| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
8.2 chain-derived peptides in the context of MHC. How this regulation affects the fate of target V8.2+ T lymphocytes in vivo that mediate experimental autoimmune encephalomyelitis has remained unknown. The present study using immunoscope and CFSE-labeling analysis demonstrates that the expansion of regulatory CD4 and CD8 T cells in vivo results in apoptotic depletion of the dominant, myelin basic protein-reactive V8.2+ T cells, but not subdominant V13+ T cells. The elimination of only activated T cells by this negative feedback mechanism preserves the remainder of the naive V8.2+ T cell repertoire and at the same time results in protection from disease. These studies are the first in clearly elucidating the fate of myelin basic protein-specific encephalitogenic T cells in vivo following regulation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The presence of regulatory T cells with the capability of controlling self-reactive T cells has been shown in various animal models of autoimmunity (2, 3, 4, 5, 6). The mechanisms of regulation and the fate of pathogenic lymphocytes in vivo are still not well understood. Several studies have shown that CD4+ T cells secreting TGF-, IL-4, or IL-10 can down-regulate or suppress a variety of autoimmune diseases including experimental autoimmune encephalomyelitis (EAE),3 murine inflammatory colitis, and diabetes in the nonobese diabetic mouse (7, 8, 9). A distinct population of T cells with regulatory/suppressor activity and CD4+/CD25+ surface markers exists in the naive repertoire of mice (2, 4). The exact mechanism(s) by which these T cells mediate their regulatory function in vivo remains to be clarified.
EAE is a prototypic CD4 T cell-mediated autoimmune disease induced by immunization with myelin Ags, for example myelin basic protein (MBP). It is characterized by inflammation and demyelination in the CNS, resulting in paralysis. EAE is considered to be an instructive model for the human demyelinating disease multiple sclerosis because they share many pathological and immune dysfunctions (10). A majority of MBP-primed potentially pathogenic CD4 T cells in H-2u mice recognize the immunodominant N-terminal peptide MBPAc1–9 and predominantly use the TCR V8.2 gene segment (11, 12). Although the regulation and function of individual cytokines are complex, most experimental observations are consistent with the idea that myelin Ag-reactive Th1 cells are encephalitogenic, whereas the Ag-reactive Th2 response is protective (9, 13, 14).
Generally, MBPAc1–9-induced EAE is monophasic in B10.PL mice, and spontaneous recovery is mediated by the combined action of physiologically primed regulatory CD4 and CD8 T cells (Treg) (15, 16, 17, 18, 19, 20, 21). The regulatory CD4+ T cells recognize a framework 3 region determinant from the V8.2 chain (B5 peptide, aa 76–101), while regulatory CD8+ T cells are reactive with a complementarity-determining region (CDR) 1/2 determinant (peptide 41–50). Both of these Treg populations are necessary: e.g., adoptive transfer of regulatory CD4 T cell clones into CD8+/+, but not into CD8-/- mice prevents disease (15, 16, 17, 18, 22). Expansion of Treg populations in vivo following immunization of H-2u mice with TCR peptides B5 or p41–50, with recombinant single chain TCR V8.2 proteins, with V8.2 plasmid DNA, or recombinant adenovirus vector expressing the V8.2 TCR protects mice from Ag-induced EAE (22, 23, 24, 25, 26, 27). Furthermore, mice depleted of Treg contract severe disease and do not recover normally (17, 20, 21).
Recently, we have found that a dominant pathogenic public T cell clone, which uses V8.2-J2.7 gene segments with a characteristic CDR3 length of 9 aa, expands in lymphoid organs and infiltrates CNS tissue during the course of disease, and is lost from these tissues during spontaneous recovery.4 There is a concomitant expansion of a dominant TCR peptide B5-reactive regulatory CD4 T cell clone bearing V14-J1.2 with a characteristic CDR3 length of 7 aa in each animal recovering from EAE (see below and unpublished data). In the present study, we have examined the molecular mechanisms by which regulatory T cells control dominant pathogenic V8.2-J2.7 T cells. We show in this study that the activation of these Treg interferes with the expansion of the dominant pathogenic MBPAc1–9-reactive V8.2-J2.7, but not V13-J2.5 T cells. Using CFSE-labeled MBP-reactive V8.2+ T cells, we demonstrate that pathogenic lymphocytes are primed normally, but are eliminated in vivo by regulatory T cells. Furthermore, this apoptotic depletion of V8.2+ T cells requires a type 1 priming of V14+ CD4 Treg and the presence of CD8 T cells. These findings are the first to provide a molecular mechanism by which regulatory T lymphocytes control MBP-reactive encephalitogenic T cells in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B10.PL and PL/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MBP-specific V8.2 TCR transgenic PL/J mice (28) were a kind gift from J. Lafaille, New York University Medical Center (New York, NY). All the mice were bred under specific pathogen-free conditions in our own colony at La Jolla Institute for Allergy and Immunology. Aged-matched female mice (from 6–14 wk of age) were used in all experiments.
