# Presentation: 598626

## An Overview of 

**Candidate Immunologic Biomarkers **
**and Surrogate Markers**** **
**Alan Landay, Ph.D.**

## T-Lymphocyte Development and Function

**Naive****T cell**

**Memory****T cell**

**Cell death**

**Thymic****maturation**

**Progenitor cells**

**+ antigen**

**Effector****activity**

## Tissue

***Tissue***

***Blood***

***Bone Marrow***

- Pluripotent hematopoietic stem cell

- Lymphoid progenitor

- Myeloid progenitor

- Megakaryocyte

- Erythroblast

- Plasma cell

- Effector T cell

- Mast cell

- Macrophage

- B cell

- T cell

- Neutrophil

- Eosinophil

- Basophil

- Monocyte

- Platelets

- Red blood cells

- Provided by John Schmitz

## Rare Event Enumeration - CD34

**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.

**Smith KY, et al. *****J Infect Dis*****. 2000.**

## Evaluation of TCR Diversity:  CDR3 Size Analysis

- Provided byCrystal Mackall

## Age Associated Reduction inTCR Repertoire Diversity

- Provided byCrystal Mackall

## Evaluation of Thymic Output by TREC Analysis

**Evaluation of Thymic Output by TREC Analysis**

## Replicative Senescence: Aging of Cells

**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

- Centenarian

- HIVInfectedAdult

## Kaushal et al

**Kaushal et al**
**Clinical Immunology,**
**vol92 1999**

## Apoptosis - PI Assay

**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

**+ antigen**

**effector**
**activity**

**CD34****+****Thyl****+**

**CD45RA****+ ****CD62L****+**
**CD45RA****+**** CD45RO****-**
**CD45RO****-**** CD27****+**** CD95****Low**

**TREC****Telomere**
**CD103**

**CD95**
**Annexin V**
**Tunel**
**PI**

**CD45RO+ RA-**

**DTH**

**Naive****T cell**

**Memory****T cell**

**Cell death**

**Thymic****maturation**

**Progenitor cells**

## Host Components of the Immune Response

**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)**

## Basic Elements of Immune Response

**CD4**
**Helper**

**CD8**
**CTL**

**MØ**

**or**

**Dendritic**

**IL-12**

**Basic Elements of Immune Response**

## Activation	Maturation	Functional
HLA DR	CD45RO+RA-	CD95
CD38	CD45RA+62L+	CD28
CD25
CD71
CD69

- HLA DR	CD45RO+RA-	CD95
- CD38	CD45RA+62L+	CD28
- CD25
- CD71
- CD69

- CD4		CD8
- Subsets

## IL-2

**IL-2**

- 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

**T cell**

**B cell**

**macrophage**

**NK cell**

**INF******

**Cross regulation**

**IL-4, IL-10**

**Type 1 help**

**Type 2 help**

**Cellular**
**immunity**

**Humoral**
**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**

## 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

- 3

- 2m

- 1

- 2

- 3

- 2m

- 1

- 2

- 3

- 2m

- 1

- 2

- 3

- 2m

**Avidin**

**Enzymatic Biotinylation**
**Oriented T cell epitope**
**Single Peptide Ligand**
**Specificity Altered****at Will**

**Provided by****John Altman**

## Flow Cytometry:Gating on Subpopulations of Cells

**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)**