Murine [DX5.sup.+]NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing [CD4.sup.+][CD62L.sup.high] T Cells through Fas Ligand.
It is well established that T cells, in particular naive [CD4.sup.+] T helper (Th) cells, play a key role in mediating immune responses and especially many aspects of autoimmune diseases [1-3]. In line with this hypothesis, liver damage in autoimmune hepatitis, for instance, is likely orchestrated by naive [CD4.sup.+] T cells recognizing an autoantigenic liver peptide . In mice, it has been shown that transfer of enriched [CD4.sup.+][CD62L.sup.high] T cells into severe-combined-immunodeficient (SCID) mice induced chronic colitis [5-8]. For autoimmunity to occur, the antigen must be presented by antigen-presenting cells to naive [CD4.sup.+] T helper (Th0) cells. Once activated, Th0 cells can differentiate into Th1, Th2, or Th17 cells, initiating a cascade of immune reactions that are determined by the cytokines they produce .
In order to prevent effector cells to initiate and perpetuate tissue damage, subsequently resulting in autoimmune disease, there are several immune cell populations involved that tightly regulate their activation, such as regulatory T cells (Treg)  and NKT cells . For instance, NKT cells prevent the development of experimental crescentic glomerulonephritis by inhibiting proliferation of mesangial cells  and they are able to inhibit the onset of type one diabetes by impairing the development of pathogenic T cells specifically targeting pancreatic beta cells . There are also different cellular mechanisms involved, like the induction of programmed cell death to regulate respective immune responses in order to prevent self-endangering activities .
The acquisition of a distinct cytokine profile by naive [CD4.sup.+] T (Th0) cells and their proliferative capacity is modulated by specific cytokines. Th1 [CD4.sup.+] T cell differentiation is mediated by IL-12 and IFN-[gamma] that lead to the expression of the Th1 lineage specification transcription factor T-bet [15, 16]. Th2 cell differentiation depends on the activities of IL-4 and the transcription factor GATA3 . Differentiation into each lineage is also opposed by cytokines; thus, IFN-[gamma] promotes Th1 while suppressing Th2, IL-4 promotes Th2 and suppresses Th1, while TGF-[beta] suppresses Th1 and Th2 cell differentiation .
Natural killer T (NKT) cells represent a subset of T lymphocytes that express NK cell markers such as NK1.1 and CD94, as well as T cell receptors (TCR) [alpha]/[beta] with a restricted repertoire [17, 18]. These cells use a precisely rearranged homologous TCR variable (V) [alpha] and junctional (J) [alpha] segments. In mice, the invariant T cell receptor a chain V[alpha]14, encoded by J[alpha]18 with a conserved CDR3 region, is preferentially associated with V[beta]8.2, V[beta]7, or V[beta]2 gene segments [17, 19]. In contrast to other T lymphocytes, the TCR of NKT cells only recognizes glycolipids presented by CD1d, which is a MHC class I-like glycoprotein that belongs to a group of CD1 molecules associated with [beta]2-microglobulin [20-22]. Activation of NKT cells via CD1d antigen presentation initiates the production of both Th1 (IFN-[gamma]) and Th2 cytokines (IL-4, IL-5, IL-13)  and increases their cytolytic activity .
Depending on CD1d reactivity and whether they either express or do not express the invariant V[alpha]14-J[alpha]18 [TCR.sub.[alpha]/[alpha]], three subsets of NKT cells have been described. First, there are CD1d-dependent invariant NKT cells (iNKT) also called type I NKT cells. Second, a population of CD1d-reactive NKT cells expressing diverse TCR referred to as type II NKT cells has also been characterized. The third group consists NKT-like cells, which are CD1d-independent and express diverse TCR .
Several surrogate markers, such as NK1.1 in C57Bl/6 mice, coexpressed with the [TCR.sub.[alpha]/[beta]], have been used for identification of NKT cells . Another frequently used marker for these cells in NK1.1- mice strains is the antibody DX5, which recognizes the [[alpha].sub.2]-integrin CD49b . DX5 was initially characterized as a marker for NK cells , and more recently, DX5-coexpressing [CD3.sup.+] lymphocytes have been described .
Previous studies, including from our group, revealed evidence for an immunoregulatory potency of [DX5.sup.+]NKT cells by the production of Th1 and Th2 cytokines [7, 28-31].
