Tyrosine-mediated inhibitory signals contribute to CTLA-4 function in vivo
Lou Ann Yi1,
Soheila Hajialiasgar1 and
Ellen Chuang1,2
1 Division of Hematology and Medical Oncology, Department of Medicine and 2 Graduate Program in Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA
Correspondence to: E. Chuang; E-mail: elc2007{at}med.cornell.edu
Transmitting editor: K. Murphy
 |
Abstract
|
---|
The ability of CTLA-4 to inhibit T cell activation may be either negatively or positively regulated by a critical tyrosine at position 201 (Y201) within the CTLA-4 cytoplasmic domain. By binding to the clathrin-associated adaptor complex AP-2 and inducing endocytosis, Y201 reduces the amount of CTLA-4 on the cell surface, thereby down-regulating CTLA-4 inhibitory function. Alternatively, Y201 may function to transmit CTLA-4 inhibitory signals, perhaps through binding to intracellular proteins that oppose TCR- and/or CD28-induced signal transduction. Results from studies performed in vitro have cast doubt on whether this second mechanism contributes significantly to CTLA-4 function. In order to determine if a role existed for Y201 in mediating CTLA-4 inhibitory signaling in vivo, we studied lymphocyte activation and homeostasis in CTLA-4/ mice that were reconstituted with a transgenic CTLA-4 receptor in which Y201 was mutated to valine (Y201V/CTLA-4/). We found that despite augmented levels of CTLA-4 on the cell surface of T cells, Y201V/CTLA-4/ mice developed a lymphoproliferative syndrome characterized by lymphadenopathy and the accumulation of T cells that secreted IL-4. Mutant T cells exhibited increased cell division when treated with suboptimal doses of mitogenic stimuli in vitro. These results demonstrate that in addition to down-modulating CTLA-4 expression on the cell surface of T cells, the Y201 residue also functions to transmit CTLA-4 inhibitory signals in vivo. Elucidating the biochemical pathways downstream of Y201 will be important for a full understanding of the molecular basis for CTLA-4 function.
Keywords: co-stimulation, T cell homeostasis, Th1/Th2
 |
Introduction
|
---|
CTLA-4 is an inhibitory receptor on T cells that functions to down-regulate TCR- and/or CD28-induced T cell activation. CTLA-4 and CD28 share the same ligands, B7-1 (CD80) and B7-2 (CD86), but the avidity of CTLA-4 for these ligands is higher compared with CD28. At the simplest level, CTLA-4 may function by competing with CD28 for ligands. If so, the relative levels of CD28 and CTLA-4 on the surface of T cells would be expected to be an important determinant of whether CD28 co-stimulatory or CTLA-4 inhibitory effects predominate during an immune response. CD28 is constitutively expressed on T cells, whereas CTLA-4 gene expression requires T cell activation. The subcellular distribution of CTLA-4 protein is regulated by protein trafficking (1,2). Although CTLA-4 is present at low levels on the cell surface relative to CD28, it is present in abundant quantities within intracellular compartments. This is due to ligand-independent endocytosis of cell-surface CTLA-4 into intracellular vesicles. We and others have shown that endocytosis of CTLA-4 occurs via an internalization motif within the CTLA-4 cytoplasmic domain, which associates with the clathrin-associated adaptor protein complex AP-2 (36). Association with AP-2 is mediated by the CTLA-4 tyrosine 201 (Y201) residue and surrounding amino acid sequences. The interaction was shown to occur with the non-phosphorylated Y201 residue and was inhibited when Y201 underwent phosphorylation. Tyrosine phosphorylation and inhibition of endocytosis may be one mechanism by which a rapid increase in the amount of CTLA-4 receptor on the cell surface can be achieved. Despite containing a similar tyrosine-based motif, the homologous receptor CD28 does not bind to AP-2 and does not undergo endocytosis (7,8). Thus, in the simplest model of CTLA-4 function, the CTLA-4 extracellular domain, through competition for CD28 ligands, would be the major functional domain of CTLA-4, with the cytoplasmic domain serving primarily to regulate CTLA-4 cell-surface expression through Y201-mediated regulation of CTLA-4 trafficking.
