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The Journal of Immunology, 2006, 177: 8542-8549.
Copyright © 2006 by The American Association of Immunologists, Inc.

CD5-CK2 Binding/Activation-Deficient Mice Are Resistant to Experimental Autoimmune Encephalomyelitis: Protection Is Associated with Diminished Populations of IL-17-Expressing T Cells in the Central Nervous System1

Robert C. Axtell*,{dagger}, Liang Xu*, Scott R. Barnum{dagger} and Chander Raman2,*

* Department of Medicine and {dagger} Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulating the differentiation and persistence of encephalitogenic T cells is critical for the development of experimental autoimmune encephalomyelitis (EAE). We reported recently that CD5 has an engagement-dependent prosurvival activity in T cells that played a direct role in the induction and progression EAE. We predicted that CD5 regulates T cell apoptosis/survival through the activation of CK2, a prosurvival serine/threonine kinase that associates with the receptor. To test this hypothesis, we generated mice expressing CD5 with the inability to bind and activate CK2 and assessed their susceptibility to EAE. We found mice deficient in CD5-CK2 signaling pathway were mostly resistant to the development of EAE. Resistance to EAE was associated with a dramatic decrease in a population of effector infiltrating Th cells that coexpress IFN-{gamma} and IL-17 and, to a lesser extent, cells that express IFN-{gamma} or IL-17 in draining lymph nodes and spinal cords. We further show that T cells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation; however, following restimulation, they rapidly develop nonresponsiveness and exhibit elevated activation-induced cell death. Our results provide a direct role for CD5-CK2 pathway in T cell activation and persistence of effector T cells in neuroinflammatory disease. This study predicts that targeting of IFN-{gamma}+/IL-17+ infiltrating Th cells will be useful for the treatment of multiple sclerosis and other systemic autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Experimental autoimmune encephalomyelitis (EAE)3 is a CD4 T cell-mediated disease model of multiple sclerosis (MS) (1, 2). The generation of effector CD4+ T cells is a critical event for the progression of EAE. Until recently, the disease was primarily considered infiltrating Th cell 1 (TH1) dependent; however, now the newly described TH17 lineage of CD4+ T cells has emerged to be the major effector cell for the development of EAE (3). It is currently thought that IL-17 up-regulates the expression of chemokines, such as CCL2, CCL20, and MIP-2, to mediate inflammation in the CNS to promote disease (4, 5). In contrast, the TH2 and regulatory T cell subsets play a protective role in EAE (6, 7, 8).

CD5 is considered primarily to be a negative costimulator of T cell activation signals (9, 10, 11, 12, 13), and we showed recently that it also plays a critical role in promoting prosurvival signals in T cells (14). T cells can adjust their threshold for activation by altering the level CD5 on the cell surface (10, 15, 16, 17, 18). The up-regulation of CD5 can also lead to the development of Ag-specific unresponsiveness. This was demonstrated by Hawiger et al. (19), when they reported the generation of a population T cells with elevated levels of CD5 that developed Ag-specific nonresponsiveness. The nonresponsiveness was dependent on CD5, because such a population could not be generated in CD5–/– mice or mice treated with anti-CD5. Conversely, by lowering CD5 expression, T cells can develop responsiveness as demonstrated recently in tumor-infiltrating lymphocytes isolated from a lung carcinoma (15).

We showed recently that CD5–/– mice had decreased severity of EAE following myelin oligodendrocyte glycoprotein (MOG35–55) (3) peptide immunization (14). Consistent with the inhibitory role of CD5, the CD5–/– mice exhibited an enhanced T cell response to MOG immunization. This initial response was followed by elevated activation-induced cell death (AICD), which conferred resistance to disease. In addition, we found the prosurvival activity in T cells provided by CD5 can be abrogated by blockade of CD5 engagement with soluble CD5-Ig in mice and is therapeutically beneficial for treatment of EAE. Another recent study demonstrated that myelin-specific T cells that escape superagonistic-induced AICD, in fact, had elevated levels of CD5 and were unresponsive to re-exposure to Ag (16). Together, these data suggest that CD5 levels are critical in determining both the responsiveness as well as the survival of T cells after antigenic stimulation.

We and others established previously that CK2, a serine/threonine kinase, is associated with the cytoplasmic tail of CD5, and upon engagement of CD5, the kinase is activated (20, 21, 22). CK2 activity has been shown to promote cell survival of lymphocytes both by directly inhibiting apoptosis and by activating or inducing expression of prosurvival molecules (23, 24). We predicted that the CD5 engagement would deliver prosurvival/antiapoptotic signals to T cells by triggering CK2 activity, a process that would have a direct effect on the development of EAE. To address this hypothesis, we evaluated EAE susceptibility in CD5–/– mice reconstituted with a T cell expression-restricted CK2 binding/activation-deficient CD5 transgene. Comparisons were made with CD5+/+, CD5–/–, and CD5–/– mice reconstituted with CD5WT transgene. In this study, we report two important observations. First, we found that CD5-CK2 binding/activation-deficient mice are resistant to EAE, which was associated with a dramatically reduced population of TH cells that express both IFN-{gamma} and IL-17 as well as a reduction in classical TH1 (IFN-{gamma}+) and the newly described TH17 (IL-17+). Second, our studies also revealed a novel mechanism for regulating T cell activation through CK2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice were purchased from The Jackson Laboratory. CD5WT transgenic mice were provided by Dr. P. Love (National Institutes of Health, Bethesda, MD) (10). CD5–/– mice were crossed into C57BL/6 for 10 generations (14, 25). The CK2-binding-deficient CD5 transgene was generated by introducing a microdeletion of S458–S461 aa residues (CD5{Delta}458-461) by site-directed mutation (Stratagene) of the coding region of CD5. The resultant cDNA was sequenced in entirety to confirm the presence of desired mutation and absence of nondesired mutations. The mutated cDNA was then cloned into the CD2 minigene construct provided by Dr. Love (10). Founder lines were generated in the C57BL/6 mice, bred into CD5–/– mice, and screened using forward primer (5'-atggactcccacgaagtgctg-3') and reverse primer (5'-cttgtagaggatggtgcca-3'). This same set of primers was also used to genotype the CD5WT transgenic mice. All animals were housed and treated in accordance with National Institutes of Health and University of Alabama at Birmingham Institutional Animal Care and Use Committee guidelines.