Antibodies
All Abs were purchased from BD PharMingen (San Diego, CA). Anti-CD4 and anti-CD3 conjugated to either FITC or PE or CyChrome 5, anti-V8.2 FITC, anti-CD8 (clone 2.43) mAb, purified and azide free, were used for deleting CD8 T cell populations from PL/J mice.
Peptides
TCR peptides were synthesized by S. Horvath (Caltech, Pasadena, CA), as reported earlier (15). TCR V8.2 chain peptides correspond to the sequence predominantly used in the MBP-specific response in B10.PL mice (11) and are as follows: TCR peptide, B1 (1–30L)-EAAVTQSPRNKVAVTGGKVTLSCNQTNNHNL; B5 (76–101)-LILELATPSQTSVYFCASGDAGGGYE.
Induction of EAE
Mice were immunized s.c. with 150 µg Ac1–9 (AcASQKRPSQR) emulsified in CFA. A total of 0.15 µg of pertussis toxin was injected in PBS 24 and 72 h later. Mice were observed for EAE daily. Disease was scored on a 5-point scale, as described earlier (15): 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, whole body paralysis; 5, death.
Immunoscope analysis
Repertoire analysis using immunoscope was performed utilizing a modified protocol to that described by Pannetier et al. (29). Total mRNA was extracted from immunized mice using the RNeasy mini kit from Qiagen (Valencia, CA). Spleen cells were cultured with the immunizing peptide (40 µg/ml) for 3 days before extracting the total mRNA. The total mRNA was subjected to cDNA synthesis with an oligo(dT16–18) primer. An equal amount of cDNA was subjected to PCR using TCR V14 (V14, 5'-ACGACCAATTCATCCTAAGCAC-3') and C (C145, 5'-CACTGATGTTCTGTGTGACA-3') primers; V8.2 (5'-CATTATTCATATGGTGCTGGC-3') and C primers; or V13 (5'-AGGCCTAAAGGAACTAACTCCCAC-3') and C primers. After runoff extensions with 12 J fluoresceinated primers, the runoff products were separated on the basis of their length on an automated 310 genetic analyzer ABI PRISM, with POP-4 polymer and a 5–47 cm x 50-µm capillary (PE Applied Biosystems, Foster City, CA). The results were analyzed using GeneScan 2.0 software. All the primers used in this study were purchased from Operon Technologies (Alameda, CA).
The relative index of stimulation (RIS) value was calculated as the area under the experimental peak divided by the area under the control peak found within a Gaussian distribution. Control peaks were obtained from either CFA-immunized animals or naive animals, which typically gave equivalent RIS values. An RIS value >3 for a particular peak is considered a specific expansion of T cells with that CDR3 length in this study.
CFSE labeling and cell transfer
Naive V8.2 TCR transgenic CD4 T cells were purified using MACS beads (Miltenyi Biotec, Auburn, CA) by the following protocol: splenocytes were subjected to positive selection by MACS beads against B220+ (B cells), CD8
+ (CD8+ T cells), and CD11b+ (macrophages) markers to negatively select the unbound CD4 T cells. After subsequent positive selection, populations of CD4 T cells were 90–95% pure. The purified CD4 T cells were stained for V8.2 TCR (100%) and other activation markers such as CD45RB (100% high) and CD69 (>5%) to check the purity and naive phenotype of the TCR transgenic T cells. The purified CD4+ TCR transgenic T cells were labeled with CFSE, as follows: briefly, 10 x 106 cells in PBS/0.1% FBS were labeled with 1 µl of 5 mM CFSE, prepared in DMSO, for 10 min at 37°C. Cells were washed thoroughly with cold PBS/0.1% FBS. A total of 5 x 106 CFSE-labeled cells was injected i.v. into normal PL/J mice. A total of 10 µg of Ac1–9 in 200 µl PBS was given i.v. to the CFSE-labeled cell recipients. Day 2, 3, or 5 after Ac1–9 challenge, spleen, lymph nodes, liver, and lungs were isolated and CD4+ T cells were purified from these organs using anti-CD4 MACS beads. Flow cytometry was performed to detect CFSE+ TCR transgenic T cells.
