IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 11. Cell Interactions in the Humoral Immune Response

INTRACELLULAR SIGNALS DURING LYMPHOCYTE ACTIVATION

The TCR complex is capable of transmitting signals into The Cell interior. This signaling involves the CD3 complex (γ-, δ-, ε-, ζ-, and η-chains) and the p56^lck enzyme, which is associated with the intracellular domains of CD4 or CD8 molecules (p56^lck is an abbreviation for lymphocyte-specific Tyrosine kinase with a molecular mass of 56 kDa). B Cells also express a family of signaling molecules that includes Igα, Igβ, and membrane-bound IgM and IgD (see Ch. 6).

Lymphocyte activation is initiated by tyrosine Kinases

Recognition of the antigen–MHC molecular complex by the T-cell receptor and the binding of costimulatory molecules trigger signal Transduction into T cells. The earliest Intracellular Signaling event consists of tyrosine phosphorylation mediated by src-family tyrosine kinases, specifically lck (associated with CD4) and fyn; both phosphorylate target sequences found in the CD3 ζ-chain as well as in Igα, Igβ, and FcγR molecules. These sequences are termed immunoreceptor tyrosine-based activation motifs, or ITAMs. The ZAP-70 tyrosine kinase binds to ITAMs and becomes activated, in turn activating phospholipase C and thereby initiating the classical signaling pathway. Signals from the TCR and CD28 are integrated (Fig. 11.12), activating latent cytoplasmic METABOLISM/31.html">Transcription factors such as NF-AT or NF-κB, which subsequently migrate to The Nucleus. Here they act on genes required for T-cell activation, including the IL-2 and IL-2 receptor genes. The resulting IL-2 production binds to its receptor and drives Cell Division.

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Fig. 11.12. T-cell activation involves signal transduction from both the T-cell receptor and the CD28 molecule. The CD4 coreceptor, associated with the TCR complex, interacts with the lck kinase. Such kinases are activated through dephosphorylation, potentially mediated by the phosphatase domains of the common leukocyte antigen CD45. Activated lck kinase phosphorylates the ITAM domains of the CD3 ζ-chains, enabling them to associate with other kinases, including fyn and ZAP-70. The fyn kinase activates phospholipase C (PLC), which indirectly induces the release of intracellular calcium. Calcium binds to calcineurin and activates transcription factors. Concurrently, the ZAP-70, fyn, and PI-3 kinases (PI-3K, associated with CD28) Relay integrated signals via cytoplasmic kinase cascades; these signals also activate transcription factors. Upon entering the nucleus, these factors turn on genes including immediate-early cell division genes and the IL-2 Gene.

B- and T-cell activation processes proceed in a similar manner

The function of the T-cell CD3 molecule in B cells is performed by Igα and Igβ molecules, which also contain ITAMs in their cytoplasmic domains. Cross-linking of surface Igs leads to the activation of src-family kinases, which in B cells are represented not only by fyn, but also by lyn and blk. The Syk kinase, analogous to the T-cell ZAP-70 kinase, binds to phosphorylated Igα and Igβ. This results in the Activation of a kinase cascade and the nuclear translocation of transcription factors (similar to the process in T cells) (Fig. 11.13).

Fig. 11.13. B- and T-cell activation occurs via comparable pathways. Cross-linking of membrane-bound Igs (mIg), for example by a T-independent antigen (Ag), activates tyrosine kinases including lck, lyn, fyn, and blk. They phosphorylate the ITAM domains in the Igα and Igβ chains of the receptor complex, endowing them with The ability to bind another kinase, Syk, which in turn activates phospholipase C. The latter acts on membrane PIP2, generating IP3 and diacylglycerol (DAG), which activates protein kinase C. Signals transmitted by other kinases activate nuclear transcription factors. (mIg, membrane Ig.)

B-cell activation is significantly enhanced by the "coreceptor complex," which consists of three Proteins: Complement receptor 2 (CR2, CD21), CD19, and the target molecule for antiproliferative Antibodies (TAPA-1; CD81) (Fig. 11.14). Follicular dendritic cells are known to retain antigen on their surface for extended periods in the form of immune complexes (iccosomes). Antigen within such a complex can bind both to surface Ig on B cells and (via its attached complement component C3d) to CR2. This binding leads to phosphorylation of the cytoplasmic domain of the CD19 molecule, which also associates with kinases. These kinases, positioned close to the B-cell receptor, likely facilitate B-cell activation, especially when antigen concentration is low.

