Non-classic properties of human cytolytic lymphocytes. basic and clinical aspects

The immune system comprises a complex network of specialized cells and organs that function together to recognize and remove potentially harmful "non-self", i.e. pathogens, malignant cells and –by extension– also transplanted allogeneic cells. Innate –evolutionary ‘old’ – immune mechanisms function in a non-clonal germlineencoded manner, whereas the hallmark of the adaptive immune system is clonal expansion of cells after rearrangement of germline-encoded immune receptors. Natural killer cells (NK cells) represent an important component of the innate immune system, whereas CD4+ (‘helper’) and CD8+ (‘cytotoxic’) T cells are key players of adaptive immunity. Rare NK cell-deficient humans suffer from overwhelming herpes viral infections, indicative of their important role in infection biology. Mechanistic insight into their antiviral effector function, however, remains largely based on murine data. Intriguingly, the best molecular understanding of NK cell function in humans is derived from studies of the ‘man-made’ setting of allo-stem cell transplantation. By contrast, in solid organ transplantation NK cells have, until the recent emergence of exciting murine data, been largely ignored. To begin to understand the role NK cells may play in human solid organ transplantation, we initiated a prospective cohort-study of kidney allo-transplant recipients. Blocking the IL-2 receptor α-chain (CD25) with mAb is an immunomodulatory treatment modality used in various clinical situations, including allo-transplantation. Of note, in patients with multiple sclerosis and uveitis administration of anti-CD25 mAb has been linked to robust expansion of CD56bright NK cells, which has been suggested to mediate its effect. On this background we first we enumerated NK cell-subsets in kidney transplant-recipients receiving anti-CD25 mAb (n=16) before transplantation, and at week 12, 26 and 52 post-transplantation, and in healthy controls (n=5). In healthy controls NK cell-subsets remained stable over time. In transplant-recipients, by contrast, overall median frequencies of both CD56bright and CD56dim NK cells transiently decreased after transplantation. Expansion of CD56bright NK cells was observed in only 6/16 patients. These data dissociate expansion of CD56bright NK cells and in vivo blockade of CD25 in kidney allograft recipients, thus dismissing expansion of CD56bright NK cells as unifying clinical hallmark of its effect across all clinical situations (G. Zenhaeusern et al. Submitted). Aiming next at studying functional allo-specific properties of NK cells, we developed a simple, paraformaldehyde fixation-based protocol that provided an accurate and robust means for assessing alloreactivity, avoiding an irradiation-induced network of various cytokines complicating interpretation of results (G. Zenhaeusern et al. JIM ‘07). Using this protocol, we found that a subset of NK cells was activated rather than silenced when interacting with cells expressing normal levels of autologous MHC I. Instead of inducing an inflammatory phenotype, however, activation led to the secretion of the regulatory cytokines TGF-β and IL-10. Together these data ascribe a novel pattern of reactivity to NK cells with potential implications both in autologous and allogeneic systems (G. Zenhaeusern et al. in revision).
The molecular understanding of the various cellular interactions involving NK cells is only emerging, whereas recognition by T cell receptor (TCR) of cognate antigen presented by HLA molecules has been analyzed in depth. On the other hand, relatively little attention has been given to basic T cell physiological properties, such as their random crawling activity (required to screen for antigen) or their mitochondrial energy efficiency. In order to begin to tackle these important issues, I established an experimental in vitro system allowing us to quantify random crawling activity of various T cell subsets, and monitor crawling under conditions mimicking the intra- and extravascular environment. Using these assays, we identified robust differences between phenotypically distinct subsets of CD8+ T cells with regards to their random movement activity and the frequency-distribution of crawling cells. Specifically, using migration-assays and time-lapse microscopy we found (i) that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrated more efficiently in trans-well assays and (ii) that these same cells were characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu –but not when examined on endothelial cells. With this finding at hand, we hypothesized that increased random movement activity ought to be linked to higher energy consumption. To test this hypothesis we measured mRNA expression of genes key to mitochondrial energy metabolism (PGC-1β, ERRα, Cytochrome C, ATP Synthase, and the uncoupling proteins UCP-2 and UCP-3), quantified cellular ATP contents and performed micro-calorimetric analyses. Much to our surprise, ATP contents were consistently higher in CCR7+ CD8+ T cells, the subset of T cells that showed less crawling activity, and genes involved in mitochondrial biosynthesis and ATP production (PGC-1β, ERRα, Cytochrome C, ATP Synthase) were significantly upregulated. Intriguingly, we also observed that these CCR7+ CD8+ T cells expressed uncoupling proteins UCP-2 and UCP-3 at significantly higher levels than CCR7- CD8+ T cells, which at least partly explained the higher heat flow measured in the lymph node homing T cell subset. (G. Zenhaeusern et al. Blood 2008, e-pub ahead print). Together these assays identified a phenotypically distinct ‘high crawlingfrequency’ CD8+ T cell population, and differentially regulated heat production among non-lymphoid vs. lymphoid homing CD8+ T cells.