The Role of Drak2 in T Cell Function and Autoimmunity

The immune system utilizes many regulatory mechanisms to limit immune responses and ensure that immune cells target foreign pathogens and not healthy cells of the body. However, some immune cells can escape these checkpoints and attack the body’s healthy cells, leading to tissue destruction and devastating autoimmune disorders. For example, multiple sclerosis (MS) occurs when immune cells attack the myelin sheath surrounding neurons of the central nervous system (CNS). Likewise, the destruction of pancreatic islet cells by dysregulated immune cells leads to type 1 diabetes (T1D). Remarkably, there are more than 80 types of autoimmune diseases. An estimated 50 million Americans suffer from autoimmune disease, and the prevalence continues to increase. These diseases are chronic and potentially life threatening, with associated healthcare costs estimated at $100 billion annually. Current therapies to limit autoimmune diseases often include immunosuppressant medications that also increase susceptibility to infections and tumors. Therefore, therapeutic treatments which specifically inhibit autoreactive immune cells, while sparing immune cells required for pathogen or tumor clearance would significantly improve treatment options. Drak2, a serine-threonine kinase, expressed abundantly in T and B cells, is a negative regulator of T cell activation. However, unlike other negative regulators, Drak2 plays an important role in eliciting autoimmunity, rather than preventing it. This is demonstrated by the finding that Drak2-/- mice are resistant to autoimmune disease in mouse models of T1D and MS. This resistance is due to reduced accumulation of Drak2-/- autoreactive T cells in the pancreas and CNS compared to wildtype mice. The decreased accumulation of autoreactive T cells in the target organs of Drak2-/- mice is partly due to diminished survival. Interestingly, despite Drak2-/- T cells being more sensitive to death, pathogen clearance and tumor surveillance are maintained in Drak2-/- mice. Therefore, inhibiting Drak2 is a potential alternative therapeutic approach to inhibit autoreactive T cells without suppressing the entire immune system. Thus, there is major interest in identifying the mechanisms by which Drak2 inhibits autoimmunity. This dissertation discusses the current knowledge of Drak2, its role in autoimmunity, and its potential as an inhibitory target to treat disease. We utilized several in vivo T cell adoptive transfer models to show that resistance to T1D was due to the absence of Drak2 in T cells rather than pancreatic β-cells, and that regulatory T cells (Tregs) were required to elicit resistance. Further analysis revealed that in the absence of Drak2, IL-2 signaling and Treg development increased and likely contributes to disease resistance. We also determined that Drak2 is not a negative regulator of TGF-β signaling in primary T cells, opposing a previous report. Thus it is unlikely that alterations in the TGF-β signaling pathway mediate autoimmune disease resistance in the absence of Drak2. Finally, to advance our understanding of how Drak2 contributes to T cell accumulation, and ultimately to T1D and MS, we established in vitro culture methods to recapitulate the survival defect observed in the absence of Drak2 in vivo. Interestingly, we discovered that Drak2 modifies the actin polymerization pathway either directly or indirectly, and that Drak2-/- T cells exhibited defects in cell cycle progression, proliferation, and other actin-mediated T cell functions that impair T cell accumulation. Together, these data highlight novel insights into the roles of Drak2 in T cell function and autoimmunity, and suggest that subtle changes within these diverse processes may cooperate to contribute to autoimmune disease resistance in the absence of Drak2

Drak2 Does Not Regulate TGF-β Signaling in T Cells.

