Cytotoxic T cell Helper T cell Tc TH T cell receptor Class I MHC protein T cell receptor Fragments of foreign TH Class II Helper T cell Cytotoxic T cell infected target cell Antigen presenting cell

Helper T cells: • Express CD4 molecule on the cell surface. • Recognize antigen peptides bound to class II MHC molecules (exogenous or ingested antigens). • Provide help to B cells, macrophages and other T cell effector responses though the production of cytokines Cytotoxic T cells: • Express CD8 molecule on the cell surface. • Recognize antigen peptides bound to class I MHC molecules (endogenously produced antigen such as viral antigen). • Kill infected target cells.

IL-2 Gene NFAT Z Construct lac Z TK promoter Hygromycin Resistance gene NF-AT IL-2 TATA -70 +1 +47 NFAT Z Construct NFIL-2D NFIL-2C NFIL-2B NFIL-2A IL-2 Gene -286 -257 -256 -242 -208 -188 -158 -145 -93 -66

Detection of ß-Galactosidase: Upon addition of its chromogenic substrate, chlorophenol red galactoside (CPRG), ß-galactosidase hydrolyzes CPRG, turning it from a yellow color to purple at alkaline pH. The color changs thus provide a convenient measure of gene transcription activities during T cell activation.

IL-2 Receptor Ca IP3 Ca2+ NF-ATc IL-2 DAG PKC NF-ATn Ras, Rac, PKC T cell receptor activation IP3 Ca2+ NF-ATc Phosphoinositol pathway IL-2 DAG PKC NF-ATn Ras, Rac, PKC NF-ATn NF-ATc PO3 Ca 2+ Calcineurin NF-AT kinase CsA/Cph CRAC IL-2 Receptor IP3R

Con A EGTA Ionomycin thapsigargin Ca CsA/Cph PMA IL-2 Receptor Ca 2+ CRAC Ionomycin Ca 2+ thapsigargin IP3R Ras, Rac, PKC CsA/Cph PMA NF-ATc PO3 Calcineurin NF-AT kinase NF-ATn NF-ATc IL-2

This week’s experiments 1. Mammalian cell culture: Goal: learn how to count cells using a hemacytometer Learn to grow mammalian cell lines 2. Signal transduction lab: Examine the effects of various inhibitors and activators on IL-2 expression.

How to count cells using a hemacytometer? Cell unit per box: 1 x 104 cells/ml Blue cells : 3 x 104/ml