - - - - K - - - "
4/17
"
- - - - - - - - - - - - - - - - - V - - - - - - - - - - Table I. SL9-epitope and flanking amino acid sequences. Predicted amino acid sequences of the SL9-epitope and the 10 amino acids flanking each epitope are
shown. Sequences were derived from plasma RNA at the indicated timepoints.
Antibody Responses
General Properties of Anti-viral
Antibodies
•Can be generated to any accessible portion of the virus.
•Effective in blocking entry (neutralizing) if directed to viral
receptors such as gp120 of HIV.
•Can block fusion (neutralizing) if antibody (Ab) binds to fusion
protein such as gp41 of HIV.
•Can effect clearance of virus if it binds the virus and then
binds Fc receptors on monocytes and macrophages.
•Can also bind complement and kill enveloped viruses.
•Most effective if they are present at the site of viral entry.
HIV-1 derived gp120
Gp120 is presented
as a trimer. The
monomer does
not present the
proper epitopes.
CD4 binding site
is devoid of glycosylation
and relatively conserved
between isolates but is
masked by V1V2 loops
and is in a depression.
gp120
CD4 binding site
gp41
C
Gp41
C
N
Inner
N
CD4bs
V5
Outer
V4
CD4bs
Bridging Sheet
V1V2
V3
Coreceptor bs
Non-nuetralizing
face
2G12
CD4bs
Trimerization
Bridging Sheet
Co-R bs
Neutralizing face
Glycosylation:
Silent face
ANTIBODY RESPONSE TO
HIV
Neutralizing antibodies are made primarily to gp120.
Group 1: Arise later in infection, recognize gp120 from a broad
range of isolates. Interfere with binding to CD4. Recognize
discontinuous epitopes known as the CD4bs epitopes.
Group 2: Are directed to epitopes induced by gp120 binding to CD4.
These are located near the conserved gp41 structures important for chemokine receptor interactions. However, these
epitopes are poorly exposed prior to CD4 binding. (17b)
(2F5 is the only confirmed antibody to bind gp41)
Group 3: Recognizes a conserved epitope most likely conserved
carbohydrate in the outer domain. Rare but broadly
neutralizing. (2G12)
Antibody mediated inhibition of
Fusion is difficult to achieve.
Changes in gp120 glycosylation allow HIV escape from Nab
responses
Richman et al. PNAS 2003 vol. 100:4149
Changes in the ability of HIV infected
individuals to neutralize HIV over time.
Richman et al. PNAS 2003 vol. 100:4149
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*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
HIV and aPC’s
•APC’s may exhibit altered:
chemotaxis
IL-1 production
antigen presentation
oxidative burst response
antimycobacterial activity
•Antigen presenting cells can act as
Trojan horses.
Dendritic Cells and
DC-SIGN
DC-Specific, ICAM-3 Grabbing, Nonintegrin.
Interaction of DC-SIGN with ICAM-3 establishes the initial
contact of the DC with a resting T-cell.
This is important because of the low number (100-1000
copies/cell) of MHC-peptide ligands on the DC. This
enhanced binding allows the T-cell to scan the surface of
the DC.
DC-SIGN also binds the glycan-rich HIV-1 envelope in the
absence of CD4.
Proposed pathways for the
Transmission Of HIV-1 to T-cells.
R. Steinman Cell 2,000 100:491-494
Why does the immune response
fail to clear HIV?
•HIV integrates into the host genome.
Therefore, to eliminate HIV, infected cells must
be killed.
•Host factors can paradoxically enhance
HIV replication. Therefore, by
responding to HIV, CD4+ T-cells can be
destroyed.
Why does the immune response
fail to clear HIV?
•HIV can mutate and escape immune mediated
opposition.
•Suboptimal CTL responses can be elicited.
Why does the immune response
fail to clear HIV?
•Sugar coating (glycosylation) and folding of
gp120 protects against Ab recognition.
•Critical binding sites on gp41 are revealed for
only a short period of time.
Why does the immune response
fail to clear HIV?
•APC’s may exhibit altered functions diminishing their
ability to elicit immune responses.
•Antigen presenting cells can act as Trojan horses,
spreading HIV to CD4+ T-cells as they begin to
respond to antigen.
Why does the immune response
fail to clear HIV?
Role of viral genes:
Tat: Extracellular Tat stimulates CD4+ and CD8+ T-cells.
Nef: Intracellular Nef appears to activate cells to promote
viral replication. Affect on cellular function?
Intracellular Nef downregulates CD4 and MHC class I
molecules. In vivo significance?