EAE induction

Mice were immunized with 150 µg of MOG35–55 peptide (Biosynthesis), which was modified from a previously described protocol, and EAE symptoms were monitored daily for 30–35 days using a standard clinical score ranging from 0 to 6 (14). Briefly, mice received a single immunization of peptide in CFA on day 0, followed by pertussis toxin (List Biological Laboratories) on days 0 and 2. Animals with a clinical score <2 as defined by loss of tail tone are considered free of clinical disease. Infiltrating cells were isolated from spinal cord as previously described (26). Briefly, spinal cord homogenates were obtained from 8 to 10 perfused animals and incubated with collagenase (2 mg/ml) and DNase (5 U/ml) for 1 h at 37°C; mononuclear cells were purified by two-step Percoll gradient centrifugation.

Flow cytometry

Single-cell suspensions of axillary and brachial draining lymph node (DLN) and spinal cord cells were incubated with anti-CD16/32 (2.4G2, FcR block) before staining with anti-CD8 (53-6.7) or anti-CD4 (L3T4) and anti-CD69 (H1.2F3), all from eBioscience, conjugated to various fluorochromes as needed. For intracellular cytokine staining, cells were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences) for 4 h before surface staining with anti-CD4-FITC and intracellular cytokine staining with anti-IFN-{gamma}-cyanine 5 and anti-IL-17-PE (BD Biosciences). For the measurement of cell cycle in vivo, mice were injected with 1.5 mg of BrdU 12 h before tissues were harvested. Cells were stained with anti-CD4-allophycoyanin before intracellular staining with anti-BrdU-FITC and analyzed by flow cytometry as described in the manufacturer’s instructions (BD Biosciences).

In vitro stimulation assays

To assess primary T cell responses, spleen cells were cultured in 1 µg/ml anti-CD3 (145-2C11) for 72 h. Frequency of CD4 and CD8 cells in cycle was determined by pulse labeling cells with BrdU (10 µM) for 90 min before flow cytometric analysis of BrdU incorporation as describe above. Cell divisions were assessed by tracking the dilution of FL1-fluorescence of CD4 or CD8 cells labeled with CFSE (0.2 µM; Invitrogen Life Technologies) by flow cytometry. The kinetics of proliferation was assessed in triplicate at 24, 42, and 72 h of stimulation and addition of [3H]thymidine for the last 18 h of incubation. Incorporation of radioactivity was measured using a liquid scintillation counter (Packard Instrument). To assay for early apoptotic events in T cells, we measured loss of mitochondrial membrane potential by incubating cells with dihexyloxacarbocyanine (DiOC6; Invitrogen Life Technologies) at a final concentration of 20 nM for 15 min at 37°C before staining for with anti-CD4-allophycocyanin, anti-CD8-PE, and 7-aminoactinomycin D (Invitrogen Life Technologies) (14, 27).

T cell recall response was assessed by culturing spleen cells for 24 h with 1 µg/ml anti-CD3. Cells were washed and rested by culturing in fresh medium for an additional 72 h. Live cells were separated from dead cells by Ficoll density gradient centrifugation, restimulated with 1 µg/ml anti-CD3 and assayed and BrdU incorporation as described above. Early apoptosis was assayed as described above.

Statistics

Results were analyzed for statistical significance using two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation and characterization of the mice expressing a CK2 binding-deficient CD5

We demonstrated recently that CD5–/– mice had a delay in onset and decrease in severity of EAE, which was due to the inability of activated T cells to persist (14). Because CD5 engagement activates CK2, a kinase known to regulate cell survival (21, 24), we predicted that the attenuation of EAE in CD5–/– mice was due to the loss of CD5-CK2 signaling. To test this hypothesis, we reconstituted CD5–/– mice with a CD2 promoter-regulated transgene encoding a CK2 binding/activation-deficient CD5 (CD5{Delta}458-461) with restricted expression in T cells (Fig. 1A). This microdeletion of 4 as (serine-serine-aspartic acid-serine) completely abrogates the interaction of CK2 with CD5, which is necessary for the activation of the kinase following engagement of the receptor (21). As controls, we used CD5–/– mice reconstituted with wild-type CD5 transgene (CD5WT) (10). In both of these transgenic lines CD5 expression is under the control of the same regulatory elements. Both CD5WT and CD5{Delta}458-461 mice contain similar frequencies and numbers of CD4 and CD8 T cell populations in the thymus and peripheral lymphoid organs (Fig. 1, B and C, and data not shown). The expression level of CD5 on mature T cells in the thymus and in the periphery is similar in both lines of CD5 transgenic mice. However, the expression of CD5 is lower on immature thymocytes (double negative and double positive) in CD5{Delta}458-461 mice, compared with CD5WT mice (Fig. 1, D and E). The significance for this finding is currently unclear and is being explored.


