First printed in R&D Systems' 1997 Catalog.
The terms stimulation and activation are often used synonymously when describing experimental effects. However, in the field of lymphocyte biology, the descriptions take on very different meanings. It was demonstrated in the early 1960s that normal T cells would develop in vitro into cytotoxic T cells when exposed to allogeneic (same species, mismatched antigen) leukocytes. The initial explanation for this phenomenon was that foreign (leukocyte) antigens interacted with receptors on select T cells, resulting in their activation. This suggested a "one-signal" T cell activation model. However, problems existed with this model. Cells stimulating normal T cells had to be viable. Mismatched allogeneic non-lymphoid cells (e.g., fibroblasts and platelets) also elicited no T cell response. Additionally, when xenogeneic (different species) mismatched lymphocytes were used, normal T cells showed little or no response.1
By 1970, it was proposed that two signals were required to activate T cells.2 In this model, a restricted phenotype stimulator cell (allogeneic leukocyte) would provide first, a foreign antigen to the T cell receptor, and second, an "inductive" or co-stimulator molecule to the T cell in general, which would initiate its activation.1 Subsequent studies have shown that stimulator cells (antigen presenting cells, APCs) do, indeed, initiate T cell activation by a two-signal mechanism, and that T cell receptor stimulation in the absence of a co-stimulus fails to induce T cell activation. The molecules on the APC that initiate the co-stimulus are now known to be members of the B7 family of ligands, and the co-stimulus receptor on T cells has been identified as CD28.3 Recent reviews covering the B7 and CD28 ligand/receptor families can be found in references 3-8.
|Figure 1. Hypothetical B7/CD40 Pathway for B Cell Activation.|
The ligand(s) for the CD28 receptor has been identified as the B7 family of antigens.9 The "B" designation for the ligand is a reflection of early studies with monoclonal antibodies that were used to identify previously unknown B cell antigens.10, 11 Following the cloning of B7, at least one and possibly two additional molecules were found to be related to B7. At this time, the original B7 is now known as B7-1 (CD80). The second family member is designated B7-2 (CD86 or B70, for its identification as a 70 kDa, B cell membrane glycoprotein). The presumptive third family member is called B7-3 (or BB-1 for B lymphoblast antigen-1).9, 12-14 cDNAs for both B7-1 and B7-2 have been isolated from human sources.15, 16 The mature form of B7-1 is a type I (extracellular N-terminus) transmembrane glycoprotein of 262 amino acid (aa) residues that has a native molecular weight of 44 kDa. The cytoplasmic region consists of only 19 aa vs. 216 aa residues in the extracellular domain. The extracellular segment contains two immunoglobulin-like (Ig-like) domains, the most external of which is reminiscent of a variable Ig domain, while the membrane proximal Ig-like domain resembles an Ig constant region.15 The cDNA for B7-2 predicts a mature protein of 306 aa residues with a short cytoplasmic tail (approximately 60 aa residues) and an extensive extracellular region (approximately 220 aa residues).
Like B7-1, the 60-80 kDa B7-2 extracellular segment also contains Ig-like variable and constant regions, indicating that both B7-1 and B7-2 are members of the Ig-superfamily of molecules (IgSF).15, 16 Overall aa sequence identity between human B7-1 and B7-2 is only about 25% and this is reflected in a low, region-specific homology between the two Ig variable-like regions (24%) and the two Ig constant-like regions (30%).16, 17 Although a cDNA for the purported B7-3 antigen has not been isolated, its existence is suggested by studies on B7+ and B7- B cells.9 The antibody BB-1 that was initially assumed to be specific for B7-1 has now been proposed to actually recognize the undefined B7-3.9, 18 The genes for B7-1 and B7-2 have been localized to the same region in the human genome (3q13-q23). Even though there is low aa identity between the antigens, the suggestion has been made that the genes are the result of a duplication event.19
Cells known to express B7-1 include cultured (but not resting) peripheral blood dendritic and Langerhans cells,20, 21 B cells,9, 22, 23 macrophages,23 keratinocytes19 and T cells.24 Cells reported to express B7-2 include cultured peripheral blood dendritic and Langerhans cells,20, 21, 25 B cells,9, 22, 23 macrophages and Kupffer cells,25 activated monocytes and NK cell clones14 and vascular endothelial cells.26 Cells possibly demonstrating B7-3, based on immunohistochemistry, include keratinocytes18, 27 and nervous system microglia.28 Finally, there is marked species cross-reactivity for the components of the B7/CD28/CTLA-4 system.17, 29, 30
