It is now well established that the ligand for CD2 is the structurally related molecule CD58, or LFA-3, both of which are members of the immunoglobulin superfamily of molecules, and possess two immunoglobulin domains (Shaw et al. 1986; Selvaraj et el. 1987; Peterson and Seed, 1987; Wallner et ai. 1987). The binding between the two molecules takes place via the NH2 terminal
domain of each protein (Somoza et ai. 1993; Osborn et al. 1995). It has also been reported that an unrelated molecule, CD59, is an additional ligand for CD2, but direct interaction between the two molecules has not been demonstrated (Deckert et al. 1992; Hahn et al. 1992a). LFA-3 is a 40-70 kD glycoprotein which is expressed on a wide range of haematopoetic and non- haematopoetic cells, including epithelium, endothelium and stromal cells. It is also present on monocytes and T cells, and is present on these cells at increased levels following activation. The molecule exists in two membrane anchorage isoforms, a transmembrane form, and a glycophosphatidylinositol (GPI) linked form, which are generated by alternative splicing (Dustin et al.
1987b). The GPI linked form is more laterally mobile in the membrane, and this is thought to be important in the formation of contact zones between T cells and target cells, at which LFA-3 molecules accumulate (Hollander, 1992). The gene encoding LFA-3 has been cloned, but the promoter region has not been defined and the mechanisms of regulation of LFA-3 expression are not known (Seed, 1987).
ICAM-1 B7-1
Target cell
Class I HLA >_>____ LFA-3 LFA-1 CD28I cell
CD2 CD3/TCR CD8Figure 1.3. Interactions between a CD8+ve T cell and a virally infected target cell.
The basic structures and ligand interactions of the molecules mediating adhesion and recognition of virally infected cells by CD8+ve T cells are shown.
Class I HLA interacts with the I cell receptor (TCR) while the interaction is stabilised by the CD8 molecule binding to another part of the class I HLA
molecule. LFA-3 and CD2 interact via their membrane distal immunoglobulin domains. ICAM-1 interacts with the a subunit of LFA-1 via its 5th immunoglobulin domain. The molecules are drawn approximately to scale. (Adapted from Davis and van der Merwe, 1996).
CD2
CD2 is a member of the immunoglobulin superfamily which is expressed on all T lymphocytes and NK cells. The molecule is associated with signalling function, due to the positive effect of anti-CD2 antibodies on CD3 mediated T cell activation, and the activation of T cells by certain combinations of anti-CD2 antibodies. One of these antibodies must recognise the CD2R epitope which is poorly expressed on resting T cells but is induced following binding of a second CD2 antibody specific for another epitope (Hunig et al. 1987). On T cells CD2 forms a loose association with the TCR complex, and the presence of CD3 is required for activation to take place, unless CD2 is artificially over-expressed. The regulation of the interaction between CD2 and LFA-3 is regulated by the density of expression of each molecule, and by increased avidity of CD2 following engagement of CD3 (Hahn etal. 1992b; Hahn and Bierer, 1993). The CD2/LFA-3 interaction
The CD2/LFA-3 interaction enhances class I and class II MHC-restricted T cell antigen recognition (Sanchez-Madrid et al. 1982; Krensky et al. 1984), and is also important in non-MHC restricted killing by NK cells and lymphokine activated killer (LAK) cells (Siliciano et al. 1985; Bolhuis et al. 1986; Zarcone et al. 1992). Two important features of the interaction between LFA-3 and CD2 have been identified which may be responsible for enhancing CTL killing; initiation and stabilisation of the interaction between the infected cell and the lymphocyte by adhesion, and the delivery of costimulatory signals to the T cell via the CD2 molecule (Tiefenthaler et al. 1987; Dustin et al. 1987a; Hahn et al.
1992b). It is thought that the adhesion function is of principal importance in enhancing the responsiveness of the cell to antigen. It has been shown that a non-signalling mutant of the CD2 molecule enhances antigen responsiveness of T cells, but only if expressed on the same cell as the TCR/CD3 complex (Moingeon et al. 1989). The requirement for both molecules to be expressed on the same cell was confirmed by others, showing augmentation of the proliferative response to HLA-DR transfectants by co-transfection of LFA-3 molecules (Greenlaw et al. 1992). The molecular dimensions of the CD2 and LFA-3 molecules are similar to those predicted for the TCR and MHC molecules (Bjorkman et al. 1987; Davis and Bjorkman, 1988; Jones et al.