In experiments performed to determine the effect of induction of TCR peptide B5-reactive T cells on the proliferation of CFSE-labeled V8.2 TCR transgenic T cells, 10 µg TCR peptide B5 or control peptide B1 was given i.v. 1 wk before the transfer of CFSE-labeled cells into PL/J mice. To skew the deviation of TCR peptide B5-reactive T cells toward the secretion of IL-4, 6- to 8-wk-old PL/J were nasally instilled with 10 µg of the TCR peptide B5 or control peptide B1, as described earlier (30).
CD8 T cells were depleted from PL/J mice with 100 µg of CD8 mAb (clone 2.43)/mouse in PBS, given i.p. 1 day before the TCR peptide administration and on the day of TCR peptide administration. Typically, normal mouse IgG-treated PL/J mice have 16–18% CD8+ T cell population in their spleen. After the in vivo depletion regime, the level of CD8+ T cells drops down to 
3% in the spleen.
Flow cytometry
To detect CFSE-labeled TCR transgenic T cells, CD4+ T cells, purified from different organs of recipient mice, were labeled with anti-V8.2 PE (FL-2)-labeled mAb and anti-CD4 cytochrome (FL-3). CD4+- and V8.2+-gated T cells were analyzed for the CFSE label (FL-1).
To detect apoptotic TCR transgenic T cells from spleen and lymph nodes of the recipient mice, annexin V and 7-amino actinomycin D (7-AAD) staining were performed on the total spleen and lymph node cells using the annexin V-PE apoptosis kit-I (BD PharMingen), according to the instructions provided in the manual. Briefly, spleen cells and lymph node cells from TCR transgenic T cell recipient mice were labeled with annexin V-PE in the 1x annexin V-binding buffer for 10 min in the dark at room temperature. Cells were stained with vital dye 7-AAD and analyzed immediately on FACSCalibur (BD Biosciences, San Jose, CA). The 7-AAD cells were excluded, and the CFSE+ cells positive for annexin V staining were analyzed. The FACSCalibur used in all the experiments is equipped with an Argon lamp (488 nm) and a red diode (635 nm).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
8.2-J2.7 T cell clones
Using functional CD4 T cell lines, clones, and hybridomas reactive to TCR peptide B5, we have recently established that CD4 Treg predominantly use V14-J1.2 gene segments with a characteristic 7-aa CDR3 length (L. Madakamutil, E. Sercarz, and V. Kumar, mauscript in preparation). Using immunoscope analysis, we found that exactly the same V14-J1.2+ CD4 Treg population expands naturally during recovery from EAE and can be expanded in vivo in the absence of disease following injection of H-2u mice with the TCR peptide B5 (Fig. 1b).
|
8.2-J2.7 expansion through immunoscope analysis.4 We examined the fate of these pathogenic V8.2-J2.7 clones in vivo following expansion of CD4 Treg. Groups of TCR peptide B5- or B1 (control)-vaccinated mice were challenged after 1 wk with MBPAc1–9/CFA/PT (PT, pertussis toxin) for the induction of EAE. Mice in both groups were monitored daily for clinical signs of disease, and 15–20 days later, splenocytes were isolated from both groups of mice for immunoscope analysis. None of the animals in the B5-vaccinated group developed EAE, while all mice in the control (TCR peptide B1-vaccinated group) contracted disease, with clinical scores ranging from 1 to 4 (Fig. 1a).
Immunoscope analysis for the pathogenic V8.2-J2.7 T cells revealed that each animal in the control group had a significant expansion of this clone (individual relative index of stimulation = RIS, values ranging from 3 to 11, with a mean RIS value of 6.2). In contrast, mice in the B5-vaccinated group showed no significant expansion of the pathogenic V8.2+ T cell clone (individual RIS values ranged between 1 and 2.1, with a mean RIS value of 1.2) (Fig. 2, a and c).
|
8 MBP-reactive T cells, immunoscope analysis was performed on V13 TCR expansions. We have shown earlier that V13+ T cells represent a minor Ac1–9-reactive T cell population in B10.PL mice (11). Unlike the dominant public expansion of the V8.2-J2.7+ T cell clone seen in every B10.PL mouse with EAE, V13-J2.5 TCR expansions were frequently seen, although not in all animals. As shown in Fig. 2, d and f, these expansions remained similar in both the B1- and B5-vaccinated groups (RIS values ranged from 4 to 6). These data demonstrate that expansion of Treg following B5 vaccination results in the specific inhibition of the pathogenic V8.2-J2.7 T cell expansion, but does not affect the expansion of non-V8 T cells reactive to MBP. Furthermore, we found that the population of V8.2+ T cells as a whole was not affected by expansion of Treg. Thus, the percentage of V8.2+ T cells, as determined by staining with the anti-V8.2 mAb, F23.2, in the spleen and lymph nodes did not differ between the B1- and B5-vaccinated groups (data not shown) (15). Likewise, anti-V8.2 Ab-induced proliferation of specific T cells was similar in the two groups (15). These data indicate that only MBP-reactive V8.2+ T cells that are only a minor part of the total V8.2+ population are inhibited by this regulation, while nonactivated V8.2+ T cells are spared.