Alternatively, the Y201 residue may function to transmit intracellular signals that inhibit T cell activation. Tyrosine 201 can associate with and become phosphorylated by the src kinases Lck and Fyn (4,5), and phosphorylation may lead to intracellular signaling events that result in T cell inhibition. The phosphorylated YVKM motif of CTLA-4 forms a consensus binding site for SH2 domain-containing proteins and CTLA-4 has been shown to bind to phosphatidylinositol 3-kinase (PI3K) (9) and the Src homology 2-containing tyrosine phosphatase (SHP)-2 (10). Consistent with a role for Y201 in mediating intracellular signal transduction, one group found that a Y201 mutant expressed in a T cell hybridoma line was partially defective for CTLA-4 function (11). However, evidence against a role for Y201 in mediating CTLA-4 inhibitory signaling was provided by other groups. In those studies, expression of a CTLA-4 Y201 mutant in cell lines or T cell clones did not impair the ability of CTLA-4 to inhibit TCR-induced T cell proliferation and IL-2 production in vitro (1214). Whether Y201 mediates CTLA-4 inhibitory signals in vivo is not known. However, our previous studies demonstrating that expression of the extracellular domain of CTLA-4 without the cytoplasmic tail was unable to completely rescue the CTLA-4/ phenotype suggested that specific amino acids within the cytoplasmic domain may indeed be important for conferring CTLA-4 inhibitory function in vivo (15).
Thus, although it is accepted that Y201 can regulate CTLA-4 cell-surface expression, the role of Y201 in mediating CTLA-4 inhibitory signals is not yet established, either in vitro or in vivo. In order to examine whether such a role for Y201 existed in vivo, we studied T cell activation and T cell homeostasis in CTLA-4/ mice that were reconstituted with a mutant CTLA-4 receptor in which the Y201 residue was changed to valine (15). We found that despite increased levels of CTLA-4 expression on the cell surface of T cells, older Y201V/CTLA-4/ mice developed an indolent lymphoproliferative syndrome characterized by enhanced IL-4 secretion and increased T cell division. These studies demonstrate that in vivo, Y201-associated inhibitory signals contribute to CTLA-4 function. Increased cell-surface expression that resulted from impaired endocytosis was unable to compensate completely for loss of these signals. Elucidation of the components of CTLA-4 Y201-associated intracellular pathways will be important for a complete understanding of the molecular basis for CTLA-4 inhibitory function.
 |
Methods
|
---|
Mice
CTLA-4+/ mice and CTLA-4 transgenic mice were obtained from Dr C. Thompson (University of Pennsylvania, Philadelphia, PA). Transgenic mice expressing either the full-length, unmutated CTLA-4 receptor (FL CTLA-4) or the mutant CTLA-4 Y201V receptor under the proximal Lck promoter and the CD2 enhancer (15) were bred onto the C57BL/6 background for a minimum of seven generations (Y201V/CTLA-4/ founder 1) or five generations (Y201V/CTLA-4/ founder 2 and FL/CTLA-4/). All mice used for these experiments were heterozygous for the transgene in that they were obtained from Tg+CTLA-4/ x CTLA-4+/ breedings. C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were kept under specific pathogen-free conditions, and utilized in agreement with the Institutional Animal Care and Use Committee according to NIH guidelines.
Antibodies
The hamster mAb specific for mouse CD3 (2C11), mouse CTLA-4 (4F10) and mouse CD28 (PV-1) were provided by Dr J. Bluestone (University of California at San Francisco, San Francisco, CA). PV-1 was also purchased from Southern Biotechnology Associates (Birmingham, AL). Hamster IgG was from ICN (Costa Mesa, CA). Phycoerythrin (PE)-conjugated anti-B220, PE-conjugated anti-CD25, PE-conjugated anti-CD62L, PE-conjugated anti-CD69, PE-conjugated anti-CD45RB, PE-conjugated anti-CTLA-4, FITC-conjugated anti-CD8a, CyChrome-conjugated anti-CD4, biotin-conjugated anti-CD25 and streptavidinPE were purchased from PharMingen (San Diego, CA).
Detection of CTLA-4 expression
Cell-surface and intracellular CTLA-4 expression was assessed as previously described (1). Briefly, cells were incubated with anti-TCRCyChrome and either anti-CTLA-4PE or a control hamster IgGPE on ice for 30 min for surface staining. For intracellular staining, cells were incubated with anti-TCRCyChrome, fixed with 4% paraformaldehyde, permeabilized with 0.3% saponin/PBS and stained with anti-CTLA-4PE on ice for 30 min. Stained cells were analyzed on an Epics XL flow cytometer (Beckman Coulter, Hialeah, FL).