Although, all these data describe typical characteristics of NKT cells, there remains an ongoing discussion as to whether [DX5.sup.+]NKT cells belong to the class of CD1d-dependent NKT cells [32, 33]. However, less attention has been given to their cytotoxic potential so far.
In the present study, we further characterized an anti-proliferative effect of [DX5.sup.+]NKT cells on colitis-associated [CD4.sup.+][CD62L.sup.high] cells. For this purpose, [CD4.sup.+][CD62L.sup.high] of the spleen of Balb/c mice was isolated and coculture experiments were set up with either [DX5.sup.+]NKT or [CD8.sup.+] T cells. [DX5.sup.+]NKT cells had an antiproliferative effect and induced apoptosis in [CD4.sup.+][CD62L.sup.high] cells. Furthermore, we could identify Fas ligand (FasL) to be a key player in the cytotoxic and proapoptotic function of [DX5.sup.+]NKT cell potentials.
Consequently, we were able to show that the proapoptotic effect of DX5-NKT cells against [CD4.sup.+][CD62L.sup.high] T cells is directed by FasL. Our observation therefore confirms previous reports about the cytotoxicity of type I NKT cells and extends these to DX5-NKT cells [34, 35].
2.1. Cell Harvesting and Isolation. Different lymphocyte subsets were purified from splenic mononuclear cells isolated from Balb/c mice (Charles River Laboratories, Wilmington, MA, USA). If necessary, further isolation was performed by magnetic activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) or by FACS (FACSAria I, BD Biosciences, San Jose, USA).
Briefly, cell suspension of the spleen was prepared by cutting small pieces and gently pressing through a 100 jm wire mesh. [DX5.sup.+] cells were purified using antimouse-[DX5.sup.+] MicroBeads (Miltenyi Biotec). Cells were passed through a MACS column (type LS) attached to a MidiMACS magnet (Miltenyi Biotec). [DX5.sup.+] cells were collected in the positive fraction. [DX5.sup.+] splenocytes were labeled with FITC-conjugated anti-mouse CD3 molecular complex (clone: 17A2, rat IgG2b) and PE-conjugated anti-mouse CD49b (clone: DX5, rat IgM) (all from BD Biosciences) for further [DX5.sup.+]NKT cell isolation by FACS sorting. [CD4.sup.+][CD62L.sup.high] and [CD4.sup.+][CD62L.sup.low] cells were purified using the [CD4.sup.+][CD62L.sup.+] Isolation Kit (Miltenyi Biotec) and [CD8.sup.+] cells by using anti-mouse-[CD8.sup.+] MicroBeads (Miltenyi Biotec).
2.2. Antibodies and Flow Cytometry. The following reagents were used for cell labeling in multiparameter flow cytometric analysis (FACS Calibur, BD Biosciences): PE or FITC-conjugated anti-mouse CD4 (clone: RM4-5, Rat IgG2b) Alexa 648 or FITC-conjugated anti-mouse CD3 (clone: 17A2, rat IgG2b), Alexa 648 or FITC-conjugated antimouse-CD8a (clone: 53-6.7, rat IgG2a), PE-conjugated anti-mouse-CD49b (clone: DX5, rat IgM), FITC- or PE-conjugated anti-mouse-CD62L (clone: MEL-14, rat IgG2a), and FITC- or PE-conjugated anti-mouse-CD178 (Fas ligand) (clone: MFL3, hamster IgG1); all are from BD Biosciences. APC-conjugated anti-mouse-CD49b (clone: DX5, rat IgM) and FITC-conjugated anti-mouse-CD49b (clone: DX5, rat IgM) all are from Miltenyi Biotec. APC-conjugated anti-mouse-CD4 (clone: RM4-5, rat IgG2b) is from Caltag (Towcester, UK).