Figure 1
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  		FIGURE 1. Generation and phenotypic characterization of CD5{Delta}458-461 mice. A, CD5WT and CD5{Delta}458-461 CD2 minigene constructs. The CD5WT transgenic mouse (denoted by an asterisk) as well as the CD2 minigene construct used to generate CD5{Delta}458-461 construct were provided to us by Dr. P. Love (National Institutes of Health, Bethesda, MD) (10 ). B and C, Frequencies of CD4+ and CD8+ lymphocytes in thymus and spleen of 6- to 8-wk-old mice. The percentage of cells in each quadrant is given. D and E, Expression levels of CD5 on T cell subsets in the thymus and spleen. DN, double negative.

 
CK2 binding/activation-deficient CD5 transgenic mice are resistant to EAE

To address the role of CD5-CK2 signaling in susceptibility to EAE, we compared the progression of disease in CD5+/+, CD5–/–, CD5WT, and CD5{Delta}458-461 mice following immunization with MOG35–55 peptide. As we reported previously, CD5–/– mice exhibit a delayed onset and decreased severity of EAE (Fig. 2). Reconstitution of CD5–/– mice with CD5WT transgene restored the disease phenotype to that of CD5+/+ mice. Most remarkable is that CD5{Delta}458-461 mice are almost completely resistant to the development of EAE, and, in fact, disease severity in these mutant mice is significantly lower than in CD5–/– mice (Fig. 2A). The cumulative disease index and maximum clinical score is CD5WT = CD5+/+>CD5–/–>CD5{Delta}458-461 mice (Fig. 2, B and C). Although incidence of disease is >90% in the CD5+/+, CD5WT, and CD5–/– mice, only 20% (3 of 15) of the CD5{Delta}458-461 mice develop disease (Fig. 2D).


Figure 2
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  		FIGURE 2. CD5-CK2 binding/activation-deficient mice are resistant to EAE. A, Clinical course of MOG35–55-induced EAE in CD5+/+ (n = 18), CD5–/– (n = 16), CD5WT (n = 9), and CD5{Delta}458-461 (n = 13) mice. EAE was induced in mice following s.c. immunization with 150 µg of MOG35–55 as described in Materials and Methods. Results are the mean clinical score (bars, SEM). B, Mean cumulative index is the mean of the sum of the daily clinical scores observed between days 1 and 30. C, Mean maximum clinical score was calculated from the maximum clinical score of individual animals between days 1 and 30. D, Disease incidence represents the percentage of mice developing EAE. Mice with a clinical score ≥2 for two consecutive days are considered to have clinical disease. *, p < 0.05 with Student’s t test.

 
CD5{Delta}458-461 mice are responsive to MOG immunization but fail to maintain CD4+ T cells in the CNS

To begin defining the underlying mechanism of resistance of CD5-CK2 binding/activation-deficient (CD5{Delta}458-461) mice to EAE, we analyzed DLN and spinal cords for changes in numbers and activation state of CD4+ T cells at various time points during the acute phase of the disease and compared it with that of CD5+/+, CD5WT, and CD5–/– mice. The absolute number of DLN cells from each of the four CD5 genotypes varied between 0.5 and 0.7 x 106 per node at all time points analyzed and, therefore, is not significantly different from each other (data not shown). The proportion of CD4+ T cells and activated CD4+CD69+ T cells in the DLN of CD5+/+, CD5WT, and CD5{Delta}458-461 mice is also similar at all time points (Fig. 3A and data not shown). Only in the DLN of CD5–/– mice did we observe an early increase in CD4+ T cells and activated CD4+ T cells (CD4+CD69+; day 4) followed by a decrease (day 12) as described previously (Fig. 3A and data not shown) (14). The results indicate that initial T cell priming is not compromised in CD5-CK2 activation-deficient mice.


Figure 3
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  		FIGURE 3. Activated and infiltrating mononuclear cell populations in MOG-immunized mice during acute EAE. A, CD69+ TH cells in DLN of CD5+/+, CD5WT, CD5–/–, and CD5{Delta}458-461 mice. DLN were analyzed for changes in frequency of CD4+CD69+ T cells at various time points after s.c. immunization with MOG35–55 peptide representing early stages of acute phase of EAE. The graphs represent percent CD69+ ± SD T cells within CD4-gated lymphocytes population. B, CD4+ T cells in spinal cords of CD5+/+, CD5WT, CD5–/–, and CD5{Delta}458-461 mice on days 9–17 following immunization with MOG35–55 peptide. Results represent proportion of CD4+ T cells within the mononuclear cell (CD45+) gate. C, CD8+ and CD11b+ populations in spinal cords 12 days following immunization. Mononuclear cells were prepared from animals perfused with PBS as described in Materials and Methods.

