Flow cytometry (FC) has become an important component in the diagnosis and monitoring of patients with a diverse array of diseases. While the basic principals underlying FC remain relatively unchanged, the technology and array of available reagents have continued to evolve, thus expanding the list of applications in laboratory medicine. This review provides an overview of the basic theory of FC including instrumentation, sample processing, and analysis. Additionally, current and future clinical applications of FC are discussed. Among these, applications of FC in solid organ and hematopoeitic stem-cell transplantation, transfusion medicine, hematopathology, and infectious diseases are presented. Finally, applications employed primarily in clinical research are presented in anticipation of their eventual use in the clinical laboratory setting.

The pathogenesis of rheumatoid arthritis (RA) is determined by a complex interaction of genetic and environmental factors. Of all risk factors, age has the largest impact. RA occurs most often during the postmenopausal period of life, with incidence rates peaking in the eighth decade. While age is generally accepted as an etiologic factor for failure of immunocompetence, much less is understood about the role of T-cell senescence in autoimmunity. We have hypothesized that senescent T cells are particularly prone to be activated in specialized microenvironments, such as the synovial membrane. CD4 T cells in the senescence program were identified by the loss of CD28. Gene expression profiling documented that CD28- T cells have acquired a spectrum of regulatory receptors that are usually seen only on NK cells. Such regulatory receptors include stimulatory and inhibitory members of the killer immunoglobulin-like receptor (KIR) family, the stimulatory c-type lectin receptor NKG2D, and CX3CR1, the receptor for the chemokine fractalkine. Synovial fibroblasts express the relevant ligands, thus providing stimulatory signals to tissue-infiltrating T cells. The signaling pathways of these regulatory receptors are complex and dependent on the individual T cells, some of which express important adapter molecules such as DAP10 and DAP12. Inhibitory KIRs on T cells are often only partially functional. Our data suggest that, by virtue of altered receptor profiles, conventional tolerance mechanisms can be evaded in the aging host. By acquiring a new set of regulatory receptors, senescent CD4 T cells become responsive to novel environmental cues and find ideal stimulatory conditions in the synovial microenvironment.

IntroductionImmune homeostasis is tightly regulated by negative regulatory signals providing a counterbalance to activating stimuli. 1,2 Negative regulatory receptors have been described on all hematopoietic cells; a prime example that illustrates their importance is natural killer (NK) cell function. Self tolerance of NK cells is ensured by a set of inhibitory cell surface receptors that bind to self-major histocompatibility (MHC) class I ligands. 3 In humans, these receptors include the family of killer immunoglobulin-like receptors (KIRs).Inhibitory KIRs have long cytoplasmic tails with 2 immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Upon ligation of an inhibitory KIR, the ITIMs are phosphorylated and bind to the src homology 2 (SH2) domains of the phosphatase SHP-1. As a result of this binding, the catalytic site of SHP-1 is released from autoinhibition. 4 In NK cells, cognate binding of inhibitory KIRs to their human leukocyte antigen (HLA) ligands is enough to facilitate receptor clustering, ITIM phosphorylation, and SHP-1 activation. 5 The direct target of SHP-1 appears to be the guanine nucleotide exchange factor Vav1. 6 Dephosphorylation of Vav1 leads to inhibition of Rac1, 6 thereby preventing cytoskeletal rearrangement. One consequence is complete inhibition of NK cell activation. In fact, the formation of an activation platform between NK cells and target cells is completely prevented. 7 In addition to NK cells, KIRs are also expressed on T cells. 8,9 Naive and memory T cells are equipped to support their transcription; 10 however, expression is only found on senescent or enddifferentiated CD4 ϩ and CD8 ϩ T cells that have lost the expression of CD28. 11 The biologic function of inhibitory KIR expression on T cells is difficult to envision and likely different from NK cells. In NK cells, they are the basis for the missing self hypothesis 12 (ie, NK cells only respond to cells that have lost MHC class I expression). Otherwise, the inhibitory receptors keep NK cells completely unresponsive. Such a model would not be meaningful for T cells, the activation of which is dependent on MHC-restricted T-cell-receptor (TCR) triggering. Not surprisingly, functional studies of inhibitory receptors on T cells have come to conflicting results. 8,13,14 Inhibitory KIRs on selected tumor-specific CD8 ϩ T cells in patients with melanoma 15 and renal carcinoma were shown to inhibit tyrosine phosphorylation of early signaling proteins, lipid rafts, TCR/CD3 clustering, and the reorganization of the actin cytoskeleton; they also completely shut down T-cell activation. Consequently, the tumor-specific immune response was dampened, supporting a model of KIR expression on T cells as a cause of defective immunosurveillance. 8 Other studies of inhibitory receptors, such as immunoglobulin-like transcript 2 (ILT-2) on human CD8 T cells and GP49B1 on murine CD8 T cells, have only G.H. designed research, performed research, analyzed data, and wrote the paper; K.S. performed research and analyzed data; D.C. performed rese...

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