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An Overview of Candidate Immunologic Biomarkers
and Surrogate Markers
Alan Landay, Ph.D.Alan Landay, Ph.D.
T-Lymphocyte Development and Function
NaiveNaiveT cellT cell
MemoryMemoryT cellT cell
Cell deathCell death
ThymicThymicmaturationmaturation
Progenitor cells
+ antigen
Effectoractivity
Tis
sue
Blo
od
Bo
ne
Mar
row
Pluripotent hematopoietic stem cell
Lymphoid progenitor Myeloid progenitorMegakaryocyte Erythroblast
Plasma cell Effector T cell Mast cell Macrophage
B cell T cellNeutrophil Eosinophil Basophil Monocyte
Platelets Red blood cells
Provided by John Schmitz
Rare Event Enumeration - CD34
0.12%
Flow Cytomertry Lab UNC Hospital
Thymic Function in HIV Infection
The thymus is disrupted in HIV infection.
Does sufficient thymic function remain to allow reconstitution of immune function with HAART– Thymic scans – Naïve T cell markers– TCR V beta repertoire– Telomere Length – TREC’s
Smith KY, et al. J Infect Dis. 2000.
Evaluation of TCR Diversity: CDR3 Size Analysis
V CDR3 J C
V specific amplification
Labelled CElectropheresis
BV5Provided byCrystal Mackall
Age Associated Reduction inTCR Repertoire Diversity
0
2
4
6
8
10
104 105 106 107
22yo Cord blood
46yo
Me
an
Pe
ak
Nu
mb
er/
BV
Fa
mily
CD4 INPUT CELL NUMBER Provided byCrystal Mackall
Vn Vn Rec V2 Dn Jn C J Jn C
V2
Dn Jn
C
VJ CodingJoint TREC
V
Vn
Rec-J
Jn
VJC
Vn Vn Rec-J Jn C
Rec-J Signal Joint TREC
Evaluation of Thymic Output by TREC Analysis
Age6050403020
CD
4 T
RE
C (
cop
ies/
ug
) 300000
200000
100000
0
-100000
r= -0.530 p= 0.003
Age6050403020
CD
8 T
RE
C (
cop
ies/
ug
) 300000
200000
100000
0
-100000
r= -0.385 p= 0.006
Replicative Senescence: Aging of Cells
Telomere: are extreme ends of chromosomes consisting of (TTAGGG)n repeats and are essential for chromosomal stability. Telomere are 5-15 kb long in human lymphocytes and approximately 50-100 bp/yr are lost in vivo
Telomere lengths is maintained by a highlyspecialized ribonucleoprotin called telomerase
Telomere
(TTAGGG)n
Replicative Senescence on AIDS:In AIDS patients there is an increase in number of CD28-CD8+ cells shortening of telomeres in CD8 cells suggest extensive replicative history as a result of clonal expansions
(Effros et al AIDS 10F17, 1996)
Replication cells
Shortening of telomeres
HealthyAdult
CentenarianHIV
InfectedAdult
Kaushal et alClinical Immunology,vol92 1999
Apoptosis - PI Assay
Darzynkiewicz, et.al. Chapter 38 Man. Clin. Lab. Immunol., 5th Ed., 1997.
Apoptosis - HIV Infection
Apoptosis is a morphologic finding resulting from the process of programed/activation induced cell death.
Characterized by condensation of the nucleus and cytoplasm and a distinctive pattern of chromosomal DNA fragmentation.
This process is of interest in HIV infection because it may be an important mechanism of CD4 T cell depletion. In HIV infection there are increased numbers of apoptotic cells.
Several methods including flow cytometry can be used to enumerate these cells.
