The Role of Integrin LFA-1 in CD8+ T cell Activation and Cell Fate

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2016.Successful development of T cell effector function and long-lasting immune memory requires a prolonged interaction with antigen presenting cells upon cognate antigen recognition and precise regulation of migration and localization of dividing T cells in immune niches. The integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with APCs, as well as chemokine-mediated migration. Recent evidence has suggested that lineage commitment of effector and memory T cells occurs as early as the first T cell division, using an evolutionarily conserved mechanism known as asymmetric cell division. Although LFA-1 is a signature marker of T cell asymmetric division, the mechanisms by which LFA-1 regulates T cell differentiation during early T cell division in secondary lymphoid organs have not been fully characterized. We hypothesized that asymmetric LFA-1 expression mediates T cell migration and APC interaction during early T cell activation, regulating subsequent localization and cell differentiation in the secondary lymphoid organs. Using our novel fluorescent CD11a (CD11a-mYFP) knock-in mice on a T cell receptor transgenic background, we discovered that naïve T cells reserve a significant intracellular store of LFA-1 located in the rear of the cell during chemokinemediated T cell migration. Furthermore, polarized redistribution of intracellular LFA-1 occurs in an antigen-dependent manner and is a key step in the execution of asymmetric T cell division. Both in vitro and in vivo imaging revealed that asymmetric segregation of LFA-1 during the first cell division induces bimodal patterns of cell migration and interactions with antigen-presenting cells that lead to distinct localization patterns of the daughter T cells in the lymph node. During influenza infection, the bimodal behavior of the daughter T cells that results from asymmetric LFA-1 inheritance is critical for the differentiation of CD8+ memory T cells. In addition, we identified Rab27 as a key regulator of LFA-1 redistribution during T cell activation. Collectively, these results suggest that LFA-1 plays a crucial role in regulating the migration of asymmetrically divided CD8+ daughter T cells in the lymph node and their subsequent differentiation into memory T cells

Regulation of T Cell Motility <i>In Vitro</i> and <i>In Vivo</i> by LPA and LPA2

<div><p>Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1–6) in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, <i>lpa2−/−</i> CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration <i>in vitro</i> were unaffected by loss of <i>lpa2</i>. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.</p></div

Mouse CD4+ T cells differentially express LPA1–6 over the course of T cell activation and polarization.

<p>(<b>A</b>). Naïve mouse CD4+ T cells were collected and activated by plate-bound anti-CD3 and anti-CD28 antibodies for 24, 48, or 72 hours. At each time point, cells were harvested and the mRNA expression of LPA1,2,3,4,5,6 was determined by semi-quantitative real-time PCR, performed in triplicate. Expression levels were normalized to mouse GAPDH using the 2∧-deltaCt method. Relative Value Units (RVU) = 2∧-deltaCt×1000. Data are mean of three independent experiments. (<b>B</b>). Protein lysates were collected at 0, 24, and 72 hours post-activation and protein expression of LPA1, LPA2, and LPA3 was measured by Western blot. GAPDH was used as a lane loading control. Data are representative of one-two experiments. (<b>C</b>). Naïve mouse CD4+ T cells were collected and activated by plate-bound anti-CD3 and anti-CD28 antibodies and cultured under Th1 (IFN-γ, anti-IL-4), Th2 (IL-4, anti-IFN-γ), or Th17 (TGF-β, IL-6, anti-IL-4, anti-IFN-γ) polarizing conditions for 72 hours. Cells were harvested and the mRNA expression of LPA2 and LPA6 was determined by semi-quantitative real-time PCR, performed in triplicate. Expression levels were normalized to mouse GAPDH and compared to naïve CD4+ T cells using the 2∧-deltadeltaCt method. Data are mean +/− SEM of three independent experiments.</p

LPA induces the migration, but not chemotaxis of naïve CD4+ T cells.

<p>(<b>A.</b>) Naïve mouse CD4+ T cells were added to the upper chamber of a Transwell and LPA (1∶1 mixture of 18∶1 and 16∶0 LPA at 1 µM final concentration) was added to the bottom chamber or top chamber as indicated or (<b>B.</b>) top chamber at various concentrations. Cells were allowed to migrate for 2 hours at 37°C and the number of cells that migrated to the bottom chamber was quantified by hemocytometry. CCL21 (100 ng/ml) was added to the bottom chamber to induce chemotaxis, as a positive control. Data are mean +/− SEM of 3–5 experiments. *p<0.05; **p<0.01; ****p<0.0001.</p

Naïve CD4+ T cells from <i>lpa2</i>−/− mice still migrate in response to LPA.

<p>Naive mouse CD4+ T cells from wild-type C57BL/6 mice (open bars) or <i>lpa2</i>−/− mice (dark bars) were added to the upper chamber of a Transwell (5 µm pore size) in the presence or absence of LPA (1 µM; 18∶1 and 16∶0). Cells were allowed to migrate for 2 hours at 37°C and cell migration analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101655#pone-0101655-g002" target="_blank">Figure 2</a>. Data are expressed as Migration Index, or the number of cells that migrated in response to LPA relative to the number of cells that migrated in the presence of serum-free media only. Data are mean +/− SEM of four independent experiments. *p<0.05. n.s. = not significant.</p

LPA induces enhanced migration of naïve CD4+ T cells.

<p> Naïve mouse CD4+ T cells from wild-type C57BL/6 mice were added to microchambers coated with ICAM-1 and CCL21 in the absence of LPA or with 1 µM or 10 µM LPA. T cell migration was imaged every 15 s for 15 min and tracked by Volocity software. (<b>A</b>). Spider-plots of individual cell tracks over 15 min without LPA or with 1 µM LPA. (<b>B</b>). Mean track length, (<b>C</b>). Mean displacement, (<b>D</b>). Mean velocity with (1 µM and 10 µM) LPA or without LPA. Data are mean +/− SEM and representative of three independent experiments. **p<0.01, ****p<0.0001.</p

LPA2 does not play a crucial role in steady-state T cell homing to lymph nodes.

<p>Wild-type (CD90.2+, CD45.1+) and <i>lpa2</i>−/− (CD90.2+, CD45.1−) CD4+ T cells were adoptively transferred through tail vein injection into wild-type recipient mice. Forty-two hours post-transfer, the inguinal, brachial, cervical lymph nodes and spleen were harvested and the number of donor CD4+ T cells were enumerated by flow cytometry. Data are mean +/− SEM of two independent experiments, n = 5 recipient mice/experiment.</p