A study of chemokine receptors and the immunological synapse

Chemokine receptors play a central role in directing leukocytes to various tissues around the body by recognising chemotactic cues called chemokines. Upon entry into a tissue, infiltrating leukocytes from the blood stream encounter tissue resident cells, and in the case of T lymphocytes scan antigen presenting cells (APCs). Following antigen recognition, signalling components can be recruited into micro-scale domains at the interface of the APC-T cell conjugate, forming a structure known as the immunological synapse (IS). The chemokine receptors CCR5 and CXCR4 have been implicated in promoting IS formation through their recruitment to the T cell side of human APC-T cell interfaces. However, this is based primarily on studies with immortalized cell lines transfected with GFP-tagged chemokine receptors and does not take into account what happens on the APC side of the IS. Hence, the behaviour of endogenous CXCR4 and CCR5 during the formation of IS between primary T cells and APCs needs to be clarified.

Live cell microscopy and flow cytometry were employed to examine a previously uncharacterised macrophage-CD4+ T cell IS, formed in vitro between primary human blood isolated T cells and monocyte-derived macrophages (MDMs) pulsed with a superantigen (sAg). Under these conditions, MDM-CD4+ T cell conjugates displayed elevated T cell intracellular calcium responses. A fraction of these conjugates showed T cell receptor capping as well as ICAM1 and relocation of the Golgi apparatus towards the cell–cell interface, which suggests that the interactions between MDMs and CD4+ T cells lead to the formation of synapses and possibly kinapses.

Endogenous T cell surface CCR5 and CXCR4 molecules were found to cap at the MDM-CD4+ T cell IS, albeit infrequently. Yet, intracellular pools of T cell CCR5 receptors were consistently reported facing the IS interface and showed partial overlap with a marker of the trans-Golgi network, implying that intracellular CCR5 may translocate to the IS as a cargo in the Golgi. In contrast, on MDMs, cell surface CCR5 did not accumulate at the IS interface. FRAP analysis revealed that CCR5 molecules are relatively mobile at the surface of MDMs, and that CCR5 movement is affected by depletion but not sequestration of membrane cholesterol. These findings suggested that the non-accumulation of MDM CCR5 at the IS interface was not due to the restriction of lateral movement. Importantly, this work highlights cell-type differences in the behaviour of endogenous CCR5 receptors.

This thesis brings new insights into the roles of endogenous chemokine receptors in human leukocytes during antigen-dependent interactions and informs future research aiming to bridge the gap between mouse and human systems to study immune synapses.