Lymphocyte Function Tests

Certain functions of isolated lymphocyte populations can be determined by a number of methods:

■ Determination of the number of cells producing antibodies, e.g., the hemolytic plaque assay in which antibody production is tested by adding antigen-coupled erythrocytes. In the vicinity of antibody-secreting cells, the erythro-cytes are covered with antibodies and can be lysed by addition of complement. Today, ELISA methods are more often used than erythrocytes (ELISPOT).

■ ELISPOT ASSAY: used to measure antibody-producing, or IL-releasing, lymphocytes. The antigen or anti-IL antibody is fixed on a plastic surface. Lymphocytes are then placed over this, within a thin layer of agar medium. When the cells are incubated at 37 °C, they may secrete the antibodies or IL recognized by the corresponding test substances. After a certain period of time, the cell layer is shaken off and the preparation is thoroughly washed. The bound material can then be developed using an overlaid semisolid agar, as for the ELISA method. The enzyme reaction generates spots of color, each of which corresponds to a cell, and which can be counted (Fig. 2.30).

■ Measurement of the release capacity of cytokines, or detection of mRNA, is also possible with the ELISPOT assay.

■ Lymphocyte stimulation assay: isolated lymphocytes are incubated with antigen in culture medium. Measuring the 3H-thymidine incorporation,

Fig. 2.31 This device analyzes cells by means of fluorescent-labeled antibodies directed against cell surface antigens - or for permeabilized cells, directed against internal cell antigens. In the example shown, peripheral blood lymphocytes (PBL) are incubated with monoclonal antibodies specific for CD4or CD8, resulting in the distribution of fluorescence intensity as indicated in a. In b the labeling of different cell populations with anti-CD4 or anti-CD8 is shown. By this means, the percentages of the subpopulations in the total population can be determined. The fluorescence-activated cell sorter shown in (c) makes use of this data. By means of vibration, the cell stream is broken up into fine droplets which, depending on the fluorescence and sorting settings used, are charged just before they are separated and ideally contain one cell each. Certain parameters are measured for each cell with the help of a laser beam, where-upon the droplets are deflected into the intended containers by the + and - plate fields. ►

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

interleukin release, or a pH transition, can determine whether antigen-specific lymphocytes are present or whether polyclonal T-cell responses (con-canavalin [ConA], phytohemagglutinin [PHA]) or B-cell responses (lipopoly-saccharide [LPS], pokeweed mitogen [PMA]) were induced.

■ Mixed lymphocyte reactions are used to measure alloreactivity (prolifera- 2 tion, cytotoxicity), mainly between recipients and donors of organ or bone

— Fluorescence-Activated Cell Sorter (FACS) -

Histogram

Histogram

Fluorescence intensity (log 10)

Fluorescence intensity (log 10)

Buffer solution

Application of a charge

Cells

Two-channel analysis of peripheral blood lymphocytes

Application of a charge

Polarized field for droplet deflection

Two-channel analysis of peripheral blood lymphocytes

Anti-CD8

Anti-CD8

Red fluorescence

Red fluorescence

Medical Microbiology

Green fluorescence

Green fluorescence

90° deflection r , (granulation)

scattered light (cell size)

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

Cell Function Tests

Fig. 2.32 The tetramer assay is an antigen-specific binding test for living T cells. Complexes comprising biotin-coupled MHC class I heavy chains, b2 microglobulin, and specific peptide are properly folded, washed, then bound toavidin (which contains four binding sites for biotin). The resulting tetrameric complexes are then incubated with a population of T cells. Those T cells expressing the appropriate T-cell receptor will bind to two or three of the exposed MHC class I-peptide complexes present on each tetramer. Labeling of the avidin component with fluorescein, phycoerythrin, or other fluorescent substances then permits FACS analysis of tertramer binding T cells.