Loss of activated MBPAc1–9-reactive V8.2+ T cells in vivo following expansion of Treg
We then sought to determine whether expansion of Treg prevented priming of the pathogenic V8.2 T cell response, or whether pathogenic V8.2 T cells were primed normally, but eliminated. Naive CFSE-labeled pathogenic V8.2 T cells isolated from TCR transgenic mouse expressing the V8.2 TCR of the encephalitogenic MBP-reactive T cell clone were adoptively transferred into PL/J mice that had been previously vaccinated with either B1 or B5 TCR peptides (10 µg/mouse in PBS). MBPAc1–9 (10 µg/mouse)-induced proliferation of the CFSE-labeled V8.2 T cells was examined in these recipients. As shown in Fig. 3a (right panels), CFSE-labeled V8.2 T cells underwent several cell divisions in response to Ac1–9 in vivo. However, in B5-vaccinated recipients, although CFSE-labeled V8.2 T cells became activated and divided following Ac1–9-challenge, there was a marked reduction in their accumulation at each division. In contrast, in control mice, CFSE-labeled V8.2+ cells proliferated and accumulated normally following each division. This was also observed in the lymph nodes of recipient mice. Profiles of CFSE-labeled V8.2 T cells in recipients not challenged with Ac1–9 remained identical in both TCR peptide B1- and B5-vaccinated mice (Fig. 3a, left panel). In four independent experiments (see Fig. 3c), we have examined the total number of CFSE-labeled T cells recovered from spleens of mice in the B5-vaccinated group. There was a 81–89% reduction in the absolute number of labeled V8.2+ T cells recovered from animals in this group in comparison with mice in the PBS- or B1-vaccinated groups. Interestingly, expansion of Treg does not significantly eliminate the adoptively transferred V8.2+ T cells by day 2 after Ac1–9 immunization (Fig. 3d). Accordingly, the total number of CFSE-labeled T cells recovered from spleens of mice in the B5 (20056 CFSE+ cells/106 total spleen)- or the B1 (20168 CFSE+ cells/106 total spleen)-vaccinated group did not differ significantly, suggesting that V8.2 T cells are targeted for removal only after activation and multiple cell divisions in vivo. Collectively, these data suggest that the V8.2+ T cells are primed and activated normally in both groups of mice, but that following activation and cell divisions they are eventually cleared in vivo in mice in which Treg are expanded. In parallel, we also transferred purified, CFSE-labeled B cells into B1- or B5-vaccinated mice, and recipients were analyzed for changes in the number of labeled cells following expansion of Treg. As shown in Fig. 3b, there was no change in the number of B cells in any of the groups of mice. These data further confirm that only activated V8.2+ T cells are specifically eliminated by expansion of Treg populations.
|
8.2+ T cells in B5-vaccinated mice was caused by migration of CFSE-labeled T cells into other organs, their presence was sought in liver, lungs, and blood in both groups of mice. No significant changes in CFSE-labeled cells were found in these tissues in either group (data not shown). Furthermore, all activated T cells were not lost following regulation, as the expansion of Treg does not significantly influence a polyreactive allogenic T cell response of purified CFSE-labeled CD4 T cells from C57BL/6 mice when transferred into PL/J recipients (data not shown).
Elimination of V8.2+ T cells is dependent upon the presence of CD8 T cells
We have shown earlier that expansion of CD4 Treg following immunization of H-2u mice with TCR peptide B5 results in the recruitment of a CD8 Treg population reactive to a distinct determinant, p41–50, from the CDR1/2 region of the V8.2 chain (15, 18, 22, 24). Furthermore, we have shown that both CD4 and CD8 Treg populations are required for the regulation of EAE. To directly test whether the loss of CFSE-labeled V8.2 T cells is dependent upon the recruitment of an appropriate CD8 population, B5-vaccinated recipients were depleted of CD8 T cells using anti-CD8 mAb. CD8 T cell depletion in animals does not affect the priming of the V14-J1.2 CD4 Treg population (data not shown). However, data in Fig. 4 show that TCR peptide B5-vaccinated mice could not mediate clearance of the proliferating CFSE-labeled V8.2 T cells in the absence of CD8 T cells. These data reaffirm that both regulatory CD4 and CD8 T cell populations are required for the elimination of MBP-reactive V8.2+ T cells (22).
|
8.2+ T cells in vivo following appropriate priming/expansion of Treg population
As mentioned above, s.c. or i.v. challenge with TCR peptide B5 in PBS or in adjuvants results in the expansion of a predominantly Th1 CD4 Treg (V14-J1.2) population and subsequent protection of recipients from MBP-induced EAE. In contrast, nasal instillation of B5 results in the expansion of a predominantly Th2 CD4 Treg (V14-J1.2) population and exacerbation of Ag-induced EAE (30). We have determined whether elimination of CFSE-labeled MBP-reactive T cells is influenced by Th1- or Th2-like priming of CD4 Treg.