 
In contrast with DLN, we observe differences in spinal cord CD4+ T cell populations between CD5{Delta}458-461 mice and CD5+/+, CD5WT and CD5–/– mice. On day 9 after induction of EAE, the frequency of CD4+ T cells in the spinal cords of the CD5{Delta}458-461 mice is similar to CD5+/+, CD5WT, and CD5–/– animals (9.9, 8.0, 9.2, and 7.0%, respectively). The proportion of the CD4+ T cells in spinal cords of CD5+/+, CD5WT, and CD5–/– mice, but not the spinal cords of the CD5{Delta}458-461 mice, increases sharply with time (Fig. 3B). Because CD5{Delta}458-461 mice cords do contain significant numbers of CD4+ T cells, we performed histological analysis to determine whether the pattern of infiltration is different from CD5WT mice. On day 17, the peak of the acute phase of the disease, CD5WT cords show that both the gray matter and myelin-rich white matter is infiltrated with inflammatory mononuclear cells. In contrast, CD5{Delta}458-461 mice contained mononuclear cells only in the gray matter (Fig. 4A). Staining of sections with Luxol fast blue reveal that white matter infiltration correlated with demyelination in the CD5WT cords; such demyelination is not observed in the CD5{Delta}458-461 mice (Fig. 4B). The proportion of CD8+ T cell infiltrates and activated macrophages/microglia (CD11bhigh) in spinal cords of all four groups of mice is comparable (Fig. 3C).


Figure 4
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  		FIGURE 4. Histology of spinal cord sections from MOG35–55 peptide-immunized CD5WT and CD5{Delta}458-461 mice. Serial sections of spinal cord obtained 17 days after immunization and stained with H&E (A) and Luxol fast blue (B). H&E-stained section shows infiltration of mononuclear cells in gray matter of both CD5WT and CD5{Delta}458-461 mice and white matter of CD5WT mice (arrow). Luxol fast blue stain shows areas of intact myelin (blue) and demyelination (pink). Arrows indicate demyelination in CD5WT cord, which includes sites associated with infiltration of mononuclear cells within myelin-containing white matter.

 
Two recent reports now establish that, in EAE, the CNS is a site for encephalitogenic T cell amplification following re-exposure to Ag as well as recruitment and activation of new T cell clones by a process known as epitope spreading (28, 29, 30). The lack of expansion of CD4+ T cells in spinal cords of CD5{Delta}458-461 mice may reflect an inability of cells to proliferate in the inflamed tissue following re-exposure to CNS Ags (19, 31, 32). To address this possibility, we injected CD5WT and CD5{Delta}458-461 mice with BrdU 12 h before sacrifice on day 17 and assessed BrdU incorporation as a measure of cells in cycle. Although the numbers of CD4+ T cells are lower in CD5{Delta}458-461 cords than in CD5WT, a greater proportion of them incorporated BrdU (Fig. 5A). The greater frequency of BrdU+ cells may reflect differences in peripheral activation followed by recruitment into the CNS rather than in situ proliferation. However, we do not believe this is the case, because by day 17, DLN in both groups of mice contained very small percentage of CD4+ T cells that were BrdU+ (Fig. 5B). The proportion of CD8+ T cells in cycle is comparable in CD5{Delta}458-461 mice and CD5WT mice. The low frequency of CD4+ T cells in the spinal cords of CD5{Delta}458-461 mice in the presence of greater proliferation suggests a lower capacity to persist rather than an inability to respond to Ag in the CNS.


Figure 5
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  		FIGURE 5. Spinal cords of MOG35–55 peptide immunized CD5{Delta}458-461 mice contain greater frequency of cycling (BrdU+) CD4+ T cells. To determine the proportion of cycling CD4+ and CD8+ T cells in spinal cord (A) and DLN (B), MOG-immunized CD5WT and CD5{Delta}458-461 mice were injected with 1.5 mg of BrdU i.p. 12 h before sacrifice on day 17, and BrdU incorporation was determined by flow cytometry. Dot plots represent CD4 and CD8 staining of cells within the mononuclear cell gate. Histograms represent BrdU incorporation in CD4+ or CD8+ gated cells. Data are representative of two to three experiments.

 
Altered effector TH populations in DLN and spinal cords of CD5{Delta}458-461 mice

The presence of infiltrating of CD4+ T cells in the CNS at day 9 seen in CD5{Delta}458-461 mice is similar to that observed in IL-23 p19–/– mice immunized with MOG35–55 peptide (Fig. 3C) (3, 4, 33). Those recent studies led to the discovery of a population IL-17-expressing TH cells, now described as TH17 cells, as essential for the immunopathogenesis in EAE. The TH17 cells are distinct from TH1 cells that express IFN-{gamma} (34). To address the possibility that resistance of CD5{Delta}458-461 mice to EAE may be associated with altered populations of TH effector cells, we analyzed DLN for the presence of T cell populations expressing IFN-{gamma} (TH1) and IL-17 (TH17) on days 4, 9, and 17 of acute EAE. On day 4, the DLN from CD5{Delta}458-461 mice contain a greater frequency of CD4+ T cells that express IFN-{gamma} or IL-17 than from CD5+/+ and CD5WT mice; whereas only IFN-{gamma}-expressing CD4+ T cells are elevated in the CD5–/– mice (Fig. 6). A small but detectable population of cells that coexpress both IFN-{gamma} and IL-17 (THIFN-{gamma}+IL-17+) is detected in DLN of CD5{Delta}458-461 and CD5–/– mice but not in DLN of CD5+/+ or CD5WT mice. Because the absolute number of DLN cells is similar in each of the four groups of mice, it indicates that, early in the immune response, a greater number of effector cells are generated in mice unable to activate CK2 through CD5. Analysis of day 9 DLN reveals that the proportion of TH17 cells is mostly similar in all groups of mice. In contrast, the frequencies of TH1 (IFN-{gamma}+) cells is at least 6-fold lower in CD5{Delta}458-461 mice than in CD5+/+, CD5WT, and CD5–/– mice (Fig. 6). Interestingly, the population of THIFN-{gamma}+IL-17+ is ~5-fold lower in DLN of CD5{Delta}458-461 mice than in DLN of CD5+/+ and CD5WT mice. The levels of this dual cytokine-expressing TH cells in DLN of CD5–/– mice is intermediate between that of CD5+/+ and CD5{Delta}458-461 mice; correlating with disease severity (Fig. 2A). The frequency of effector TH cell populations in DLN, especially on day 9, is greater than one would expect if only MOG-reactive T cells are activated. Because we use CFA to prepare the immunogen, we believe a large proportion of the reactive T cells are in response to mycobacterial Ags.