1992). It is thus proposed that the CD2/LFA-3 interaction positions the membranes at the appropriate distance to favour MHC interactions with the TCR-CD3 complex (van der Merwe et al. 1995; Dustin et al. 1996), providing a
mechanism by which the small proportions of MHC molecules expressing the appropriate peptide can be sampled by the TCRs.
In addition to reports of enhancement of T cell responsiveness by CD2 ligands, there have also been reports of inhibition of T cell function by the binding of certain anti-CD2 antibodies (Palacios and Martinez-Maza, 1982; Yssel et al.
1987). Inhibition of I cell function by recombinant LFA-3 fusion proteins has also been shown, but this may be due to the nature of the recombinant proteins used, and may not be relevant in vivo (Guckel at ai. 1991; Miller at a/. 1993). It has been shown that the CD2 molecule expressed on energised cells underwent a temporary conformational change, which prevented LFA-3 binding. The reversion of CD2 to its former state following IL-2 treatment facilitated the binding of LFA-3, and the re-acquisition of the ability to respond to alloantigen in the presence of LFA-3 (Boussiotis at ai. 1994; Bell and Imboden, 1995). CD2 may therefore have a further unique role in reversal of anergy which is not shared by other adhesion molecules.
CD2 knockout mice have been constructed, and develop apparently normal T cell dependent immune responses, suggesting that CD2 does not have any unique, essential functions which cannot be compensated for by other molecules (Killeen at at. 1992). However, it is unlikely that a gene would have survived in a functional form, and with such a high degree of sequence conservation between species if it was truly redundant (Marshall at al. 1994). It is therefore likely that the phenotypic change due to the loss of CD2 is so subtle that it is not detectable in the knockout mouse system. A role in the reversal of anergy as has been suggested is a possibility. It is also possible that a decrease in the size of the T cell repertoire, due to non-selection of lower affinity T cell clones during thymic selection, will become apparent in further studies, as has been shown for CDS deficient mice (Tarakhovsky atal. 1994). 1.2.9.2 ICAM-1 and LFA-1
ICAM-1
ICAM-1 derives from a protein precursor of around 55kD in its non glycosylated form, and is processed via an intracellular precursor of 73kD, to a final molecular weight of 90-114kD. The ICAM-1 sequence has 7 potential glycosylation sites, which accounts for this heterogeneity in molecular weight seen between different cell types (Simmons at al. 1988). The ICAM-1 gene was cloned by two separate groups in 1988, and was identified as a member of the immunoglobulin superfamily based on similarities with other members of this
family (Yang et al. 1986; Simmons at al. 1988). It possesses 5 immunoglobulin domains of which the domain most distal from the membrane interacts with the a subunit of LFA-1 (Staunton at al. 1990). The majority of cell surface ICAM-1 molecules are expressed in a dimeric form, which has a greatly enhanced affinity for LFA-1 compared to the monomeric molecule (Reilly at al. 1995; Schulz at al. 1995). ICAM-1 has a wide tissue distribution, and is found on both haemopoietic and non-haemopoietic cells. It is induced by IL-ip, TNFa and IFNy on various cell types (Dustin at al. 1986b; Rothlein at al. 1988; Simmons
at al. 1988), and by IL-4 on dermal fibroblasts (Piela-Smith at al. 1992). The
induction of surface expression of ICAM-1 requires da novo mRNA and protein synthesis (Dustin at al. 1986b). A single NF-kB site is essential for the ICAM-1 promoter to respond to inflammatory cytokines, lipopolysaccharide, or phorbol esters in human endothelial cells (Voraberger at al. 1991 ; Ledebur and Parks, 1995). ICAM-1 is important in many aspects of the immune response, as it is a ligand for LFA-1 (present on all leukocytes) and Mac-1 (present on neutrophils). LFA-1
LFA-1 (CD11a/CD18) is a member of the p2 family of integrins, and consists of an a and p chain which are non covalently linked (Hynes, 1987). The ligands for LFA-1 are ICAM-1, -2 and -3 (Marlin and Springer, 1987; Staunton at al.