Mice were primed with TCR peptides, B1 or B5, in either a Th1 or Th2 mode. In both cases, expansion of B5-reactive CD4 Treg was similar, as determined by proliferative responses to B5 in spleens of primed mice (stimulation indices ranged from 12 to 17) (30). One week later, 5 x 106 CFSE-labeled MBPAc1–9-reactive V8.2+ T cells were adoptively transferred into these mice and were examined for MBPAc1–9-induced proliferation. As shown in Fig. 5, the transferred V8.2 T cells divided normally in mice in control groups vaccinated with B1 in a Th1 or a Th2 mode. As expected, elimination of V8.2+ T cells occurred in mice vaccinated with TCR peptide B5 in a Th1 mode. In contrast, CFSE-labeled V8.2 T cells divided and accumulated normally at each cell division in recipients nasally instilled with B5, which leads to Th2 priming. These data demonstrate that the loss of MBP-reactive V8.2+ T cells is dependent upon the secretion of proinflammatory cytokines by the CD4 Treg populations.
|
8.2 T cells in vivo was examined following expansion of Treg. During the analysis of the MBPAc1–9-induced proliferative response of adoptively transferred, CFSE-labeled V8.2 T cells, splenocytes from the recipients were also stained with vital dye 7-AAD and annexin V-PE. Cells stained with 7-AAD were excluded from the analysis, and the CFSE+ annexin V-positive cells were analyzed in mice vaccinated with TCR peptides, B1 or B5, in a Th1 or Th2 mode. As shown in Fig. 5a (right panel), a high percentage (average value, 68.7%; Fig. 5b) of proliferating CFSE-labeled V8.2+ T cells stained with annexin V, but only in recipients vaccinated with B5 in a Th1 mode. In contrast, in groups of mice either primed with B1 or primed in a Th2 mode with B5, only a smaller percentage (average value, 30.7%) of CFSE+ T cells showed annexin V staining (Fig. 5c, right panel). These data demonstrate that a central mechanism of regulation of MBP-reactive pathogenic V8.2+ T cells in vivo is the induction of apoptosis following their activation by the combined action of CD4 and CD8 Treg populations. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
14+ CD4 Treg in the presence of a CD8 population results in the apoptotic depletion of activated pathogenic V8.2+ T cells and the prevention of EAE. In addition to the requirement for activation of the target V8.2+ T cell, it is also necessary that the CD4 Treg population be raised in a Th1 environment.
It is clear from our earlier data (15, 17, 18, 22) as well as from studies presented in this work that regulation of the anti-MBP response following CD4 Treg expansion requires the presence of a CD8 T cell population. The requirement of CD8 T cells in this regulation is consistent with earlier observations that recovery as well as regulation of EAE are markedly reduced in CD8 knockout or CD8 T cell-depleted mice (15, 20, 21). The CD8 T cells are reactive to a V8.2 determinant from the CDR1/2 region, distinct from B5, and upon adoptive transfer can prevent EAE (18). Similar to the expansion of CD4 Treg, direct CD8 Treg expansion following TCR peptide 41–50 immunization in mice results in apoptotic elimination of activated V8.2+ T cells (L. Madakamutil and V. Kumar, unpublished data). It is of importance that the CD8 T cell line kills only activated V8.2+ T cells and not naive V8.2+ or non-V8.2+ T cells in vitro (6, 18). Similarly, TCR V8.2-reactive CD8 hybridomas have been shown to recognize only V8+ Th1 cells in vitro (31). We are currently investigating whether following activation, V8.2+ T cells become a target for recognition by the CD8 Treg owing to enhanced processing or presentation of V8-derived peptides in a class I context (32).
This study demonstrates that regulation of the anti-MBP response by Treg affects only T cells using TCR V8.2 gene segments, but not T cells using other TCR, for example, V13 gene segments. These data thus exclude the possibility that an activation marker alone is enough to make the target cell susceptible for elimination. This is also consistent with the observation with alloreactive T cells that are not apoptotically eliminated by Treg. We propose that specificity for the regulation could be provided by CD8 Treg recognition of V8-derived peptide/class I complexes on the surface of activated target MBP-reactive V8.2+ lymphocytes.
