An Overview of

Candidate Immunologic Biomarkers

and Surrogate Markers

Alan Landay, Ph.D.

T-Lymphocyte Development and Function

Naive Cell death

T cell
+ antigen

Thymic
Memory
maturation
T cell

Progenitor cells Effector

activity
Bone Marrow Pluripotent hematopoietic stem cell

Megakaryocyte Erythroblast
Lymphoid progenitor Myeloid progenitor

Monocyte
Blood

Neutrophil Eosinophil Basophil

B cell T cell

Platelets Red blood cells

Tissue

Plasma cell Effector T cell Mast cell Macrophage

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 C
Electropheresis

Provided by
BV5 Crystal Mackall
 Age Associated Reduction in
TCR Repertoire Diversity
10
Cord blood
22yo
8 46yo
Mean Peak Number/BV Family

0
104 105 106 107
CD4 INPUT CELL NUMBER Provided by
Crystal Mackall
 Evaluation of Thymic Output by TREC Analysis
Dn Jn

Vn Vn Rec V2 Dn Jn C J Jn

C

V2 C

Rec-J Signal
Joint TREC
Vn

Vn Vn Rec-J Jn C

Rec-J
V
Jn

VJ Coding
Joint TREC

VJC

300000 300000

CD8 TREC (copies/ug)

CD4 TREC (copies/ug)

r= -0.385 p= 0.006
200000 r= -0.530 p= 0.003 200000

100000 100000

0 0

-100000 -100000
20 30 40 50 60 20 30 40 50 60
Age Age
 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
Replication cells
lymphocytes and approximately 50-100 bp/yr
are lost in vivo
Telomere
Telomere lengths is maintained by a highly (TTAGGG)n
specialized ribonucleoprotin called telomerase
Shortening of telomeres

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 Healthy Centenarian
HIV
Infected
Adult Adult
(Effros et al AIDS 10F17, 1996)
Kaushal et al
Clinical 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 Development
and Function
TREC CD45RA+ CD62L+
Telomere
CD45RA+ CD45RO-
CD103
Naive
CD45RO- CD27+ CD95Low
T cell
CD95
Annexin V

Cell death Tunel

+ antigen PI

Thymic
maturation

Memory CD45RO+ RA-

T cell
Progenitor cells
effector
activity

CD34+Thyl+ DTH
 Host Components of
the Immune Response

B Cell NK Cell

Dendritic Cells Macrophage

CD4 CD8
 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)
Basic Elements of Immune Response

Dendritic

or CD4 CD8
Helper CTL
MØ Interferon-
IL-12
IL-2
 CD4 CD8
Subsets

Activation Maturation Functional

HLA DR CD45RO+RA- CD95
CD38 CD45RA+62L+ CD28
CD25
CD71
CD69
 Cell Division
TH0 Cell
CD28
CD3 IL-2 R
TCR CD4
CD40L

IL-2

IL-1
IL-6
TNF
B7 IFN 
MHC II
CD40
M Thomas F. Lint, Ph.D.
Rush Medical College
 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

Type 1 help Cross regulation Type 2 help

IL-2 IL-4
IL-12 IL-4, IL-10 IL-5
IL-6
INF
IL-10
IL-15 IL-13

Cellular Humoral
immunity immunity
 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 by
Louis Picker
Provided by
Louis Picker
 Cytotoxic
Mechanisms
 Perforin
– (Granule exocytosis)
 Granzymes
 Cytokines
– (IFN  + TNF  or TNF )
 Fas - Fas Ligand (TNF-like)
– apoptosis
 Cytotoxicity Assays

test cpm - spontaneous cpm

•Percent specific lysis = x 100
max cpm - spontaneous cpm
•Precursor frequency

Handbook of Human Immunology Ch 10

 MHC Tetramer Design Features

1 2 2 1
 Enzymatic Biotinylation 2m 3 3 2m
 Oriented T cell epitope
 Single Peptide Ligand Avidin

 Specificity Altered
at Will 2m 3 3 2m
1 2 2 1

Provided by
John Altman
 Flow Cytometry:
Gating on Subpopulations of Cells

Mamu-A*01/p11C
250 10000 10000
CD3 CD8+ +
14.6% 2.44 0.55
200
1000 1000
SSC

150
CD8 24.1 72.9
Lymphocytes 100 100
100
35.1% 10 10
50

0 1 1
0 50 100 150 200 250 1 10 100 1000 10000 1 10 100 1000 10000

FSC CD3 CD62L

Provided by
John Altman
 Measures of HIV or Pathogen Specific
Immunity

APC IL12/IL10 Intracellular 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)