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)