This study also suggests that there is a hierarchy in disease-causing potential among MBP-reactive T cells. One of the dominant, high avidity public T cell clones bears the TCR V8.2-J2.7 (33), and control of this Ac1–9-reactive T cell subset initially results in protection from EAE. This is consistent with our findings that the loss of this public clone from the CNS as well as from the periphery of B10.PL mice results in spontaneous recovery from EAE, although many private expansions of predominantly Th2 cells remain.4 Thus, the immune system effectively focuses on one or several, but not all self-reactive T cells as targets for regulation in vivo. It is also relevant to mention that these residual, in vivo primed MBP-reactive Th2 cells may slowly expand in the absence of the dominant disease-causing high avidity V8.2-J2.7 Th1 cells, resulting in an overall immune deviation of the anti-MBP response (23, 26, 34). Secretion of IL-4, IL-10, or TGF- by these Th2 cells in a bystander fashion may be able to suppress other T cells subsequently recruited via determinant spreading (13, 35). This could explain why this regulatory mechanism is more effective in preventing EAE than depletion of all V8.2+ T cells by anti-V8 Ab.
Our data further suggest that once the dominant CD4 T cell clone(s) is eliminated, the remaining self-reactive type 2 CD4 T cell repertoire need not be regulated and does not lead to clinical disease in H-2u mice. As mentioned earlier, these MBP-reactive Th2 cells are rather protective and may be required for efficient bystander regulation. However, in the relapsing and remitting model of EAE, for example in SJL/J mice, it is possible that the down-regulation of one or more dominant disease-causing clones may be inefficient, owing to either poor apoptotic depletion or insufficient bystander suppression, permitting newer recruits to cause new waves of disease. For example, in PLP-reactive T cell clone-induced EAE, the dominant encephalitogenic clone always remains at a predominant level in the CNS following relapses (36, 37). It remains to be shown whether if this clone were efficiently eliminated, new relapses might not occur.
From our earlier studies, it is clear that CD4 Treg provide necessary help for the recruitment/activation of CD8 Treg that are the ultimate effectors of regulation (15). The priming of the CD4 Treg must occur in a Th1 milieu for the effective regulation of the anti-MBP response and protection from EAE (23, 27, 30). If CD4 Treg are forced to deviate in a Th2 direction, disease is exacerbated because of the absence of natural regulation of MBP-reactive T cells (23, 27, 30). Data presented in this study clearly demonstrate that physiological or Th1 priming of CD4 Treg is required for the apoptotic elimination of activated V8.2+ T cells. We have recently used B10.PL-IFN-
-/- mice to show that secretion of IFN- by CD4 Treg is necessary for the regulation and apoptosis of V8.2+ T cells (B. Pedersen, L. Madakamutil, and V. Kumar, unpublished data). Consistent with these data, the failure to suppress expansion of activated MBP-reactive CD4 T cells in IFN- knockout mice leads to exacerbation of EAE (38, 39). Furthermore, it has been shown that myelin Ag-reactive effectors undergo apoptosis in vivo in the periphery as well as in the CNS (40, 41, 42). In addition to Ag-induced cell death (43), apoptosis of myelin-reactive T cells mediated by regulatory T cells is responsible for the recovery from Ag-induced EAE. We are currently investigating whether Fas/Fas ligand (FasL) or perforin pathways are involved in regulatory T cell-induced apoptosis of pathogenic lymphocytes. Initial observations with gld (FasL-/-) and lpr (FasR-/-) mice suggest the involvement of Fas/FasL interaction in the TCR peptide B5-mediated depletion of transferred CFSE-labeled V8.2+ T cells. Studies are ongoing to dissect the exact role of the Fas/FasL pathway during this TCR-mediated regulation process. Thus, our studies demonstrate that pathogenic V8.2 T cells are eliminated during the recovery phase of Ac1–9-mediated EAE by an active negative feedback mechanism involving CD4+ V14+ regulatory T cells in combination with certain CD8+ T cells. This conclusion is based on three key observations about the nature of this feedback regulatory mechanism that results in clearance of pathogenic T cells: 1) loss of the public V8.2-J2.7 T cell clone as determined by immunoscope analysis; 2) loss (81–89%) in the absolute number of CFSE-labeled V8.2+ T cells recovered following regulation; and 3) a significant increase in the annexin V staining of V8.2 T cells in vivo following TCR peptide B5 vaccination. These findings are also relevant to human disease in that it has recently been suggested that a failure in apoptosis of T lymphocytes may be involved in multiple sclerosis (44).