Figure 6
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  		FIGURE 6. Populations of effector TH cells in DLN and spinal cords of MOG35–55-peptide immunized mice. Intracellular staining of CD4+ gated T cells for IFN-{gamma} and IL-17 in DLN (days 4 and 9) and spinal cord mononuclear cells (days 9 and 17) from immunized CD5+/+, CD5WT, CD5–/–, and CD5{Delta}458-461 mice. An initial lymphocyte gate was used for all analyses. The numbers in each quadrant for DLN (days 4 and 9) and spinal cord (day 9) represent the frequency of CD4+ T cells expressing IFN-{gamma} and/or IL-17. For day 17 spinal cord, the unbracketed numbers represent normalized values to take into consideration the differences in absolute numbers of CD4+ T cells between the four groups of mice. The normalized numbers are the frequency of cytokine expressing CD4+ T cells within the lymphocyte gate. The bracketed numbers represent nonnormalized values. Each data point represents a pool of three to eight mice from two to three experiments.

 
We next assessed the TH effector cell populations in spinal cord on day 9 (before the onset of clinical symptoms) and on day 17 (peak of acute EAE). On day 9, TH subsets in CD5+/+ and CD5WT mice are similar (Fig. 6). In contrast, cords of CD5–/– mice and CD5{Delta}458-461 mice contain fewer dual cytokine-producing THIFN-{gamma}+IL-17+ cells. The cords of CD5{Delta}458-461 mice also contain fewer TH1 cells than any of the other three groups of mice. By day 17 following MOG-peptide immunization, all of the CD5+/+, CD5WT, and CD5–/– mice, but none of CD5{Delta}458-461 mice, exhibited symptoms of clinical EAE. At this time point, we observe significantly fewer TH1, TH17 and THIFN-{gamma}+IL-17+ cells in cords of CD5{Delta}458-461 mice, compared with the other three CD5 genotypes (Fig. 6). CD5–/– mice, which exhibit lower disease severity than CD5+/+ and CD5WT mice, also contain lower proportion of THIFN-{gamma}+IL-17+ cells, albeit higher than that in CD5{Delta}458-461 mice. Overall, the results show that the resistance of CD5-CK2 binding/activation-deficient mice to EAE is associated with decrease in generation and/or persistence of TH effector cells. An intriguing finding suggested by our results is that, in addition to TH17 cells (IFN-{gamma}–), the proportion of THIFN-{gamma}+IL-17+ cells closely correlates with severity of EAE, an aspect that will be addressed further (see Discussion).

CD5-mediated activation of CK2 regulates the T cell activation in a primary response

The pseudo ITAM domain of CD5 that includes Y429 and Y441 is considered to be involved primarily with the inhibitory activity of CD5 (10, 11, 12). Because this domain is intact in CD5{Delta}458-461 mice, we tested the prediction that T cells from these mice will respond to TCR stimulation to the same extent as T cells expressing CD5WT. We assayed T cell response by assessing BrdU incorporation after in vitro anti-CD3 stimulation. Surprisingly, CD4+ and CD8+ T cells from CD5{Delta}458-461 had a frequency of BrdU+ cells comparable to that of T cells from CD5–/– mice, which are both significantly higher than the frequency of BrdU+ T cells from CD5WT and CD5+/+ mice (Fig. 7A). These data indicate that greater numbers of T cells from CD5{Delta}458-461 mice enter into cell cycle after activation but do not address the possibility of S-phase arrest, a mechanism that would explain the decrease in amount of CD4+ T cells observed in the CNS of MOG-immunized CD5{Delta}458-461 mice. To address this possibility, we assessed cell division in CFSE-labeled T cells from CD5+/+, CD5WT, CD5–/– and CD5{Delta}458-461 mice after anti-CD3 stimulation. The results show that a greater frequency of CD4+ and CD8+ T cells from CD5–/– and CD5{Delta}458-461 mice have undergone at least one cell division, compared with that of T cells from CD5+/+ and CD5WT mice (Fig. 7B). Despite this fact, a greater proportion of CD4+ and CD8+ T cells from CD5+/+ and CD5WT show evidence of five or more divisions than that from CD5–/– and CD5{Delta}458-461 mice. In general, CD8+ T cells respond more vigorously to anti-CD3 stimulation than CD4+ T cells from all four CD5-genotype mice. We also determined the kinetics of T cell proliferation over 96 h of stimulation by [3H]thymidine incorporation. The results show that T cells from CD5–/– and CD5{Delta}458-461 mice initially hyperproliferate reaching a peak within 48 h and then decline in their ability to incorporate [3H]thymidine (Fig. 8A). In contrast, proliferation of T cells from CD5+/+ and CD5WT mice reach maximal proliferation after 72 h of stimulation. By 96 h, few T cells from all the four different genotypes were proliferating. The results from CFSE dilution assay and kinetics of [3H]thymidine incorporation indicate that T cells lacking CD5 or lacking ability to bind and activate CK2 are hyperactive and have increased AICD. To directly assess AICD, we quantitated cells poised for apoptosis by evaluating mitochondrial depolarization using the dye DiOC6. After 24 h of anti-CD3 stimulation, a greater proportion of CD4+ T cells from both CD5–/– and CD5{Delta}458-461 mice are undergoing early stages of apoptosis (DiOC6low), compared with that from CD5+/+ and CD5WT mice (Fig. 8B). A similar trend is also observed within the CD8+ T cell population. Overall, these data indicate that T cells deficient for CD5-dependent CK2 activation (1) are hyperproliferative and (2) have decreased ability to respond to prolonged stimulation.


