CD4⁺NKG2D⁺ T Cells Exhibit Enhanced Migratory and Encephalitogenic Properties in Neuroinflammation

<div><p>Migration of encephalitogenic CD4⁺ T lymphocytes across the blood-brain barrier is an essential step in the pathogenesis of multiple sclerosis (MS). We here demonstrate that expression of the co-stimulatory receptor NKG2D defines a subpopulation of CD4⁺ T cells with elevated levels of markers for migration, activation, and cytolytic capacity especially when derived from MS patients. Furthermore, CD4⁺NKG2D⁺ cells produce high levels of proinflammatory IFN-γ and IL-17 upon stimulation. NKG2D promotes the capacity of CD4⁺NKG2D⁺ cells to migrate across endothelial cells in an in vitro model of the blood-brain barrier. CD4⁺NKG2D⁺ T cells are enriched in the cerebrospinal fluid of MS patients, and a significant number of CD4⁺ T cells in MS lesions coexpress NKG2D. We further elucidated the role of CD4⁺NKG2D⁺ T cells in the mouse system. NKG2D blockade restricted central nervous system migration of T lymphocytes in vivo, leading to a significant decrease in the clinical and pathologic severity of experimental autoimmune encephalomyelitis, an animal model of MS. Blockade of NKG2D reduced killing of cultivated mouse oligodendrocytes by activated CD4⁺ T cells. Taken together, we identify CD4⁺NKG2D⁺ cells as a subpopulation of T helper cells with enhanced migratory, encephalitogenic and cytotoxic properties involved in inflammatory CNS lesion development.</p> </div

CD4⁺NKG2D⁺ T cells exert pro-migratory, cytolytic and pro-inflammatory properties.

<p>(<b>A</b>) Flow cytometry: The dot plot shows a representative example of NKG2D expression on CD3⁺CD4⁺CD8^-CD56⁻ T cells derived from the peripheral blood of a healthy donor (HD). The bar graph represents the mean frequency of CD4⁺NKG2D⁺ T cells in the peripheral blood of HDs (<i>n</i> = 20), stable RRMS (<i>n</i> = 15) and active RRMS patients (<i>n</i> = 14). (<b>B</b>–<b>E</b>) Mean fluorescence intensity (MFI) of different markers indicative for migratory capacity (<b>B</b>), activation (<b>C</b>), or cytolytic capacity (<b>D</b>, <b>E</b>) of CD4⁺NKG2D⁺ and CD4⁺NKG2D⁻ T cells from the peripheral blood of HDs (n = 6) or active RRMS patients (<i>n</i> = 6). (<b>F</b>) Percentages of naive (CD45RA⁺CD62L⁺), T central memory (Tcm, CD45RA^-CD62L⁺), T effector memory (Tem, CD45RA^-CD62L⁻) and T effector memory RA (Tem-RA, CD45RA⁺CD62L^-) cells in the CD4⁺NKG2D⁺ and CD4⁺NKG2D⁻ T cell compartment assessed by flow cytometry (<i>n</i> = 6 HDs). (<b>G</b>) Carboxyfluorescein succinimidyl ester (CFSE) proliferation assays of CD4⁺NKG2D⁺ T cells and CD4⁺NKG2D⁻ T cells under CD3/CD28, CD3/NKG2D, MOG_35-55 (10 µg/ml or 100 µg/ml), MBP_1-11 or PLP_190-209 stimulation (<i>n</i> = 8 HDs). (<b>H</b>) Intracellular cytokine staining for IFN-γ and IL-17 of CD4⁺NKG2D⁺ and CD4⁺NKG2D⁻ T cells derived from the peripheral blood of HDs (<i>n</i> = 7). The dot plots depict a representative example of IFN-γ- and IL-17-positive cells upon CD3/CD28 stimulation. The bar graphs show the frequencies of IFN-γ or IL-17 positive cells of unstimulated, CD3/CD28- or CD3/NKG2D-stimulated cells. (<b>I</b>) Comparison of the proportions of IFN-γ or IL-17 positive CD3/CD28-stimulated CD4NKG2D⁺ T cells derived from frozen PBMCs of HDs (<i>n</i> = 6) or active RRMS patients (<i>n</i> = 6). *P < 0.05. ns, not significant; unstim., unstimulated.</p

Inhibition of the NKG2D-signaling pathway ameliorates the disease course of experimental autoimmune encephalomyelitis (EAE).