1989; De Fougerolles at al. 1991; De Fougerolles and Springer, 1992). T cells typically express from 10"^ to 10® LFA-1 molecules, but do not spontaneously adhere to cells or surfaces bearing ICAM-1. This is because LFA-1 is present in an inactive form on resting leukocytes and becomes activated following activation through the CDS molecule, crosslinking of the TCR, or by phorbol ester stimulation. Integrin activation occurs rapidly (minutes) and is transient (30 minutes to 2 hours), providing a mechanism for adhesion and de-adhesion of leukocytes and other cells (Rothlein and Springer, 1986; Dustin and Springer, 1989). The interaction of the cytoplasmic domain of LFA-1 with cytoskeletal elements appears to be important in both the maintenance of a low avidity state, and the generation of a high avidity state (Peter and O'Toole, 1995). Clustering of integrins increases the adhesiveness of the cell to integrin ligands (Detmers atal. 1987). This appears to be modulated by calcium binding (van Kooyk at al. 1991 ). However, the individual LFA-1 molecules also exist in high and low affinity states, which are regulated by the availability of particular divalent cations (Dransfield at al. 1992; Lollo at al. 1993; van Kooyk at al.
1994). Magnesium is particularly important in the regulation of LFA-1 affinity at the single molecule level (Dransfield and Hogg, 1989; Dransfield atal. 1992).
Activation of cells with phorbol ester, or crosslinking of TCR/CD3 complexes, while producing equivalent increases in the binding of cells to immobilised ICAM-1, did not result in mAb 24 expression or soluble ICAM-1 binding. Adhesion by these processes was found to be increased compared to resting cells due to increased cell spreading and cytoskeletal changes, in contrast to the induction of a high avidity state of LFA-1 (Stewart et al. 1996). The cytoplasmic domain of the p subunit of LFA-1 is associated with the cytoskeleton, a factor which may be important in the maintenance of a high or low affinity state, as aggregates of adhesion molecules have higher affinity for their ligands (Pardi at al. 1992; Peter and O'Toole, 1995). However, as none of these activation stimuli are physiological stimuli, the relevance of these differences in vivo is not clear.
The ICAM-1/LFA-1 interaction.
The interaction between ICAM-1 and LFA-1 is important in many leukocyte functions, including T cell mediated cytotoxicity, T cell proliferation in the mixed lymphocyte response, and T cell dependent B cell activation (Davignon at al.
1981; Makgoba atal. 1988; Krensky atal. 1984). The expression of ICAM-1 on HLA-DR transfectants is critical for effective class II restricted and allospecific T cell activation (Altmann at al. 1989). It is also critical for NK cell lysis or MHC- unrestricted lysis of target cells (Zarcone at al. 1992; Chong at al. 1994). Adhesion and migration of NK cells is also reliant on the interaction of ICAM-1 with both LFA-1 and Mac-1 (Allavena at al. 1991; Somersalo at al. 1992). ICAM-1 expressed on a separate cell provides costimulatory function in the activation of naive CD4+ve T cells, demonstrating that the costimulatory signal is not required to be on the same cell, as is thought to be the case for LFA-3 costimulation (Dubey at al. 1995). The LFA-1 molecule has signalling function, and becomes phosphorylated upon T cell activation, but the main function is thought to be mediation of adhesion during both leukocyte migration and interactions with target cells (Haverstick and Gray, 1992; Pardi at al. 1992). Mutational analysis has shown that phosphorylation of LFA-1 is not required for binding to ICAM-1 (Hibbs atal. 1991).
Studies in patients lacking the P2 integrin chain have shown the importance of
LFA-1 in the immune response. The main feature of this condition, leukocyte adhesion deficiency is the lack of neutrophil infiltration into tissues, leading to recurrent life threatening bacterial and fungal infections. These patients do not suffer form severe viral infections, suggesting that an alternative pathway compensates for the lack of LFA-1 expression. However, in vitro studies have
shown that such patients have diminished allospecific CTL responses and NK cell activity, which correlated with the degree of severity of the lack of LFA-1 expression in different patients (Kohl etal. 1984; Krensky etal. 1985).