2.3. Coculture Experiments. Ninety-six well culture plates (Sigma-Aldrich, St. Louis, USA) were coated with antimouse-CD3e (clone: 145-2C11, BD Biosciences) at 10 jg/ ml and stored overnight at 4[degrees]C. As described in previous studies in more detail, after isolation, 2 x [10.sup.5] [CD4.sup.+][CD62L.sup.high] and [CD4.sup.+][CD62L.sup.low] cells were coincubated with either 2 x [10.sup.5] [DX5.sup.+]NKT cells or [CD8.sup.+] T cells in 200 pl RPMI culture medium (Gibco, Paisley, UK) in either coated or uncoated wells . For further stimulation, 5 pg/ml antimouse-CD28 (clone: 37.51, BD Biosciences) and 2000IU/ ml IL-2 (PeproTech, Rocky Hill, USA) were added . Control cultures of [CD4.sup.+][CD62L.sup.high], [CD4.sup.+][CD62L.sup.low], [DX5.sup.+]NKT cells, and [CD8.sup.+]T cells only were incubated at 4 x [10.sup.5] cells in 200 pl RPMI under the same conditions.
2.4. CFSE Proliferation Assay. After isolation, [CD4.sup.+-] [CD62L.sup.high] and low cells were labeled using the Vybrant CFDA SE Cell Tracer Kit (Molecular Probes, Eugene, USA). In brief, cells were incubated with 0.5 [micro]M CFSE solution for 15min at 37[degrees]C. Pellet was washed once with culture medium to stop the staining reaction and then incubated for 60 min at room temperature to release excessive CFSE.
2.5. Intracellular Cytokine Staining. After cell isolation, cocultures were set up as mentioned above. Additionally, 50 ng/ml PMA (InvivoGen, San Diego, USA) was added from the beginning, 750 ng/ml ionomycin (Sigma-Aldrich, St. Louis, USA) for the last 4h, and 1 [micro]g/ml GolgiPlug (BD Biosciences) was added 2 h before cell harvesting. Culture supernatants were harvested and stored at -20[degrees]C for IFN-[gamma] ELISA. Cells were fixed in 1 ml Fix/Perm (eBioscience, Hatfield, UK) for 60min at 4[degrees]C. After incubation with permeabilization buffer (eBioscience), cells were stained intracellular with PE-conjugated anti-mouse-Abs (IL-2, clone: JES6-5H4/IFN-[gamma], clone: XMG1.2/TNF-[alpha], clone: MP6-XT22) from BD Biosciences and with PE-conjugated anti-mouse-IL-13 (clone: eBio13A) and FITC-conjugated anti-mouse-IFN-[gamma] (clone: XMG1.2) all eBioscience.
2.6. Intracellular Caspase-3 Staining. After cell isolation, cocultures were set up as mentioned above. For 48 h coincubation, [CD4.sup.+][CD62L.sup.high] and [CD4.sup.+][CD62L.sup.low] cells were additionally labeled with CFSE. After the indicated time, cells were fixed in 1 ml Fix/Perm (eBioscience) for 60 min at 4[degrees]C. After incubation with permeabilization buffer (eBioscience), cells were stained intracellular with Alexa648-conjugated anti-mouse-caspase-3 (clone: C92-605, BD Biosciences). For FasL blocking (Kayagaki, Yamaguchi et al. 1997), [DX5.sup.+]NKT cells were preincubated with either 50 pg/ml purified mouse-anti-FasL (clone: MFL4; BioLegend, Cambridge, UK) or 50 pg/ml isotype control for 1 h and then cocultures were set up with [CD4.sup.+][CD62L.sup.high] cells as indicated above.
2.7. Statistics. All in vitro experiments were repeated at least 3 times, and data are presented as the mean value [+ or -] SEM. Statistical analyses were performed using either a Student's t-test or the Mann-Whitney U test. Differences were considered significant at P < 0.05.
3.1. [DX5.sup.+]NKT Cells Have an Antiproliferative Effect on Colitis-Inducing [CD4.sup.+][CD62L.sup.high] Cells. Lymphocyte subsets, such as [CD4.sup.+][CD62L.sup.high] and [CD8.sup.+]T cells, were isolated from the spleen of Balb/c mice by MACS. [DX5.sup.+]NKT cells were isolated using MACS followed by FACS sorting (Figure 1(a)). First, we analyzed the proliferation of [CD4.sup.+][CD62L.sup.high] cells in coculture experiments with [DX5.sup.+]NKT and [CD8.sup.+]T cells (Figure 1(b)) using CFSE labeling. [CD4.sup.+][CD62L.sup.high] cells began to proliferate 48 h after stimulation with anti-CD3, anti-CD28, and IL-2. As shown in Figure 1(c), after 96 h coincubation with [DX5.sup.+]NKT cells, proliferation of [CD4.sup.+][CD62L.sup.high] cells significantly decreased compared to single cultures (proliferation index: 1.39[+ or -] 0.07 vs. 1.76[+ or -] 0.12; P = 00079). The antiproliferative effect of [CD8.sup.+]T cells was less distinctive and statistically not significant.
