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CHAPTER 17 Cytotoxic T Lymphocytes and Natural Killer Cells 251

activity is then measured by the release of label into the culture
The Long-Term Maintenance of Memory Cells supernatant or the loss of target cell viability directly. Although
The production of long-lasting antigen-independent memory such an approach provides a powerful quantitative assay for
cells is essential for a rapid response should reinfection occur. CTL activity, it has the disadvantage of requiring prestimulation
CTL memory provides a more vigorous response than the primary of the CTL population for 1–2 weeks to expand the numbers of
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challenge for both quantitative and qualitative reasons. Quan- antigen-specific CTLs to detectable levels. In mouse models, this
titatively, because of the substantial clonal expansion during a limitation was overcome by the development of animals that
primary infection, the precursor frequency of antigen-specific were monoclonal for the TCR. These mouse strains express
CTLs is vastly higher in immune individuals than in naïve subjects, transgenic TCRs that recognize either MHC class I (CD8)– or
thus allowing for a stronger response. Qualitatively, memory MHC class II (CD4)–specific epitopes. Such T cells, which are
CTLs exhibit striking efficiency in elaborating the effector func- specific for a single peptide, have proven extremely useful in the
tions associated with the rapid production of IFN-γ. This study of CTL responses, as antigen-specific cells can be easily
enhanced response is the result of reprogramming of gene detected.
expression profiles by epigenetic changes in DNA methylation TCR transgenic mouse models do, however, have some limita-
or chromatin structure. tions, as they do not recapitulate the diversity of the normal
The CTL memory compartment is composed of three cell immune response and represent an approach that cannot be
types: effector memory (T EM ) cells, central memory (T CM ) cells, used in human studies. The development of labeled tetramers
and tissue-resident memory (T RM ) cells. These subsets differ in of MHC class I–peptide complexes that bind to the endogenous
their surface molecule expression and in their ability to exhibit TCR has made a major contribution to overcoming these previous
effector functions (see Table 17.1). Like their naïve counterparts, limitations, as it allows for the detection of antigen-specific CTLs
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T CM cells express high levels of CD62L and CCR7 and reside within a polyclonal population. This technique is simple and
primarily in secondary lymphoid organs. T CM cells are capable broadly applicable. It can accurately detect rare antigen-specific
of prolonged homeostatic self-renewal in the absence of antigen. populations from patient material or animal tissue (Fig. 17.3).
T EM cells, in contrast, are characterized by low expression of The ability to prospectively identify antigen-specific CTLs has
CD62L and CCR7 and are distributed throughout the body, been combined with single-cell functional assays, such as ELISpot
including peripheral tissues, such as the lung and the gut, where or intracellular cytokine assays. The finding that the lysosomal
they can immediately confront invading pathogens. T RM cells compartment proteins lysosomal-associated membrane protein
are the most recently identified memory population that resides 1 (LAMP-1/2; CD107a/b) can be detected on the cell surface of
in peripheral tissues long after an infection is cleared, providing degranulating CTLs provides another powerful flow cytometry–
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potent early response to reinfection of the same tissue. CD4 based single-cell assay to investigate CTL lytic function.
T-cell help and cytokines, including IL-15 and IL-7 and their
receptors, have been identified as crucial for the survival and NATURAL KILLER CELLS
maintenance of the memory T-cell pool. 5,8
Properties of NK Cells
CD4 T-CELL HELP NK cells are a lymphoid lineage, which, because of the absence
of antigen–receptor rearrangements, belongs to the innate immune
The final player in the initial activation of CTL is the “help” response. The development of B and T lymphocytes has been
provided by CD4 T cells specific for an antigen linked to the extensively studied, and anatomical sites and major mediators
CTL epitope (Chapter 16). The processes by which help is are well characterized, whereas NK cells have proven more elusive.
provided are poorly understood. It is likely that cytokines, such Part of the problem lies in their relative scarcity, amounting to
as IL-2 and IL-21, are involved and that the CD4 T cells influence less than 2% of cells in bone marrow or spleen and to approxi-
both DCs and CTLs. 9,10 These cytokines, provided by the CD4 mately 5% of the lymphocytes in blood. The lack of appropriate
T cells, promote the survival, proliferation, and programming markers to distinguish NK cells, coupled with phenotypic het-
of memory CTLs. CD4 T-cell–deficient mice have been developed erogeneity between species and within mouse strains, has also
to study these CTL responses. Interestingly, “helpless” CD8 T complicated their analysis. In recent years, it has become apparent
cells resemble CTLs in chronic infections and cancer, in which that NK cells develop in bone marrow from a lymphoid progeni-
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the targets are not cleared despite a robust CTL response. PD-1 tor. IL-15 is essential for most aspects of NK-cell biology,
(programmed death 1), an inhibitory member of the TNFR including differentiation, survival, proliferation, and activation
family, is expressed on both helpless CTLs and on CTL cells in vivo. The IL-15R consists of three components: the IL-2/15β
during chronic infections. Blocking the interaction of PD-1 with chain (CD122), a unique IL-15Rα chain, and the common γ
its ligands greatly enhances the numbers and functions of impaired chain, which contains the intracellular signaling component of
15
CTLs. 11,12 the receptor. The importance of this receptor complex is
emphasized by the lack of NK cells in patients with X-linked
DETECTION AND ANALYSIS OF CTL FUNCTION severe combined immunodeficiency (SCID) and in dogs and
mice that have mutations in the common γ-chain or the IL2B
Much progress in our understanding of the generation of CTLs gene. The IL-15/IL-15R complex does not function in a manner
in an immune response has resulted from the development of similar to other cytokines that are produced as soluble ligands
several accurate and sensitive assays for CTL function. Traditional and bind to their receptor in a paracrine or autocrine manner.
CTL assays were performed on bulk populations of effector cells. IL-15 is virtually undetectable in body fluids or cell culture
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In these assays, target cells are labeled, often with Cr or a supernatants despite the ubiquitous distribution of mRNA. The
nonradioactive dye, and then pulsed with peptide-antigen. solution to this paradoxical observation is that IL-15 function
Peptide-specific CTLs are incubated with the target cells. Lytic requires the presence of IL-15Rα in the same cell. The IL-15/