Regulatory T cells in other systems have been shown to bear a variety of activation markers and are present in naive animals (4). We have recently characterized TCR V gene segments used by these CD4 Treg and found that they are part of the naive repertoire and are represented by a public clone, V14-J1.2, with a 7-aa CDR3 length in both B10.PL and PL/J mice (L. Madakamutil, E. Sercarz, and V. Kumar, manuscript in preparation). In preliminary experiments, we have not been able to segregate the regulatory activity based on such activation markers as CD25, CD45R, or L-selectin (data not shown). Because CD4 Treg described in this work become activated in vivo and undergo proliferation, they must acquire activation markers in any case.
There is still much to be learned about several stages within this regulatory system, in particular the way in which the TCR determinants are processed and presented by APC and how the actual coup de grace is administered. These problems are being actively addressed. At present, the elucidation of the regulatory cells involved, their specificities, and the fate of target lymphocytes in vivo represent the most clearly defined mechanism of a regulatory feedback system yet described.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Vipin Kumar, Laboratory of Autoimmunty, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. E-mail address: vkumar{at}tpims.org
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; 7-AAD, 7-amino actinomycin D; CDR, complementarity-determining region; FasL, Fas ligand; MBP, myelin basic protein; RIS, relative index of stimulation; Treg, regulatory CD4 and CD8 T cell.
4 P. van den Elzen, E. Maverakis, D. Huffman, S. Wilson, V. Kumar, and E. Sercarz. A minority of the self-peptide-reactive T cell repertoire drives EAE. Submitted for publication.
Received for publication October 4, 2002. Accepted for publication January 7, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain controls murine EAE. J. Immunol. 154:1941.[Abstract]
)8.2 DNA vaccination results in immune deviation and protection from experimental autoimmune encephalomyelitis. Int. Immunol. 13:835.8.2 chain controls murine experimental autoimmune encephalomyelitis. J. Immunol. 154:1941.
8.2 T cell receptor in protection against experimental autoimmune encephalomyelitis using a live vaccinia virus vector. J. Immunol. 156:4940.[Abstract]
8.2 TCR is disrupted by coadministration with vectors expressing either IL-4 or IL-10. J. Immunol. In press.
8 TCR: a role of the Qa-1 molecule. Immunity 2:185.[Medline]
-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:123. gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156:5.[Abstract]
8.2+ cells from the central nervous system during recovery from experimental autoimmune encephalomyelitis induced by the passive transfer of V8.2+ encephalitogenic T cells. Eur. J. Immunol. 24:2609.[Medline]
This article has been cited by other articles:
|
|
 |
|
  T. R. F. Smith, X. Tang, I. Maricic, Z. Garcia, S. Fanchiang, and V. Kumar Dendritic Cells Use Endocytic Pathway for Cross-Priming Class Ib MHC-Restricted CD8{alpha}{alpha}+TCR{alpha}{beta}+ T Cells with Regulatory Properties J. Immunol., June 1, 2009; 182(11): 6959 - 6968. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. T. Madakamutil, I. Maricic, E. E. Sercarz, and V. Kumar Immunodominance in the TCR Repertoire of {alpha}TCR Peptide-Specific CD4+ Treg Population That Controls Experimental Autoimmune Encephalomyelitis J. Immunol., April 1, 2008; 180(7): 4577 - 4585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Tang, I. Maricic, and V. Kumar Anti-TCR Antibody Treatment Activates a Novel Population of Nonintestinal CD8{alpha}{alpha}+TCR{alpha}beta+ Regulatory T Cells and Prevents Experimental Autoimmune Encephalomyelitis J. Immunol., May 15, 2007; 178(10): 6043 - 6050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Fazilleau, C. Delarasse, I. Motta, S. Fillatreau, M.-L. Gougeon, P. Kourilsky, D. Pham-Dinh, and J. M. Kanellopoulos T Cell Repertoire Diversity Is Required for Relapses in Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis J. Immunol., April 15, 2007; 178(8): 4865 - 4875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zeiser, V. H. Nguyen, J.