3.2. Decrease of IFN-[gamma] Cytokine Secretion of [CD4.sup.+] [CD62L.sup.high] Cells in Coculture Experiments with [DX5.sup.+]NKT Cells. Next, we wanted to assess whether this antiproliferative effect was associated with differences in cytokine secretion. Therefore, the production of Th1 such as IFN-[gamma], TNF-[alpha], and IL-2 and the Th2 cytokine IL-13 in [CD4.sup.+][CD62L.sup.high] cells was compared. Isolated [CD4.sup.+][CD62L.sup.high] cells were cultured for 4 and 10 h in the presence of anti-CD3 and anti-CD28 antibodies in a single or coculture with [DX5.sup.+]NKT or [CD8.sup.+]T cells, respectively. The cells were additionally incubated with PMA, ionomycin, and GolgiPlug for intracellular cytokine staining. As shown in Figure 2(a), after 10 h, [CD4.sup.+][CD62L.sup.high] cells produced the same amount of IFN-[gamma] in a coculture with [CD8.sup.+] cells as in a single culture (5.4% [+ or -] 0.95% vs. 5.6% [+ or -] 0.76%), whereas cocultured with [DX5.sup.+]NKT cells, IFN-[gamma] production of [CD4.sup.+][CD62L.sup.high] cells was decreased (5.6% [+ or -] 0.76% vs. 1.3% [+ or -] 0.26%; P = 0.02). In contrast, looking at TNF-[alpha], IL-2, and IL-13, no interference of [DX5.sup.+]NKT cells in cytokine production of [CD4.sup.+][CD62L.sup.high] cells could be detected. Furthermore, there was no significant effect looking at cocultures with [CD4.sup.+][CD62L.sup.low] cells (Figure 2(b)).
3.3. [DX5.sup.+]NKT Cells Activate Proapoptotic Caspase-3 in Colitis-Associated [CD4.sup.+] [CD62L.sup.high] Cells. Results thus far display differences in the cytokine profile between [CD8.sup.+] cells and [DX5.sup.+]NKT cells but cannot explain the antiproliferative effect on [CD4.sup.+][CD62L.sup.high] cells. Therefore, we wanted to assess whether the cytotoxic potential of [DX5.sup.+]NKT cells is involved in this process. We measured the amount of intracellular caspase-3 after 10 and 48 h coincubation of [CD4.sup.+][CD62L.sup.high] cells with either [CD8.sup.+] cells or [DX5.sup.+]NKT cells. As shown in Figure 3, [DX5.sup.+]NKT cells significantly increased the number of caspase-3-positive cells after 48 h coculturing (38% vs. 28%; P = 0.0451) compared to the [CD4.sup.+][CD62L.sup.high] single culture.
3.4. FasL on [DX5.sup.+]NKT Cells Is Mediating the Induction of Proapoptotic Caspase-3 in Colitis-Associated [CD4.sup.+][CD62L.sup.high] Cells. Next, we wanted to assess which route [DX5.sup.+]NKT cells used to induce caspase-3. Therefore, either [CD8.sup.+] cells or [DX5.sup.+]NKT cells were cocultured with [CD4.sup.+][CD62L.sup.high] cells and stained for FasL expression. After 4 h, [DX5.sup.+]NKT cells in coculture expressed more FasL compared to [CD8.sup.+] cells (9% vs. 2%; P = 0.0026). And after 10 h incubation, even more [DX5.sup.+] NKT cells expressed FasL (16.6% [+ or -] 3.2%), whereas [CD8.sup.+] cells in coculture with [CD4.sup.+][CD62L.sup.high] cells still expressed less FasL (6.3% [+ or -] 2.4%; P = 0.0159) compared to [DX5.sup.+]NKT cells (Figure 4).