Fig. 2.32 The tetramer assay is an antigen-specific binding test for living T cells. Complexes comprising biotin-coupled MHC class I heavy chains, b2 microglobulin, and specific peptide are properly folded, washed, then bound toavidin (which contains four binding sites for biotin). The resulting tetrameric complexes are then incubated with a population of T cells. Those T cells expressing the appropriate T-cell receptor will bind to two or three of the exposed MHC class I-peptide complexes present on each tetramer. Labeling of the avidin component with fluorescein, phycoerythrin, or other fluorescent substances then permits FACS analysis of tertramer binding T cells.

marrow transplants. This test is based on the principle that T lymphocytes are stimulated to proliferate by nonself MHC class I or II antigens and to develop into cytotoxic T cells directed against class I.

■ Chromium release assay measures cytolytic activity, mainly by CD8+T cells, directed against allogeneic, virus-infected, or peptide-loaded target cells. The target cells are incubated with 51Cr which the cells incorporate. They are then cultivated with effector cells for 4-6 hours. When the target cells are lysed chromium is released into the culture medium, following which it can be quantitatively measured.

■ Assay of intracellular cytokines. Following a brief stimulatory culture (six hours), the cells are rendered permeable using a mild detergent so that specifically labeled antibodies can diffuse into the cells. Labeled cells can then be analyzed by FACS equipment (or by a microscope).

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

Table 2.13 Important CD Antigens

Designation (alternatives)

Cells which express the antigen

Functions

CD5 CD8

CD10

CD11a (a chain) CD19

Cortical thymocytes, Langerhans cells, dendritic cells, B cells, intestinal epithelium

T cells, thymocytes, natural killer cells

Thymocytes, T cells

Several groups of thymocytes, T helper cells, and inflammatory T cells (about two-thirds of the peripheral T cells), monocytes,macrophages

Thymocytes, T cells, a subgroup of B cells

Several groups of thymocytes, cytotoxic T cells (about one-third of the peripheral T cells)

B and T precursor cells, bone marrow stroma cells

Lymphocytes, granulocytes, monocytes, and macrophages

B cells

MHC class I-like molecule, associated with b2 microglobulin. Possible special significance in specialized antigen presentation

Adhesion molecule which binds to CD58 (LFA-3) and can activate T cells (LFA = lymphocyte function antigen)

Associated with the antigen receptor of T cells, and is necessary for T-cell receptor surface expression and signal transduc-tion

Co-receptor for MHC class II molecules. Binds signal transducers via cytoplasmic portion. Receptor for gp120 in HIV-1 and HIV-2

Binds to CD72

Co-receptor for MHC class I molecules, binds signal transducer via cytoplasmic portion

Zinc metal proteinase, and marker for acute lymphoid leukemia of pre-B cells a subunit of b2 integrin LFA-1 (associated with CD18). Binds to CD54 (ICAM-1), CD102 (ICAM-2), and ICAM-3 (CD50)

Forms a complex with CD21 (CR2) and CD81 (TAPA-1), Co-receptor for B cells

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

Designation (alternatives)

Cells which express the antigen

Functions

CD21 (CR2) CD22

CD23 (FceRII)

CD25 (Tac)

CD26

CD28

CD29 CD34

CD35 (CR1)

CD38

CD39

Mature B cells, follicular dendritic cells

Mature B cells

Mature B cells, activated macrophages, eosinophils, follicular dendritic cells, blood platelets

Activated T cells, B cells, and monocytes

Activated B and T cells, macrophages

Subgroup of T cells, activated B cells

Leukocytes

Hematopoietic precursor cells, capillary endothelium

Erythrocytes, B cells, monocytes, neutrophils and eosinophils, follicular dendritic cells

Early B and T cells, activated T cells, germinal center B cells, plasma cells

Activated B cells, activated natural killer cells, macrophages, dendritic cells

Receptor for the complement components (CR) C3d and the Epstein-Barr virus. Forms a co-receptor for B cells together with CD19 and CD81

Adhesion of B cells to monocytes and T cells

Low-affinity receptor for IgE, and ligand for the CD19:CD21:CD81 coreceptor a chain of the IL-2 receptor, associated with CD122 and the IL-2Ry chain

A protease which may be involved in HIV entry into host cells

Activation of naive T cells. A receptor for costimulatory signal (signal 2); binds CD80 (B7.1) and B7.2

P1 subunit of y1 integrins, associated with CD49a in VLA-1 integrin

Ligand for CD62L (L-selectin)

Complement receptor 1; binds C3b and C4b. Also mediates phagocytosis

B-cell proliferation?

