In this report we present evidence that the ICAM ligands of LFA-1 induce distinct signaling activation events that have functional consequences for human CD4+ memory T cell differentiation in vitro. We undertook a comprehensive approach to dissect the differences amongst cells treated with the ICAM ligands within the context of T cell activation and differentiation. Using multiparameter surface phenotyping in conjunction with intracellular phospho-protein profiling, extracellular cytokine profiling, and chemotactic functional assays, we found that distinct CD4+ memory T cell subsets were generated when cells were stimulated with ICAM-3/CD3/CD28 and ICAM-2/CD3/CD28, and contrasted those generated by ICAM-1/CD3/CD28, or CD3/CD28 stimulation.
Intracellular activation of p38 and Erk1/2 was sensitive to the ICAM ligand used in combination with CD3/CD28 stimulation. Both p38 and Erk1/2 are known to integrate signals to regulate both mitogenesis and differentiation in various cellular systems and these two MAPKS are integral for T cell development, activation, and differentiation [32
]. Single cell quantitation of the intracellular phosphorylation of p38 and the ratiometric measurement of phosphorylated Erk1/2 revealed that the integration of CD3, CD28 and LFA-1 were dependent on the LFA-1 ligand used for stimulation. We surmise that sub-optimal levels of active Erk1/2 result in the first stages of T cell activation (initial cell division, activation marker expression, and cytokine production), but are not sufficient to dictate full conversion into highly differentiated T cells. This can be concluded since not all ICAM stimulations promoted the full conversion of CD45RA+
cells in T cell differentiation assays in vitro
. Intriguingly, when the ICAM ligands were tested in combination, ICAM-1 and ICAM-2 enabled cell progression to the CD45RA+
stage, whereas ICAM-1 stimulation alone did not allow cells to pass the CD45RA+
stage. Also, the combination of ICAM-1 and ICAM-3 enabled some cells in the population to progress to the full CD45RO+
stage, whereas neither ICAM-1 nor ICAM-3 alone allowed progression past the CD45RA+
stage (Fig. ). These observations suggest a complex regulation of signaling events by LFA-1 dependent on the combination of ligands present. Although ICAM-1 and ICAM-3 have been observed at the immunological synapse in separate studies [34
], the dynamic interaction of among the three ICAM ligands at the synapse has not been reported. Thus, thee results presented here suggest that the combinatorial integration of ICAM ligand interactions with LFA-1 have important and unexpected functional consequences for T cell biology.
Both ICAM-2/CD3/CD28 and ICAM-3/CD3/CD28 stimulations had higher Erk1/2 signaling thresholds and correlated with less activation of caspase-3 and higher levels of intracellular BCL2 levels than other treatments. These stimulation regimes also generated highly differentiated CD4+
memory T cells that were functionally responsive to chemotactic agents. In murine models, Erk1/2 has been implicated in regulation of both positive and negative selection of developing T cells [33
]. This developmental process is dependent on activation-induced cell death mechanisms that remove autoreactive T cells from the periphery. Improper elimination of autoreactive T cells leads to several forms of autoimmunity[39
]. Inhibition of programmed cell death is also a prominent feature of various forms of T and B cell lymphomas [41
]. The relationship between intracellular signaling thresholds and cell death mechanisms in human naïve CD4+ T cells are currently not understood, however, the methodologies employed in this study can be used to resolve the interconnectedness of these two processes.
Activation of CD4 T lymphocytes in the presence of specific cytokines causes differentiation into distinct effector TH
subsets with different immunoregulatory properties. TGFβ1 is an immunosuppressive cytokine that has been observed to suppress the proliferative response of CD4+
] and inhibit the production of IFNγ [44
]. In our experiments, it was noted that CD3/CD28 and CD3/CD28/ICAM-1 stimulation generated the highest levels of TGFβ1 and consequently, these two stimulation regiments also had the lowest frequency of IFNγ producing T cells (Fig. ) and suppressed production of CD4+
memory cells (Fig. ). Therefore, the enhancement in signaling imparted by LFA-1 upon binding ICAM-2 or ICAM-3 in human CD4+ T cells can attenuate T cell differentiation. Mechanistic understanding on how LFA-1 regulates phosphatase activities may shed some light as to how ligand binding correlates with differential intracellular activities and subsequent cellular outcomes.