Figure 7
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  		FIGURE 7. CD5-CK2 binding/activation-deficient T cell hyperproliferate following in vitro stimulation. A, Frequency of CD4+ and CD8+ T cells incorporating BrdU at 72 h of anti-CD3 stimulation (1 µg/ml). Cells were pulse labeled with BrdU for the last 90 min before analysis. B, Number of cell divisions was measured by CFSE dilution assay after 72 h of anti-CD3 stimulation (1 µg/ml). The frequency of cells in each CFSE dilution peak is given.

 

Figure 8
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  		FIGURE 8. T cells from CD5-CK2 binding/activation-deficient mice and CD5 null mice have decreased viability after prolonged anti-CD3 stimulation. For these assays, spleen cells were obtained from CD5+/+, CD5WT, CD5–/–, and CD5{Delta}458-461 mice. A, [3H]Thymidine incorporation was determined at 24, 48, 72, and 96 h of anti-CD3 stimulation (1 µg/ml). [3H]Thymidine was added 18 h before analysis. B, Percentage of apoptotic (DiOC6low) CD4+ and CD8+ gated cells after 24 of anti-CD3 stimulation. Data are representative of three experiments.

 
CD5-mediated activation of CK2 regulates the persistence of T cells after restimulation

To recapitulate the consequence of Ag re-exposure on MOG-reactive T cells in the CNS, we next examined the role of CD5-dependent CK2 activation pathway in regulating T cell recall responses. We first assayed BrdU incorporation in previously activated and rested T cells from CD5+/+, CD5WT, CD5–/–, and CD5{Delta}458-461 following restimulation with anti-CD3. The results show that significantly fewer CD4+ and CD8+ T cells from CD5{Delta}458-461 mice incorporated BrdU than that from CD5+/+, CD5WT, and CD5–/– T cells (Fig. 9A). To test whether T cells expressing CD5 lacking the ability to bind/activate CK2 are more susceptible to AICD following restimulation, we assessed apoptosis by measuring mitochondrial depolarization using DiOC6. We find that a greater proportion of CD4+ and CD8+ T cells from CD5{Delta}458-461 than CD5+/+, CD5WT, and CD5–/– mice undergo apoptosis upon restimulation (Fig. 9B). These results show that CD5{Delta}458-461 T cells exhibit a low threshold for the development of anergy and are also more susceptible to AICD.


Figure 9
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  		FIGURE 9. T cells from CD5-CK2 binding/activation-deficient mice exhibit low threshold for development of nonresponsiveness and enhanced AICD following restimulation with anti-CD3. T cells from CD5+/+, CD5WT, CD5–/–, and CD5{Delta}458-461 mice were initially stimulated with 1 µg/ml anti-CD3 for 24 h and rested for 72 h before restimulation with anti-CD3 for 48 h (1 µg/ml). A, Frequency of CD4+ and CD8+ cells in cycle (BrdU+) (see Fig. 7 legend and Materials and Methods). B, Percentage of apoptotic (DiOC6low) CD4+ and CD8+ cells. Data are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We reported recently that EAE was less severe in CD5–/– mice, compared with CD5+/+ mice (14). This was due to an enhanced AICD of CD5–/– T cells leading to the discovery that CD5 had a prosurvival role in activated T cells. We predicted that CD5-dependent CK2 activation was the mechanism by which CD5 promoted survival. Consistent with our prediction, we report here that CD5-CK2 binding/activation-deficient mice, CD5{Delta}458-461, are very resistant to MOG-induced EAE (Fig. 2). An unexpected finding was that both disease severity and incidence of EAE in CD5{Delta}458-461 mice was also significantly lower than CD5–/– mice. The lower disease penetrance may reflect differences in TCR activation thresholds between T cells lacking only the CD5-dependent CK2 activation pathway and T cells with no CD5 expression. CD5–/– T cells lack both ITIM-associated and CK2 binding domain-associated regulatory pathways resulting in lower threshold for activation than CD5{Delta}458-461 T cells. In fact, immunization of CD5–/– mice with MOG35–55 peptide leads to up-regulation of CD69 on greater number of DLN T cells than in CD5WT and CD5{Delta}458-461 mice (Fig. 3A). Because CD5 is involved in thymic selection, another possibility is that the MOG-specific repertoire in CD5{Delta}458-461 mice is altered (9, 10, 35). The fact that MOG immunization in CD5{Delta}458-461 mice leads to activated T cells in DLN, and differentiation into all the effector TH populations seen in CD5+/+ and CD5WT mice argues that altered repertoire cannot be the primary explanation. Treg cells play an important role in controlling encephalitogenic T cell activity and can contribute to differences in disease susceptibility and/or severity (36). We found no differences in numbers of CD4+CD25+ Treg cells between CD5+/+, CD5WT, CD5–/–, and CD5{Delta}458-461 mice (data not shown); however, additional functional experiments need to be performed to address this question. One might suggest that enhanced resistance of CD5{Delta}458-461 mice to EAE is a transgene integration site-induced artifact. We do not believe this is the case, as we have just generated knock-in CD5{Delta}458-461 mice in which the endogenous CD5 gene was altered by gene targeting. In this model, where integration artifacts are not an issue, severity and incidence of EAE recapitulates disease progression in the transgenic CD5{Delta}458-461 mice (R. C. Axtell and C. Raman, unpublished observation).