Unknown fuction, but may mediate adhesion of B cells

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

Immunological Test Methods 137 Table 2.13 Continued: Important CD Antigens

Designation (alternatives)

Cells which express the antigen

Functions

CD40 CD40L

CD45, RO, RA, RB (leukocyte common antigen, LCA), T200, B220

CD54

CD55 (DAF)

B cells, monocytes, dendritic cells

Activated CD4 T cells Leukocytes, erythrocytes

Leukocytes

Hematopoietic and nonhematopoietic cells

Hematopoietic and nonhematopoietic cells

CD62E (ELAM-1, Endothelium E-selectin)

CD64 (FcyRI)

CD88 CD89

CD95

Monocytes, macrophages Subgroup of B cells

Polymorphonuclear leukocytes, macrophages, mast cells

Monocytes, macrophages, granulocytes, neutrophils cells, subgroups of B and T cells

Many different cell lines; unclear distribution in vivo

Receptor for the costimulatory signal for B cells; binds CD40 ligand (CD40-L)

Ligand for CD40

Binds hyaluronic acid and mediates adhesion of leukocytes

A tyrosine phosphatase which enhances signal mediation via the antigen receptors of B and T cells; alternative splicing results in many isoforms (see below)

Intercellular adhesion molecule (ICAM-1); binds the CD11a/CD18 integrin (LFA-1) and the CD11b/ CD18 integrin (MAC-1); receptor for rhinoviruses

Decay accelerating factor (DAF); binds C3b and cleaves C3/C5 convertase

Endothelial leukocyte adhesion molecule (ELAM); binds sialyl-Lewis x and mediates rolling of neutrophilic cells along endothe-lium

High-affinity receptor for IgG

Costimulators which act as ligands for CD28 and CTLA-4

Receptor for the complement component C5a

IgA receptor?

Binds TNF-like Fas ligands; induces apoptosis

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

Designation (alternatives)

Cells which express the antigen

Functions

Designation (alternatives)

Cells which express the antigen

Functions

CD102 (ICAM-2) (intercellular cell adhesion molecule)

Resting lymphocytes, monocytes, endothelial cells (in which expression is most pronounced)

Binds CD11a/CD18 (LFA-1), but not CD11b/CD18 (MAC-1)

CD106 (VCAM-1) (vascular cell adhesion molecule)

Endothelial cells

Adhesion molecule; ligand for VLA-4 (very late antigen)

CD115

Monocytes, macrophages

Receptor for the macrophage colony-stimulating factor (M-CSF)

CD116

Monocytes, neutrophils, and eosinophils, endothelium

a chain of the receptor for the granulocyte-macrophage colony-stimulating factor (GM-CSF)

CD117

Hematopoietic precursor cells

Receptor for stem cell factor (SCF)

CD118

Widespread

Receptor for alpha/beta interferons (IFNa/b)

CD119

Macrophages, monocytes, B cells, endothelium

Receptor for gamma interferon (IFNy)

CD120a

Hematopoietic and nonhematopoietic cells

Most pronounced on epithelial cells

Acknowledgment

The expert editing and translating help of Nicola Harris PhD is gratefully acknowledged.

Kayser, Medical Microbiology © 2005 Thieme

All rights reserved. Usage subject to terms and conditions of license.

Was this article helpful?

0 0
Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

Get My Free Ebook


Responses

  • TEIJA
    WHAT IS TEST CAN BE MEASURED LYMPHOCYTE FUNCTION?
    8 years ago

Post a comment