Drak2 is a serine/threonine kinase expressed highest in T cells and B cells. Drak2-/- mice are resistant to autoimmunity in mouse models of type 1 diabetes and multiple sclerosis. Resistance to these diseases occurs, in part, because Drak2 is required for the survival of autoreactive T cells that induce disease. However, the molecular mechanisms by which Drak2 affects T cell survival and autoimmunity are not known. A recent report demonstrated that Drak2 negatively regulated transforming growth factor-β (TGF-β) signaling in tumor cell lines. Thus, increased TGF-β signaling in the absence of Drak2 may contribute to the resistance to autoimmunity in Drak2-/- mice. Therefore, we examined if Drak2 functioned as a negative regulator of TGF-β signaling in T cells, and whether the enhanced susceptibility to death of Drak2-/- T cells was due to augmented TGF-β signaling. Using several in vitro assays to test TGF-β signaling and T cell function, we found that activation of Smad2 and Smad3, which are downstream of the TGF-β receptor, was similar between wildtype and Drak2-/- T cells. Furthermore, TGF-β-mediated effects on naïve T cell proliferation, activated CD8+ T cell survival, and regulatory T cell induction was similar between wildtype and Drak2-/- T cells. Finally, the increased susceptibility to death in the absence of Drak2 was not due to enhanced TGF-β signaling. Together, these data suggest that Drak2 does not function as a negative regulator of TGF-β signaling in primary T cells stimulated in vitro. It is important to investigate and discern potential molecular mechanisms by which Drak2 functions in order to better understand the etiology of autoimmune diseases, as well as to validate the use of Drak2 as a target for therapeutic treatment of these diseases

TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and <i>Drak2-/-</i> T cells.

<p>A) CD4⁺CD25^-CD44^lo naïve cells were purified from <i>OT-II</i> and <i>OT-II</i>.<i>Drak2-/-</i> mice and stimulated with irradiated splenocytes loaded with 10μM OVA₃₂₃ peptide in the presence or absence of 10-fold TGF-β titrations for three days. The number of live, divided Foxp3^-CD4⁺ cells are shown for each titration. Cells were obtained from one <i>OT-II</i> or <i>OT-II</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of five separate experiments. B) CD8⁺CD25^-CD44^loCD62L^hi naïve cells were purified from <i>OT-I</i> and <i>OT-I</i>.<i>Drak2-/-</i> mice and stimulated with splenocytes loaded with 100pM OVA₂₅₇ peptide in the presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow cytometry. The number of live, divided CD8⁺ cells are shown for each titration. Cells were obtained from one <i>OT-I</i> or <i>OT-I</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of three separate experiments. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test.</p

Smad2 translocation is not enhanced in <i>Drak2-/-</i> T cells compared to wildtype T cells.

<p>Wildtype and <i>Drak2-/-</i> CD4⁺ cells were negatively selected with Miltenyi magnetic beads and stimulated on anti-CD3-coated coverglass slides along with soluble anti-CD28 for 24 hours. Half of the cells were treated with TGF-β for the final 20 minutes of culture. Cells were fixed, permeabilized, and stained with DAPI, Phalloidin, and anti-Smad2. Images were collected via confocal microscopy. n = 2 mice per group. Data are representative of two independent experiments.</p

Enhanced susceptibility to death of <i>Drak2-/-</i> T cells compared to wildtype T cells is independent of TGF-β signaling <i>in vitro</i>.

<p>A) CD4⁺CD25^-CD44^lo or B) CD8⁺CD25^-CD44^lo naïve cells were purified from wildtype, <i>Drak2-/-</i>, <i>DNRII</i>, and <i>DNRII</i>. <i>Drak2-/-</i> mice and stimulated with anti-CD3 and anti-CD28 for 2–3 days. The percent of nonviable CD4⁺ or CD8⁺ T cells is shown. Cells were obtained from one mouse per group and tested in quadruplicate. Data are representative of four separate experiments. <i>**P < 0</i>.<i>01</i>, <i>***P < 0</i>.<i>001</i> (Mann-Whitney <i>U</i>-test).</p

Smad2 and Smad2/3 complex phosphorylation is not enhanced in <i>Drak2-/-</i> T cells compared to wildtype T cells.

<p>A) Wildtype and <i>Drak2-/-</i> splenocytes, and FACS sorted naïve CD4⁺ and CD8⁺ T cells were stimulated for 2 hours with anti-CD3 and anti-CD28, with or without 2 ng/ml TGF-β for one additional hour. Cells were lysed and analyzed by western blot with antibodies specific for Smad2, phosphorylated Smad2, and HSP90 as a loading control. Cells were pooled from 9 wildtype and 8 <i>Drak2-/-</i> mice. Data are representative of two independent experiments. B) Wildtype and <i>Drak2-/-</i> splenocytes were stimulated for 2 hours with anti-CD3 and anti-CD28 with or without increasing concentrations of TGF-β for one additional hour. The cells were harvested, stained with antibodies specific for CD4, CD8, and pSmad2/3, and analyzed by flow cytometry. The average mean fluorescence intensity (MFI) of pSmad2/3 expression is shown for 3 mice per group. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test. Data are representative of 3 independent experiments.</p

TGF-β-mediated regulatory T cell induction is not altered in the absence of <i>Drak2</i>.