T Lymphocyte Developmentand Function
+ antigen
effector
activity
CD34+Thyl+
CD45RA+ CD62L+
CD45RA+ CD45RO-
CD45RO- CD27+ CD95Low
TRECTelomere
CD103
CD95
Annexin V
Tunel
PI
CD45RO+ RA-
DTH
NaiveNaiveT cellT cell
MemoryMemoryT cellT cell
Cell deathCell deathThymicThymic
maturationmaturation
Progenitor cells
Host Components of the Immune Response
B Cell
Dendritic Cells
CD4 CD8
Macrophage
NK Cell
Assays of Immunologic Function
CD4 T cell: LPA, DTH (In vivo), ICC
CD8 T cell: CTL (bulk, LDA), ICC,
ELISPOT, MHC Tetramers
NK cell: NK cytotoxicity, ADCC
B cell Responses (Diptheria, Pertussis, Tetanus)
CD4Helper
CD8CTL
MØ
or
Dendritic
IL-12Interferon-
IL-2
Basic Elements of Immune Response
Activation Maturation Functional
HLA DR CD45RO+RA- CD95
CD38 CD45RA+62L+ CD28
CD25
CD71
CD69
Activation Maturation Functional
HLA DR CD45RO+RA- CD95
CD38 CD45RA+62L+ CD28
CD25
CD71
CD69
CD4 CD8
Subsets
TCRCD3CD28
CD4
TH0 Cell
MHC IICD40
M
IL-2 R
IL-2
Cell Division
Thomas F. Lint, Ph.D.Rush Medical College
CD40L
B7 IFN
IL-1IL-6TNF
Lymphocyte Proliferation Assay
In vitro correlate of cell mediated immunity which is important in the control of viral and intracellular bacterial infections
Quantitates the response of T cells to a variety of stimuli including mitogens, alloantigens and specific recall antigens
HIV infected patients tend to lose LPA responses in the order of recall antigen, alloantigen, mitogen
Strong LPA responses to HIV antigens associated with control of viral replication.
Lymphoproliferative Responses
HIV-1 p24 antigen
Pathogen Specific-CMV, MAC
Recall antigen-tetanus
Mitogen - PWM
Neoantigen - KLH
T cell B cell macrophage NK cell
INF
Cross regulation
IL-4, IL-10
Type 1 help Type 2 help
Cellularimmunity
Humoralimmunity
IL-2
IL-12
INF
IL-15
IL-4
IL-5IL-6IL-10IL-13
Intracellular Cytokines
Relatively new assay
Initial data has demonstrated a correlation between detectable reactivity to CMV antigen and lack of CMV end organ disease.
Likely to have better precision within and between labs
Faster turnaround time and no radioisotope use
Can study numerous cell types and cytokines relatively easily - more information than standard LPA
With appropriate peptides, can quantitate CD8 T cell frequencies
Provided byLouis Picker
Provided byLouis Picker
Cytotoxic Mechanisms
Perforin – (Granule exocytosis)
Granzymes
Cytokines – (IFN + TNF or TNF )
Fas - Fas Ligand (TNF-like) – apoptosis
test cpm - spontaneous cpm•Percent specific lysis = x 100
max cpm - spontaneous cpm •Precursor frequency
Cytotoxicity Assays
Handbook of Human Immunology Ch 10
MHC Tetramer Design Features
1 2
32m
12
3 2m
12
3 2m
1 2
32m
Avidin
Enzymatic Biotinylation
Oriented T cell epitope
Single Peptide Ligand
Specificity Alteredat Will
Provided byJohn Altman
Flow Cytometry:Gating on Subpopulations of Cells
1 10 100 1000 100001
10
100
1000
10000
24.1 72.9
2.44 0.55
CD62L
Ma
mu
-A*0
1/p
11C
0 50 100 150 200 2500
50
100
150
200
250
FSC
SS
C
35.1%
Lymphocytes
1 10 100 1000 100001
10
100
1000
10000
CD3
CD
8
14.6%CD3+CD8+
Provided byJohn Altman
Measures of HIV or Pathogen Specific Immunity
APC IL12/IL10Intracellular Flow Cytometry
CD4 CD8
IL2/IFN Class I Tetramer
Intracellular Flow Cytometry CTL ELISPOT
Class II Tetramers Perforin/Granzyme/TIA-1
Standardized Assays for Immune Assessment
In Vivo DTH (Recall) or Immunization (Recall or Neo)
In Vitro Intracellular Cytokine(APC-CD4-CD8)
Recommended