CK2 activation promotes survival by directly inhibiting proapoptotic pathways as well as by enhancing prosurvival signaling cascades (23, 24, 37, 38, 39). We therefore predicted that resistance of CD5{Delta}458-461 mice to EAE will be associated with decrease and/or absence of infiltrating cells in the spinal cord. However, our results revealed that this was not the case. Spinal cords of CD5{Delta}458-461 mice had substantial infiltrating mononuclear cells localized in the gray matter with similar numbers of CD4+ T cells early in the disease (day 9), compared with CD5WT mice (Figs. 3B and4A). However, CD4+ T cell numbers in spinal cords of CD5+/+, CD5WT, and CD5–/– mice increased as disease progressed, compared with CD5{Delta}458-461 mice, where significant clinical disease was absent. A similar phenomenon was reported in IL-23p19–/– mice, where resistance to EAE occurred in the presence of T cell infiltration in the CNS (33). A subsequent report by the same group revealed that IL-23 was necessary for the generation of IL-17-expressing T cells now called TH17, and this population was absent in spinal cords of mice resistant to EAE (3). The TH17 cells are now considered to be the critical effector population in the pathogenesis of EAE and collagen-induced arthritis (3, 4, 40). Recent reports define the TH17 population as a subset that differentiates directly from naive T cells (Tn) and expresses IL-17 but not IFN-{gamma} (41, 42, 43). The generation TH17 cells require TGF-beta and IL-6 but not IL-23 as originally believed. However, IL-23 may play an important role in the persistence of TH17 cells. Early in the acute phase of EAE (day 9), we observed that the frequency of total IL-17-producing CD4+ T cells in DLN and spinal cords was significantly lower in CD5{Delta}458-461 mice than CD5+/+ and CD5WT mice; however, this was limited primarily to a TH population that coexpressed both IL-17 and IFN-{gamma} and not the TH17 subset (Fig. 6). We also observed a lower frequency of TH1 cells (IFN-{gamma}) in DLN and spinal cords of CD5{Delta}458-461 mice. Early in the immune response to MOG, the DLN, but not spinal cords, of CD5–/– contained IL-17-expressing TH cells to the same level as CD5+/+ and CD5WT mice. Currently, we can speculate only that this contributes to the delayed onset of EAE in CD5–/– mice observed in this study and in previous studies (14). By day 17, which represents the peak of acute EAE in CD5WT-expressing mice, the spinal cords of CD5{Delta}458-461 mice contained fewer TH1, THIFN-{gamma}+IL-17+, and TH17 cells than CD5+/+, CD5WT, and CD5–/– mice. Because the frequency of cycling CD4+ cells was greater in CD5{Delta}458-461 cords, we believe the lower numbers of these TH cell subsets in CD5{Delta}458-461 mice represents a decreased ability to persist rather than failure to proliferate (Fig. 5).

The IFN-{gamma}+IL17+ TH cells probably represent a population distinct from TH17 cells and were first identified in synovial cells obtained patients with Lyme arthritis (44). This population of TH has also been observed in vitro CD4+ T cell polarization experiments in which IL-23 is added to the culture (45) and proteolipid protein peptide-stimulated CD4+ T cell cultures obtained from DLN of proteolipid protein peptide-primed mice (3). However, its potential importance in pathogenesis has not been considered. The TH17 cells, thought to be the pathogenic population in EAE, develop directly from Tn cells and represent a distinct lineage of Th cells whose differentiation is independent of the TH1 subset (4, 45). The generation TH17 cells from Tn cells is dependent on TGF-beta and IL-6 and does not require T-bet or STAT4 (41, 42, 43), two transcription factors necessary for generation of TH1 T cells. Remarkably, both T-bet- and STAT4-deficient mice are resistant to EAE (6, 46), indicating that pathogenic T cells in EAE are derived from the initial stages of TH1 differentiation which, when exposed to appropriate stimuli, including TGF-beta and IL-6, express IL-17. Our finding that resistance of CD5{Delta}458-461 mice to EAE is associated primarily with decrease in IFN-{gamma}+/IL-17+ CD4+ T cells and, to a lesser extent, to TH17 T cells is consistent with this paradigm. It is unclear whether the IFN-{gamma}+/IL17+ T cells differentiate from IFN-{gamma}+/IL17– T cells, which are also reduced in CD5{Delta}458-461 mice; this is a subject of ongoing investigation.