Fig. 11.14. The B-cell coreceptor complex consists of CD21 (complement receptor type 2), CD19, and CD81, a molecule with four transmembrane segments. An antigen (Ag) with covalently attached C3b or C3d can cross-link membrane Ig (mIg) with CD21 of the coreceptor complex. This substantially lowers the antigen threshold required for cell activation. The CD19 molecule can interact with kinases including lyn, fyn, Vav, and PI-3 kinase (PI-3K; cf. T-cell CD28). Receptor cross-linking triggers phosphorylation of the Igα and Igβ chains of the antigen-receptor complex, as well as the mobilization and activation of Syk.

The activation signal is the cumulative output of the antigen receptor and costimulatory molecules

It remains unclear precisely what constitutes an effective antigenic signal. In the case of T cells, interaction between an antigen and a single TCR is insufficient for activation; the number of such interactions required for stimulation may depend on the presence of other stimulatory signals, the T-cell type, and their activation state. It has been established that murine T-cell hybridomas (generated by fusing normal T lymphocytes with T-cell tumor lines) are easily activated. To stimulate a hybridoma, a fully functional APC (such as a macrophage) must display at least 60 complexes formed by an MHC class II molecule and an antigenic peptide. Functionally less active APCs, such as fibroblasts transfected with an MHC class II gene, require 5,000 such complexes.

Recent studies have shown that activation requires the interaction of approximately 8,000 TCR molecules with MHC–peptide complexes; precisely this number of TCRs disappears from the cell surface upon activation of T-cell clones. Because TCR–MHC interactions are characterized by low affinity, a single MHC–peptide complex is likely capable of activating A large number of TCRs. It is also possible that transformed T cells, such as hybridomas, require weaker TCR activation. In the presence of a costimulatory signal (e.g., CD28/B7 engagement), 1,500 activated and internalized TCRs are sufficient for T-cell clone activation.

Corresponding data on the action of T-dependent Antigens on B cells are lacking. Binding of a T-independent antigen to a single receptor does not lead to B-cell activation, though data comparing the effects of B-lymphocyte stimulation with the T-cell processes described above are not yet available.

The interaction of an antigen with a TCR or membrane immunoglobulin cannot serve as a positive activation signal for T or B cells on its own. While it might suffice to generate a "negative" or tolerogenic signal, even this is questionable. It is currently believed that lymphoid cell activation requires a series of interactions, each potentially possessing signaling capacity. Costimulatory molecules, such as CD2 and CD11a/CD18, are responsible for more than just adhesion; their cytoplasmic domains participate in signal transduction. For instance, experiments involving the deletion of the CD2 cytoplasmic domain have shown that such an intervention disrupts activation while leaving the adhesive function of the molecule intact.

Mitogens and superantigens can also activate lymphocytes

To study T- and B-cell activation alongside antigens, researchers also use mitogens—substances that, unlike antigens, can activate T AND B cells nonspecifically. For the majority of T cells, stimulating agents include phytohemagglutinin (PHA), extracted from red Kidney beans, and concanavalin A (ConA), extracted from jack bean seeds. The MECHANISM OF ACTION of these agents has been studied in detail; they bind to T-cell surface molecules responsible for activation, notably the TCR and CD2.

Another group of molecules, known as "superantigens"—the majority of which are of bacterial origin—also possess the ability to activate T cells nonspecifically. These include staphylococcal enterotoxins (which cause certain types of acute food poisoning), the toxin responsible for toxic Shock syndrome in Sepsis, exfoliative toxin, and several viral proteins. Superantigens bind to MHC class II molecules on APCs and are recognized by the TCR, yet not through the same mechanism operating during recognition of the MHC–antigenic peptide complex by the T-cell receptor. A superantigen binds solely to the Vβ chain of the TCR, but this is sufficient for T-cell activation (Fig. 11.15). The stimulatory effect is identical to that seen with conventional antigens: depending on experimental conditions, either an Immune Response or clonal anergy may ensue.

Fig. 11.15. To activate the TCR, antigenic Peptides typically must be processed. However, superantigens such as staphylococcal enterotoxins bypass Processing and bind directly to the MHC class II molecule and the Vβ chain of the TCR. Each superantigen activates a distinct population of Vβ-expressing T cells, depending on which Vβ gene segment encodes the receptor.