Finally, we wanted to prove that the induction of FasL expression is responsible for the activation of caspase-3 in [CD4.sup.+][CD62L.sup.high] cells. [DX5.sup.+]NKT cells were pre-incubated with a FasL blocking antibody for 1 h prior to coculturing with [CD4.sup.+][CD62L.sup.high] cells for 48 h. Additionally, cells were incubated with an isotype antibody for control reasons. As expected, coincubation with [DX5.sup.+]NKT cells increased the frequency of caspase-3-positive cells in the normal culture (34.4% [+ or -] 4.3%; P = 0.0451) as well as in the isotype control culture (38% [+ or -] 3.5%; P = 0.028) compared to a [CD4.sup.+][CD62L.sup.high] single culture (24% [+ or -] 1.5%). Interestingly, blocking of FasL on [DX5.sup.+]NKT cells reduced the frequency of caspase-3-positive [CD4.sup.+][CD62L.sup.high] cells in the coculture to that amount observed in the single culture (23.4% [+ or -] 1.2%; P = 0.008) (Figure 5).
In this study, we show that proliferation of colitis-inducing [CD4.sup.+][CD62L.sup.high] T cells is prevented by [DX5.sup.+]NKT cells. After activation, naive [CD4.sup.+] cells can differentiate into Th1, Th2, or Th17 cells, initiating different immune reactions depending on their cytokine profile . A variety of mechanisms are involved in regulation of [CD4.sup.+] T cell function, including inhibition of proinflammatory cytokines, promoting increase of certain T cell populations and suppressing proliferation of different ones  . Certain cytokines can suppress and induce differentiation, i.e., of Th17 cells   , but for some activation also, direct cell interaction with B cells is required . Toes and his group showed that [DX5.sup.+][CD4.sup.+] T cells were able to direct [CD4.sup.+] T cells towards IL-10 production through IL-4 , and in addition, these cells impair the function of [CD4.sup.+] T cells through specific inhibition of dendritic cells . [DX5.sup.+][CD4.sup.+] T cells are comparable to [DX5.sup.+]NKT cells because over 80% of isolated [DX5.sup.+]NKT cells are CD4 positive . Nevertheless, reported studies could not show an inhibition of proliferation of [CD4.sup.+] T cells by [DX5.sup.+][CD4.sup.+] T cells . This might be explained by the fact that the used cells were OVA-specific [CD4.sup.+] T cells  and therefore different compared to naive [CD4.sup.+][CD62L.sup.high] T cells. Previously published data of our group already revealed a decrease of colitis-inducing [CD4.sup.+][CD62L.sup.high] T cells after coculturing with [DX5.sup.+]NKT cells , and consistent in both settings, [DX5.sup.+] T cells decreased IFN-[gamma] production of OVA-specific [CD4.sup.+] T cells as well as of [CD4.sup.+][CD62L.sup.high] T cells as shown in our study [41, 42]. However, we used [CD8.sup.+] T cells to exclude an unspecific effect between the cultured subsets. This worked very well as a control for the IFN-gamma production assays but not so well, for example, in the proliferation assays. Furthermore, stimulation with IL-2 might also impact the responsiveness of [CD4.sup.+][CD62L.sup.high] cells, [CD8.sup.+] T cells, and NKT cells at different levels. Furthermore, as shown before, [DX5.sup.+]NKT cells express in less 20% CD25 . Therefore, a relatively small contribution of Tregs on the observed effects should be considered.
We found out that the inhibition of [CD4.sup.+][CD62L.sup.high] T cells proliferation after coculturing with [DX5.sup.+]NKT cells is associated with an increase of caspase-3 in [CD4.sup.+][CD62L.sup.high] T cells. Mature caspase-3 results from the processing of procaspase-3 and induces cell death through multiple cellular molecules. Caspase-3 is involved in both major apoptosis pathways: the intrinsic, which is mediated through the B cell lymphoma 2 (BCL-2) family, and the extrinsic pathway, which is induced through the interaction of FasL and Fas .