<p>A) CD4⁺CD25^-CD44^lo naïve cells were purified from wildtype and <i>Drak2-/-</i> mice and stimulated with 1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 days. The A) percent and B) number of Foxp3⁺ cells of electronically gated CD4⁺ cells is shown. There was no significant difference in the response of the wildtype and <i>Drak2-/-</i> cells according to the Mann-Whitney <i>U</i>-test.</p

TGF-β-mediated responses to opposing cytokines are comparable between wildtype and <i>Drak2-/-</i> T cells.

<p>CD8⁺CD25^-CD44^loCD62L^hi naïve cells were purified from <i>OT-I</i> and <i>OT-I</i>.<i>Drak2-/-</i> mice and stimulated with 100nM OVA₂₅₇–pulsed splenocytes for 2 days. Cells were harvested and replated at equal numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8⁺ cells and B) percent Annexin V⁺ of CD8⁺ cells are shown for each cytokine condition. Cells were obtained from one <i>OT-I</i> or <i>OT-I</i>.<i>Drak2-/-</i> mouse and tested in quadruplicate. Data are representative of two independent experiments. <i>*P < 0</i>.<i>05</i> (Mann-Whitney <i>U-</i>test).</p

Estimation of macular pigment optical density in the elderly: test-retest variability and effect of optical blur in pseudophakic subjects

The reproducibility of macular pigment optical density (MPOD) estimates in the elderly was assessed in 40 subjects (age: 79.1+/-3.5). Test-retest variability was good (Pearson's r coefficient: 0.734), with an average coefficient of variation (CV) of 18.4% and an intraclass correlation coefficient (ICC) of 0.96. The effect of optical blur on MPOD estimates was investigated in 22 elderly pseudophakic subjects (age: 79.9+/-3.6) by comparing the baseline MPOD, obtained with an optimal correction, with MPODs obtained with a +/-1.00-diopter optical blur. This optical blur did not cause differences in the MPOD estimates, its accuracy, or test duratio

Macular pigment optical density in the elderly: findings in a large biracial Midsouth population sample

PURPOSE: To report the macular pigment optical density (MPOD) findings at 0.5 degrees of eccentricity from the fovea in elderly subjects participating in ARMA, a study of aging and age-related maculopathy (ARM) ancillary to the Health, Aging, and Body Composition (Health ABC) Study. METHODS: MPOD was estimated with a heterochromatic flicker photometry (HFP) method in a large biracial population sample of normal 79.1 +/- 3.2-year-old adults living in the Midsouth (n = 222; 52% female; 23% black, 34% users of lutein-containing supplements). Within a modified testing protocol, subjects identified the lowest and the highest target intensity at which the flicker sensation disappeared, and the exact middle of this "no-flicker zone" was interpolated by the examiner. RESULTS: An MPOD estimate was obtained successfully in 82% of the participants. The mean MPOD in our sample was 0.34 +/- 0.21 (SD). The interocular correlation was high (Pearson's r = 0.82). Compared with lutein supplement users, mean MPOD was 21% lower in nonusers (P = 0.013). MPOD was also 41% lower in blacks than in whites (P = 0.0002), even after adjustment for lutein supplement use. There were no differences in MPOD by gender, iris color, or history of smoking. CONCLUSIONS: Older adults in the Midsouth appear to have average MPOD and interocular correlation comparable to those in previous studies. Lutein supplement use and white race correlated with higher MPOD. No evidence of an age-related decline in MPOD was seen in the sample. The HFP method for the measurement of MPOD is feasible in epidemiologic investigations of the elderly, the group at highest risk of AR