Jane Oliaro

Asymmetric T Lymphocyte Division in the Initiation of Adaptive Immune Responses

A hallmark of mammalian immunity is the heterogeneity of cell fate that exists among pathogen-experienced lymphocytes. We show that a dividing T lymphocyte initially responding to a microbe exhibits unequal partitioning of proteins that mediate signaling, cell fate specification, and asymmetric cell division. Asymmetric segregation of determinants appears to be coordinated by prolonged interaction between the T cell and its antigen-presenting cell before division. Additionally, the first two daughter T cells displayed phenotypic and functional indicators of being differentially fated toward effector and memory lineages. These results suggest a mechanism by which a single lymphocyte can apportion diverse cell fates necessary for adaptive immunity.

Asymmetric Proteasome Segregation as a Mechanism for Unequal Partitioning of the Transcription Factor T-bet during T Lymphocyte Division

Polarized segregation of proteins in T cells is thought to play a role in diverse cellular functions including signal transduction, migration, and directed secretion of cytokines. Persistence of this polarization can result in asymmetric segregation of fate-determining proteins during cell division, which may enable a T cell to generate diverse progeny. Here, we provide evidence that a lineage-determining transcription factor, T-bet, underwent asymmetric organization in activated T cells preparing to divide and that it was unequally partitioned into the two daughter cells. This unequal acquisition of T-bet appeared to result from its asymmetric destruction during mitosis by virtue of concomitant asymmetric segregation of the proteasome. These results suggest a mechanism by which a cell may unequally localize cellular activities during division, thereby imparting disparity in the abundance of cell fate regulators in the daughter cells.

DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice

As shown by analysis of mice and humans bearing DOCK8-inactivating mutations, DOCK8 plays a cell-autonomous role in survival of naive CD8 T cells, LFA-1 polarization toward the immune synapse, and CD8 T cell memory and recall responses following viral infection.

Asymmetric Cell Division of T Cells upon Antigen Presentation Uses Multiple Conserved Mechanisms

Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8+ T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.

Regulation of asymmetric cell division and polarity by Scribble is not required for humoral immunity.

The production of protective antibody requires effective signalling of naive B cells following encounter with antigen, and the divergence of responding B lymphocytes into distinct lineages. Polarity proteins have recently been proposed as important mediators of both the initial B cell response, and potentially of asymmetric cell division. Here we show that, although polarity proteins of the Scribble complex, Scribble, Dlg1 and Lgl1, are expressed and polarized during early B cell activation, their deficiency has no effect on the in vivo outcome of immunization or challenge with influenza infection. Furthermore, we find a striking correlation in the differentiation outcome of daughters of single founder B cells in vitro. Taken together, our results indicate that B cell differentiation does not require polarity proteins of the Scribble complex, and the findings do not support a role for asymmetric cell division in B cell activation and differentiation.

Ligation of the cell surface receptor, CD46, alters T cell polarity and response to antigen presentation

Lymphocyte function in vivo is dictated by multiple external cues, but the integration of different signals is not well understood. Here, we show that competition for the axis of polarization dictates functional outcomes. We investigated the effect of ligation of the immunoregulatory cell surface receptor, CD46, on lymphocyte polarity during antigen presentation and cytotoxic effector function. Ligation of CD46 on human T cells prevented recruitment of the microtubule organizing center, CD3, and perforin to the interface with the antigen-presenting cell and caused a reduction in IFN-γ production. In human NK cells, similar changes in polarity induced by CD46 ligation inhibited the recruitment of the microtubule organizing center and perforin to the interface with target cells and correlated with reduced killing. These data indicate that external signals can alter lymphocyte polarization toward antigen-presenting cells or target cells, inhibiting lymphocyte function.

A method for prolonged imaging of motile lymphocytes

With new imaging technologies and fluorescent probes, live imaging of cells in vitro has revolutionized many aspects of cell biology. A key goal now is to develop systems to optimize in vitro imaging, which do not compromise the physiological relevance of the study. We have developed a methodology that contains non‐adherent cells within the field of view. ‘Cell paddocks’ are created by generating an array of microgrids using polydimethylsiloxane. Each microgrid is up to 250 × 250 μm2 with a height of 60 μm. Overlayed cells settle into the grids and the walls restrict their lateral movement, but a contiguous supply of medium between neighboring microgrids facilitates the exchange of cytokines and growth factors. This allows culture over at least 6 days with no impact upon viability and proliferation. Adaptations of the microgrids have enabled imaging and tracking of lymphocyte division through multiple generations of long‐term interactions between T lymphocytes and dendritic cells, and of thymocyte–stromal cell interactions.

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.

Cutting edge: DNAX accessory molecule 1-deficient CD8+ T cells display immunological synapse defects that impair antitumor immunity

DNAX accessory molecule 1 (DNAM-1) is expressed on all CD8+ T cells and promotes their activation and effector function. DNAM-1 interacts with LFA-1, a critical molecule for immunological synapse formation between T cells and APCs, and for cytotoxic killing of target cells. Mice that lack DNAM-1 display abnormal T cell responses and antitumor activity; however, the mechanism involved is unclear. In this article, we show that DNAM-1 deficiency results in reduced proliferation of CD8+ T cells after Ag presentation and impaired cytotoxic activity. We also demonstrate that DNAM-1–deficient T cells show reduced conjugations with tumor cells and decreased recruitment of both LFA-1 and lipid rafts to the immunological synapse, which correlates with reduced tumor cell killing in vitro. This synapse defect may explain why DNAM-1–deficient mice cannot clear tumors in vivo, and highlights the importance of DNAM-1 and the immunological synapse in T cell–mediated antitumor immunity.

Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues

The movement of proteins within cells can provide dynamic indications of cell signaling and cell polarity, but methods are needed to track and quantify subcellular protein movement within tissue environments. Here we present a semiautomated approach to quantify subcellular protein location for hundreds of migrating cells within intact living tissue using retrovirally expressed fluorescent fusion proteins and time‐lapse two‐photon microscopy of intact thymic lobes. We have validated the method using GFP‐PKCζ, a marker for cell polarity, and LAT‐GFP, a marker for T‐cell receptor signaling, and have related the asymmetric distribution of these proteins to the direction and speed of cell migration. These approaches could be readily adapted to other fluorescent fusion proteins, tissues and biological questions.