In our study, expression of FasL was significantly increased on [DX5.sup.+]NKT cells after 10 h especially in coculture with [CD4.sup.+][CD62L.sup.high] T cells. Furthermore, blockage of FasL resulted in a decrease of caspase-3 in [CD4.sup.+][CD62L.sup.high] T cells. These results are in agreement with a previous report from our group showing that [DX5.sup.+]NKT cells reduce colitis cells through PD-L1 in vitro. However, PD1, the counterpart of PD-L1, was expressed on both [CD62L.sup.high] and [CD62L.sup.low] cells, suggesting that PD-L1 killing activity is likely mediated via a different receptor . FasL is known as a receptor on specific cytotoxic lymphocytes [43, 44], but until now, it has not been described on [DX5.sup.+]NKT cells. FasL's counterpart on the effector cell, Fas, is known to transmit signals that lead to cell death via apoptosis . Our findings suggest that the decrease in the number of colitis-inducing [CD4.sup.+][CD62L.sup.high] T cells as well as their proliferation capacity in the presence of [DX5.sup.+]NKT cells is principally executed through FasL-Fas interaction. Loss of function of the FasL-Fas pathway can cause autoimmune diseases [46-49], and it is already known that FasL-Fas is required for elimination of T cells .
Taken these data together with our findings supposes that the interaction with [DX5.sup.+]NKT cells via FasL-Fas represents one mechanism in the regulation of the development of [CD4.sup.+] T cells. Further studies should clear the importance of this effect in the origin of autoimmunity and the additional cofactors which are required to perform the function.
All data supporting the results reported in the article are generated and archived in facilities of the Department of Surgery, University of Regensburg.
Parts of the study were presented as abstract on the 15th Annual Meeting on Surgical Research 15, 22-24 September 2011, Dresden, Germany, Langenbecks Arch Surg (2011) 396: 871, doi:10.1007/s00423-011-0830-7.
Conflicts of Interest
The authors declare that no competing interests exist. HJS receives consulting and lecture fees. The remaining authors receive no external income.
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Jens M. Werner, Michael Damian, Stefan A. Farkas, Hans J. Schlitt, Edward K. Geissler, and Matthias Hornung (ID)
Department of Surgery, University Hospital Regensburg, University of Regensburg, Regensburg, Germany
Correspondence should be addressed to Matthias Hornung; firstname.lastname@example.org
Received 6 February 2018; Revised 20 August 2018; Accepted 4 September 2018; Published 30 September 2018
Academic Editor: Martin Holland
Caption: Figure 1: Flow cytometry analysis of the spleen [CD3.sup.+][DX5.sup.+]NKT, [CD8.sup.+] T, [CD4.sup.+][CD62L.sup.high], and [CD4.sup.+][CD62L.sup.low] cells of Balb/c mice after separation by MACS and FACS sorting (a). Proliferation (b) and proliferation index (c) of CFSE-labeled CD4 and 96 h of monoculture or coculture with [CD8.sup.+] T cells or [CD3.sup.+] [DX5.sup.+]NKT. Results are given as mean + SEM. Experiments were repeated at least three times (*P < 0.05).
Caption: Figure 2: Intracellular flow cytometry detection of IFN-[gamma] in [CD4.sup.+][CD62.sub.high] (a) and [CD4.sup.+][CD62L.sup.low] cells (b) after 4h and 10 h of monoculture or coculture with [CD8.sup.+] T cells or [CD3.sup.+][DX5.sup.+]NKT. Results are given as mean+SEM. Experiments were repeated at least three times (*P <0.05).
Caption: Figure 3: Intracellular flow cytometry analysis of caspase-3 in [CD4.sup.+][CD62L.sup.high] cells after 10 h and 48 h of monoculture or coculture with [CD8.sup.+] T or [CD3.sup.+][DX5.sup.+]NKT cells. Results are given as mean + SEM. Experiments were repeated at least three times (*P < 0.05).
Caption: Figure 4: Flow cytometry analysis FasL expression of CD8 T and [CD3.sup.+][DX5.sup.+]NKT cells after 4h and 10 h monoculture or coculture with [CD4.sup.+][CD62L.sup.high] cells. Results are given as mean + SEM. Experiments were repeated at least three times (*P < 0.05).
Caption: Figure 5: Intracellular flow cytometry analysis of caspase-3 in [CD4.sup.+][CD62L.sup.high] cells after 48 h of coculture with [CD3.sup.+][DX5.sup.+]NKT cells and pretreatment with either FasL block or isotype control. Results are given as mean + SEM. Experiments were repeated at least three times (*P < 0.05).
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|Title Annotation:||Research Article|
|Author:||Werner, Jens M.; Damian, Michael; Farkas, Stefan A.; Schlitt, Hans J.; Geissler, Edward K.; Hornung,|
|Publication:||Journal of Immunology Research|
|Date:||Jan 1, 2018|
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