There are two distinct receptors for the B7 family of molecules. The first is a 44 kDa disulfide-linked homodimer termed CD28. Originally identified by monoclonal antibody binding during studies investigating T cell activation by phorbol ester,31 CD28 has subsequently been found to be a type I (extracellular N-terminus) transmembrane glycoprotein which, like its ligand, is also a member of the IgSF.3, 7, 32, 33 The cDNA for human CD28 shows a 202 aa residue mature protein with a 41 aa residue cytoplasmic segment and a 134 aa residue extracellular domain. Unlike the B7 family of antigens, CD28 has only one Ig-like region that is reminiscent of a variable-like Ig domain.32 Mouse CD28 has also been isolated and found to be approximately 70% identical to human CD28. Within the variable Ig-like segment, human and mouse are 67% identical at the amino acid level.33, 34 Multiple transcripts for human CD28 exist, apparently as a result of alternate polyadenylation signal usage and deletions resulting from internal splicing events.35 Although CD28 does not exhibit classical growth factor receptor intrinsic tyrosine kinase activity (autophosphorylation), it is involved with phosphatidylinositol-3-phosphate kinase (3-PI). Following B7 ligand binding, CD28 is phosphorylated on a 3-PI binding motif in its cytoplasmic domain and this motif is then subsequently recognized by the SH2 domain of cytoplasmic 3-PI.36, 37 Cells known to express CD28 include occasional CD3- immature thymocytes, all CD3+ thymocytes, 50% of peripheral blood CD8+ and 95% of CD4+ T cells,38 plasma cells39 and gd T cells.35, 40 While the details of B7 binding to CD28 are poorly understood, it has recently been demonstrated that a hexapeptide motif in the Ig-like variable region CD28 is essential for B7-1 binding.41 Binding studies for B7-1 binding to CD28 show a Kd=200 nM.7
A second receptor for the B7 family is the CTLA-4 antigen.42 Discovered during a search for molecules involved with T cell cytotoxicity, CTLA (for cytolytic T-lymphocyte associated molecule-4) was the fourth CTLA molecule identified.43, 44 Human CTLA-4 is a type I (extracellular N-terminus) transmembrane glycoprotein with a native molecular weight of 33-37 kDa.43, 45 CTLA-4 has a mature peptide length of 186 aa residues with a 125 aa residue extracellular region and a 37 aa residue cytoplasmic domain.34 Although the human and mouse CTLA-4 molecules show 67% aa sequence identity in the extracellular region, the cytoplasmic domains exhibit absolute (100%) identity.34 Like CD28, CTLA-4 is suggested to exist as a homodimer.45 Unlike CD28, CTLA-4 binds B7-1 with considerably higher affinity (Kd = 12 nM),42 indicating that their shared hexapeptide motif is not the only determinant of binding. Cells known to express CTLA-4 include activated CD4+ and CD8+ T cells,45, 46 thymocytes,43 and B cells.47 CD28 is known to be abundantly expressed on both activated and resting T cells and shows a low avidity for B7. In contrast, CTLA-4 appears in low numbers after T cell activation, but shows a much higher avidity for B7.46
It is now generally accepted that to activate a T cell, both an antigen-specific and antigen-nonspecific signal must be provided by an activating or antigen presenting cell (APC). The antigen-specific signal is mediated by 1) antigen bound to a class II (or I) MHC molecule on the APC, and 2) the T cell receptor (TCR)/CD4 (or CD8) complex on T cells.3, 48 While the effect of this ligation is to initiate IL-2 production and IL-2 receptor (IL-2 R) expression by the T cell, these results are self-limiting unless they are amplified by a co-stimulus.49-51 This co-stimulus is provided by B7-1 binding to CD28, which stabilizes IL-2 mRNA and increases IL-2 secretion, resulting in T cell proliferation and clonal expansion.52-54 For the T cell, this two-signal combination has led to three possible outcomes relative to the engagement of the TCR complex and CD28 antigen. If both the TCR and CD28 are ligated, the T cell proliferates and is primed to become one of a limited number of functional subtypes.3, 55-58 If only the TCR is ligated, the T cell enters either a state of anergy or undergoes apoptosis.3 If only the CD28 antigen is engaged, there is no effect.3 Thus, the reinforcing activity of CD28 seems essential to normal T cell activation.
As might be expected, the interaction between the TCR and CD28 is probably more complicated. It is proposed that upon TCR ligation, the signal generated induces not only IL-2 and IL-2 R, but also CD40 ligand (CD40L) expression on the T cell.50, 59 This expression results in T cell CD40L binding to APC CD40, inducing B7-1 and B7-2 expression on APCs.59, 60 This abundant expression of B7 on the APC then insures CD28 ligation on the T cell, initiating IL-2 production and proliferation. The significance of this is reflected in the suggestion that HIV envelope glycoprotein can interrupt this cascade at the CD4/TCR level. By binding to CD4, the virus apparently interrupts the normal inductive signal(s) for CD40L and IL-2, creating the well documented T cell hyporesponsiveness seen in HIV infection.50
The biological function of CTLA-4 is not entirely clear. It does not appear to be simply an alternative B7 receptor. Depending upon the experimental conditions, it may facilitate or inhibit B7 and CD28-initiated activities.61 It has been reported that CD28 complexes to 3-PI while CTLA-4 does not. Further, CD28 activation results in IL-2 secretion while CTLA-4 stimulation does not.62 Thus, it seems to serve a distinctly different function(s). It has been suggested that CTLA-4 is a negative regulator of B7 family stimulation, given that it is essentially expressed only after T cell activation and on the same T cells as CD28. Since CD28 initiates the clonal expansion of naive T cells, CTLA-4 may well inhibit excessive expansion of now activated CD28+ T cells, serving as a negative regulator or feedback inhibitor of the clonal expansion process.63, 64