-Z. Hou, A. Beilhack, E. Zambricki, M. Buess, C. H. Contag, and R. S. Negrin Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft-versus-host disease Blood, March 1, 2007; 109(5): 2225 - 2233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Minguela, S. Pastor, W. Mi, J. A. Richardson, and E. S. Ward Feedback Regulation of Murine Autoimmunity via Dominant Anti-Inflammatory Effects of Interferon {gamma} J. Immunol., January 1, 2007; 178(1): 134 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Tang, I. Maricic, N. Purohit, B. Bakamjian, L. M. Reed-Loisel, T. Beeston, P. Jensen, and V. Kumar Regulation of Immunity by a Novel Population of Qa-1-Restricted CD8{alpha}{alpha}+TCR{alpha}beta+ T Cells J. Immunol., December 1, 2006; 177(11): 7645 - 7655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Schif-Zuck, G. Wildbaum, and N. Karin Coadministration of Plasmid DNA Constructs Encoding an Encephalitogenic Determinant and IL-10 Elicits Regulatory T Cell-Mediated Protective Immunity in the Central Nervous System J. Immunol., December 1, 2006; 177(11): 8241 - 8247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Tennakoon, R. S. Mehta, S. B. Ortega, V. Bhoj, M. K. Racke, and N. J. Karandikar Therapeutic Induction of Regulatory, Cytotoxic CD8+ T Cells in Multiple Sclerosis. J. Immunol., June 1, 2006; 176(11): 7119 - 7129. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E Fainardi, R Rizzo, L Melchiorri, M Castellazzi, E Paolino, M R Tola, E Granieri, and O R Baricordi Intrathecal synthesis of soluble HLA-G and HLA-I molecules are reciprocally associated to clinical and MRI activity in patients with multiple sclerosis Multiple Sclerosis, February 1, 2006; 12(1): 2 - 12. [Abstract] [PDF]
|
|
|
|
 |
|
 E. T. Samy, L. A. Parker, C. P. Sharp, and K. S.K. Tung Continuous control of autoimmune disease by antigen-dependent polyclonal CD4+CD25+ regulatory T cells in the regional lymph node J. Exp. Med., September 19, 2005; 202(6): 771 - 781. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Friese and L. Fugger Autoreactive CD8+ T cells in multiple sclerosis: a new target for therapy? Brain, August 1, 2005; 128(8): 1747 - 1763. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Yu, R. K. Gregg, J. J. Bell, J. S. Ellis, R. Divekar, H.-H. Lee, R. Jain, H. Waldner, J. C. Hardaway, M. Collins, et al. Specific T Regulatory Cells Display Broad Suppressive Functions against Experimental Allergic Encephalomyelitis upon Activation with Cognate Antigen J. Immunol., June 1, 2005; 174(11): 6772 - 6780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Herndon, P. M. Stuart, and T. A. Ferguson Peripheral Deletion of Antigen-Specific T Cells Leads to Long-Term Tolerance Mediated by CD8+ Cytotoxic Cells J. Immunol., April 1, 2005; 174(7): 4098 - 4104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-K. Kang, M. A. Michaels, B. R. Berner, and S. K. Datta Very Low-Dose Tolerance with Nucleosomal Peptides Controls Lupus and Induces Potent Regulatory T Cell Subsets J. Immunol., March 15, 2005; 174(6): 3247 - 3255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gonthier, R. Llobera, J. Arnaud, and B. Rubin Self-Reactive T Cell Receptor-Reactive CD8+ T Cells Inhibit T Cell Lymphoma Growth In Vivo J. Immunol., December 1, 2004; 173(11): 7062 - 7069. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. La Cava, F. M. Ebling, and B. H. Hahn Ig-Reactive CD4+CD25+ T Cells from Tolerized (New Zealand Black x New Zealand White)F1 Mice Suppress In Vitro Production of Antibodies to DNA J. Immunol., September 1, 2004; 173(5): 3542 - 3548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-C. Chen, S. P. Cobbold, P. J. Fairchild, and H. Waldmann Generation of Anergic and Regulatory T Cells following Prolonged Exposure to a Harmless Antigen J. Immunol., May 15, 2004; 172(10): 5900 - 5907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Prakken, R. Samodal, T. D. Le, F. Giannoni, G. P. Yung, J. Scavulli, D. Amox, S. Roord, I. de Kleer, D. Bonnin, et al. Epitope-specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis PNAS, March 23, 2004; 101(12): 4228 - 4233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Martin, A. Banz, B. Bienvenu, C. Cordier, N. Dautigny, C. Becourt, and B. Lucas Suppression of CD4+ T Lymphocyte Effector Functions by CD4+CD25+ Cells In Vivo J. Immunol., March 15, 2004; 172(6): 3391 - 3398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Winter, E. Fiebiger, P. Meraner, H. Auer, C. Brna, R. Strohal, F. Trautinger, R. Knobler, G. F. Fischer, G. Stingl, et al. Definition of TCR Epitopes for CTL-Mediated Attack of Cutaneous T Cell Lymphoma J. Immunol., September 1, 2003; 171(5): 2714 - 2724. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