The pseudo-ITAM/ITIM motif in CD5 cytoplasmic tail is considered to be necessary for its ability to negatively regulate T cell activation (10, 11, 12, 13). Our studies indicate that the CD5-CK2 pathway is involved in regulating survival (Figs. 8 and 9). T cells in CD5–/– mice lack both inhibitory and prosurvival activity of CD5 that may compensate for each other in response to Ag stimulation. This property could explain the intermediate phenotype of CD5–/– mice in susceptibility to EAE, compared with CD5+/+, CD5WT, and CD5{Delta}458-461 mice. By this paradigm, the lack of increase in CD4+ T cell numbers in cords of CD5{Delta}458-461 mice, compared with CD5+/+, CD5WT, and CD5–/– mice can be explained by the loss of CD5-dependent survival signals in the presence of normal CD5-inhibitory activity (Fig. 3B). However, the presence of greater proportion of different TH effector cells in DLN early in the immune response (day 4) and the results from the BrdU pulse-labeling experiment indicating that CD4+ T cells in cords of CD5{Delta}458-461 hyperproliferate provide the first indication that the CD5-dependent CK2 activation pathway may also be involved in the negative regulation of T cell activation and differentiation (Figs. 5 and 6). Results from in vitro stimulation experiments support this finding (Figs. 7 and 8). Although CD8+ T cells from CD5{Delta}458-461 mice hyperproliferate in vitro, we did not observe enhanced BrdU incorporation within the CD8+ T cell population in spinal cords. The absence of any difference in up-regulation of CD69 between CD4+ T cells from CD5+/+, CD5WT, and CD5{Delta}458-461 mice indicate that negative regulatory activity mediated by CD5-dependent CK2 activation targets pathways distal to TCR/CD3 signaling (Fig. 3A). The CD5-ITIM-associated pathway is likely to be involved in attenuating TCR-proximal signaling pathways (12). We now propose that CD5-ITIM-dependent and CD5-CK2 activation-dependent signals cooperatively regulate T cell proliferation and differentiation. Mice carrying targeted mutation of the ITIM domain will be needed to conclusively test this model.

Following MOG immunization, CD4+ T cells in CD5{Delta}458-461 mice are primed in DLN, infiltrate the CNS, and enter into cell cycle equal to or with greater efficiency than that in CD5WT mice. This leaves open the question of mechanism of resistance of CD5{Delta}458-461 mice to EAE. In this disease model, naive CD4+ T cells, after being primed in secondary lymphoid tissues migrate to the CNS where they are re-exposed to Ag presented by microglia and dendritic cells in the perivascular space (28, 30). The reencounter of Ag leads to local amplification of encephalitogenic T cells, damage to nervous tissue, expression of new Ags followed by recruitment of other T cell clones by a process known as epitope spreading (29). Encephalitogenic T cells that have poor ability to persist or those easily induced to become nonresponsive will fail to induce the pathogenic cascade in the CNS (47, 48). In experiments that, to some extent, recapitulate responses following reexposure to TCR stimulation, we observed that both CD4+ and CD8+ T cells from CD5{Delta}458-461 mice readily develop nonresponsiveness and exhibit increased sensitivity to AICD upon restimulation than T cells from CD5+/+, CD5WT and CD5–/– mice (Fig. 9). Remarkably, Tn completely lacking CD5 exhibit hyperproliferation and enhanced AICD following stimulation but not during restimulation. Although, Tn cells expressing CD5 selectively unable to bind/activate CK2 also exhibit hyperproliferation and enhanced AICD during primary simulation, and when restimulated, they rapidly develop unresponsiveness and die due to AICD. This difference could contribute to the difference in susceptibility to EAE between CD5–/– and CD5{Delta}458-461 mice. Overall, from these data, we suggest that the mechanism underlying the decreased severity to EAE in CD5{Delta}458-461 mice was not due to the failure of CD4+ T cell priming in the secondary lymphoid organs, but rather due to low threshold for development of anergy on restimulation and a decrease in the ability of effector CD4+ T cells to persist.

Autoreactive T cells play a fundamental role in the development, disease severity, and perpetuation of MS and EAE. In the course of this study, we made two significant findings. First, we report that the CD5-dependent CK2 activation pathway is an important mechanism by which CD5 regulates both T cell activation and persistence in an inflammatory disease. Second, we have determined that IFN-{gamma}- and IL-17-coexpressing CD4+ T cells are likely to be play key role in the pathogenesis of EAE. Targeting strategies that alter the generation and or the persistence of TH-IFN-{gamma}+IL-17+ cells through manipulation the CD5-CK2 activation pathway may be a useful therapeutic approach for MS and other inflammatory autoimmune diseases (49, 50, 51).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institutes of Health Grants AG16221 (to C.R.), NS46032 (to S.R.B.), T32-AR07450-23 (to R.C.A.), and by the Lupus Research Institute (to C.R.). Back

2 Address correspondence and reprint requests to Dr. Chander Raman, Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, SHEL 305, 1530 Third Avenue South, Birmingham, AL 35294-0007. E-mail address: craman{at}uab.edu Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; TH1, infiltrating Th cell 1; MOG, myelin oligodendrocyte glycoprotein; DiOC6, dihexyloxacarbocyanine; AICD, activation-induced cell death; DLN, draining lymph node; Tn, naive T cell. Back

Received for publication January 17, 2006. Accepted for publication September 30, 2006.


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