A clear understanding of the distinctive tissue distributions of the ICAM ligands, and their roles in determining function of T cells, in the human system has not been completely resolved. ICAM-1 (CD54) has a wide tissue distribution on both hematopoietic and non-hematopoietic cells, can be up regulated upon cellular activation and is viewed as the prominent LFA-1 ligand at the immunological synapse in model systems of cell-to-cell contact and of leukocyte rolling [3
]. ICAM-1 is closely related to ICAM-3 (CD50), which is constitutively expressed at high levels on leukocytes and epidermal dendritic Langherans cells and can also be up regulated upon activation on endothelial cells. These two ICAMs contrast with ICAM-2 (CD102), which is broadly expressed on leukocytes and constitutively expressed at high levels on vascular endothelium and is not up regulated upon cellular activation [45
]. Interestingly soluble forms of the ICAMs exist in human blood and have been correlated with disease indications [46
]. The physiological significance of soluble ICAMs in the blood is unknown; however, given the results presented here, one can surmise that the presence of extracellular LFA-1 ligands might potentiate the response of T cell activation and subsequent differentiation.
It is interesting to note that ICAM-1 is the only LFA-1 ligand that has been studied extensively for its adhesive contribution in the immune synapse formation and has been previously implicated to promote increased TH
1 differentiation [43
]. From biophysical experiments, ICAM affinity interactions have been calculated using recombinant proteins to suggest ICAM-1 has the highest affinity for the LFA-1 receptor [49
]. However, more recent studies have suggested a dynamic conformational change of LFA-1 that has been attributed to the discrepancy of 2D and 3D off-rate measurements and requires adjustment of the ligand and receptor densities to accurately estimate an affinity constant [50
Because structurally, the ICAMs exhibit geometrical differences, with ICAM-1 being the only ICAM reported to require dimerization for activity [51
] our data suggest further experimentation is necessary to account for the biological differences observed in human memory T cell differentiation in vitro. At present only one published study using ICAM-1-/- splenic antigen presenting cells has demonstrated a delayed response in generating pathogenic CD4+ effector cells in a murine model of diabetes [55
], however a comparative study of the different ICAMs in disease models has not demonstrated. Future work will require follow-up in T cell stimulation by selective ICAM-deficient antigen presenting cells in both murine models and human systems.
When T cells interact with antigen presenting cells (APC), these cells display multiple ICAM ligands at their surface. It has been observed that one T cell can interact with several APCs [56
], thereby altering the potential density of locally present interacting LFA-1 ligands. Thus, it is plausible that LFA-1 on any given CD4+ T cell is presented with multiple opportunities to interact with one or more of its ligands, and that the density of ligand interaction governs the intracellular events regulated by LFA-1. This coupled with the dynamic range of in vivo
peptide-MHC interactions and the number of co-stimulatory molecular interactions warrants further studies into the combinatorial matrix of influential intracellular signaling thresholds that dictate T cell fates. The studies presented here show clearly that interaction simultaneously with different LFA-1 ligands gives rise to different outcomes during memory T cell generation in vitro
. Thus, cells are capable of interpreting the presence of distinct ICAM molecules presented simultaneously and collating a response that is distinct from their response to any individual ICAM. Whether this is due to action upon all receptors simultaneously or different ICAM ligands, when presented in combination, seek out distinct LFA-1 receptors, with at present unknown modifications or abilities, on the cell surface remains to be determined.
We previously demonstrated that LFA-1 lowers T cell activation thresholds[20
] and we recently showed that signaling through LFA-1 can activate human NK cells[57
]. The present work demonstrates that LFA-1 signaling mechanisms can potentiate TH
1 development and that the combinatorial integration of ligand dependent LFA-1 signaling regulates the development of memory T cell development. Our results also illustrate that quantifiable intracellular signaling thresholds, as imposed by LFA-1, can regulate T cell commitments. We expect that such thresholds, like Erk1/2 phosphorylation, serve as signaling checkpoints that regulate TH
2 development. These may be important in T cell-dependent autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, where aberrant cell activity leads to pathological outcomes. The identity of such checkpoints, and the regulatory kinases and adapter proteins that instruct these processes, may serve as novel areas for pharmaceutical intervention for controlling autoreactive T cells in autoimmune diseases and possibly in T-cell cancers