<p>(<b>A</b>) EAE disease course was reduced in NKG2D-blocking-antibody-treated C57BL/6 mice (black arrows, days of injection, clone CX5) compared with mice treated with an isotype control (two independent experiments, <i>n</i> = 8 each). Myelin oligodendrocyte protein (MOG) recall assays and flow cytometry were performed at disease onset (gray arrow). (<b>B</b>) ELISAs for IFN-γ production and the splenocyte stimulation index indicative of cell proliferation showed no significant differences for splenocytes of antibody- and isotype-treated mice at disease onset (<i>n</i> = 5 mice per group). (<b>C</b>) Flow cytometric quantification of NKG2D⁺ CD4⁺ and CD8⁺ lymphocytes invading the brain at disease onset of EAE (<i>n</i> = 5 mice per group, clone 7 used for staining). (<b>D</b>) Representative immunohistochemical staining of spinal cord samples from isotype- or anti-NKG2D-treated mice (<i>n</i> = 10 per mouse; <i>n</i> = 5 mice per group) at the disease maximum of EAE and quantification of the area of infiltrating cells (hematoxylin and eosin [H&E] staining, highlighted area) and area of demyelination (luxol fast blue [LFB] staining, highlighted area). (<b>E</b>) In vivo blockade of NKG2D by soluble NKG2D (sNKG2D) delayed the onset of EAE when administered before disease onset (left panel). In contrast, the administration of sNKG2D after disease onset had no significant effects (right panel). (<i>n</i> = 6 mice per group). *P < 0.05. ns, not significant. </p

CD4⁺NKG2D⁺ T cells show cytolytic effects towards cultured mouse oligodendrocytes.

<p>(<b>A</b>) Upper panel: Oligodendrocytes after 48 h of differentiation (bright field). Lower panel: Immunocytochemistry of mature oligodendrocytes displaying a complex network of myelin-building processes (DAPI, blue; NogoA, green; myelin basic protein [MBP], red). (<b>B</b>) Flow cytometric quantification of MHC I expression of differentiated oligodendrocytes under basal and different inflammatory conditions (500 IU/ml IFN-γ for 24 h or 500 IU/ml IFN-γ and 500 IU/ml TNF-α for 24 h). (Representative example, <i>n</i> = 3). (<b>C</b>) Differentiated oligodendrocytes express the mouse NKG2D ligands MULT-1 and RAE-1, but not H60, under basal conditions. Upon inflammation (500 IU/ml IFN-γ and 500 IU/ml TNF-α for 24 h), the surface expression of these ligands is upregulated (<i>n</i> = 3). (<b>D</b>) The frequency of apoptotic oligodendrocytes is assessed by an immunohistochemical cleaved caspase-3 staining. Oligodendrocytes were co-cultured with CD4⁺NKG2D⁺-enriched CD4⁺ T cells (CD4⁺) for 6 h, and blocking antibodies against NKG2D, MHC I, and MHC II were added as indicated. In one assay, CD4⁺ T cells were separated by a Transwell inset (<i>n</i> = 3 independent experiments, triple values). *P < 0.05. ns, not significant.</p

NKG2D facilitates migration of CD4⁺NKG2D⁺ T cells in vitro and increased numbers are found in CSF and lesions of MS patients.

<p>(<b>A</b>) Transmigration Assay of CD4⁺ T cells from healthy donors (HDs; <i>n</i> = 5) for a migration period of 11–12 h. Migration through the membrane only (membrane) or over an endothelial cell single-layer (HBMEC) was compared. The proportions of migrated (lower chamber) or non-migrated (upper chamber) CD4⁺NKG2D⁺ T cells are shown. (<b>B</b>) Relative cell numbers of transmigrated CD4⁺NKG2D⁺ T cells and total CD4⁺ T cells through a non-inflamed or inflamed HBMEC single-layer (<i>n</i> = 5 HDs). (<b>C</b>) Expression of NKG2D ligands on non-inflamed or inflamed HBMECs (500 IU/ml IFN-γ and 500 IU/ml TNF-α for 72 h) analyzed by flow cytometry (representative example; <i>n</i> = 5). (<b>D</b>) Representative transmigration assay of FACS-sorted CD4⁺NKG2D⁻ T cells. Proportions of migrated (lower chamber) or non-migrated (upper chamber) cells are shown (<i>n</i> = 5). (<b>E</b>) CD4⁺ T cells from HDs (<i>n</i> = 6) were incubated with antibodies (5 μg/ml activating anti-NKG2D or 10 μg/ml blocking anti-NKG2D) or the respective isotype controls prior to the transmigration assay. Proportions of migrated CD4⁺NKG2D⁺ T cells after 6 h are shown. (<b>F</b>) Upper Panel: Frequencies of CD4⁺NKG2D⁺ T cells in the peripheral blood and the cerebrospinal fluid (CSF) of patients with stable (<i>n</i> = 15) and active (<i>n</i> = 14) relapsing-remitting MS (RRMS) and healthy controls (<i>n</i> = 15) assessed by flow cytometry. Lower Panel: Percentages of naive (CD45RA⁺CD62L⁺), T central memory (Tcm, CD45RA^-CD62L⁺), T effector memory (Tem, CD45RA^-CD62L⁻) and T effector memory RA (Tem-RA, CD45RA⁺CD62L^-) CD4⁺NKG2D⁺ cells in the CSF of RRMS patients (RRMS, n = 6) and healthy controls (HD, <i>n</i> = 6). (<b>G</b>) Histopathologic characterization of a representative human MS lesion (patient with RRMS) using antibodies directed against CD4 and NKG2D, a perivascular region is magnified showing CD4⁺NKG2D⁺ T cells (DAPI, blue; CD4, green; NKG2D, red). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081455#pone-0081455-t001" target="_blank">Table 1</a> for quantification. *P < 0.05. ns, not significant; unstim., unstimulated.</p