Purification of CD4+ and CD4+CD25+ T cells
Lymph nodes (LN; axillary, brachial, inguinal and mesenteric) and spleens were harvested from mice, and single-cell suspensions made. For isolation of CD4+ T cells, cells were incubated with an antibody cocktail consisting of anti-CD45R, anti-CD11b, anti-CD8, anti TER119 and anti-Gr-1 (Stem Cell Technologies, Vancouver, BC, Canada). CD4+ cells were purified by negative selection according to the manufacturers protocol. Determination of purity was performed by flow cytometry. Typical preparations yielded 92% CD4+ cells.
For isolation of CD4+CD25+ cells, purified CD4+ T cells were incubated with CyChrome-conjugated anti-CD4 (5 µg/108 cells) and biotin-conjugated anti-CD25 (15 µg/108 cells) in PBS/5% FCS for 25 min at 4°C. Cells were washed with PBS/3% BSA, incubated with PE-conjugated streptavidin (15 µg/108 cells) for 10 min at 4°C and washed. CD4+CD25+ and CD4+CD25 populations were collected by sorting on an Altra Cell Sorter (Beckman Coulter). The purity of the CD4+CD25+ fraction was routinely >92%.
In vitro cell culture and stimulation
Cells were grown in DMEM supplemented with 10% FCS, 4 mM glutamine, 50 mM HEPES, 200 mM MEM non-essential amino acids solution, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µM 2-mercaptoethanol. For stimulation of bulk LN cells, soluble 145-2C11 (0.1 µg/ml) and PV-1 (1 µg/ml) were added for either 24 or 48 h.
For stimulation of CD4+ T cells, aldehyde/sulfate polystyrene latex microspheres of 4.8 ± 0.27 µm mean diameter (Interfacial Dynamics, Portland, OR) were resuspended at 1.0 x 107 beads/ml in PBS containing 1 µg/ml of anti-CD3 and 5 µg/ml of anti-CD28. Beads were incubated with rotation at 37°C for 2 h, washed with PBS and blocked in DMEM/FCS for 30 min. CD4+ T cells (3 x 106/ml) were cultured with antibody-coated beads at a 1:3 (cell:bead) ratio.
CFSE labeling was performed as described (16). Briefly, 10 x 106 CD4+ T cells were incubated in a 5 µM solution of CFSE (Molecular Probes, Eugene, OR) in PBS for 7 min at room temperature. Cells were washed and resuspended at 106 cells/ml. Then, 2 x 105 cells were plated onto 96-well round-bottom plates precoated with
CD3 (2 or 10 µg/ml) or with
CD3 (5 µg/ml) +
CD28 (5 µg/ml) for 5 days. Cells were analyzed on an Epics XL flow cytometer (Beckman Coulter).
For regulatory T cell assays, 35 x 104 CD4+CD25+ cells purified from either wild-type or Y201V/CTLA-4/ mice were cultured at a 1:1 ratio with CD4+CD25 wild-type T cells in DMEM/FCS in triplicate cultures. An aliquot of 1 µg/ml of anti-CD3 and 11.2 x 105 wild-type splenic antigen-presenting cells irradiated with 2000 rad were added. Cultures were incubated for 48 h at 37°/5% CO2 and pulsed with [3H]thymidine (1 µCi/well; NEN, Boston, MA) 8 h prior to harvest.
ELISA
Supernatants from in vitro stimulations were harvested at 24 h (IL-2 or IL-4) or 48 h (IL-4 or IFN-
) and cytokine ELISA performed using commercial mAb pairs (PharMingen) according to the manufacturers instructions.
Statistical analysis
Statistical comparisons between groups were performed using the Students t-test.
 |
Results
|
---|
The ability of CTLA-4 to inhibit CD28 activation may be proportional to its expression on the cell surface where it can sequester ligands from CD28. Because mutation of the Y201 residue of the CTLA-4 cytoplasmic domain prevents endocytosis of CTLA-4, one consequence of this mutation may be enhanced cell-surface expression and enhanced function. The expression of CTLA-4 in resting and activated T cells obtained from C57BL/6 and CTLA-4/ mice reconstituted with mutant (Y201V) and FL CTLA-4 transgenes is shown in Fig. 1. Lymph node T cells isolated from Y201V/CTLA-4/ mice expressed low, but detectable, amounts of the Y201V receptor, whereas expression of the FL transgenic receptor was minimally detected on T cells from FL/CTLA-4/ mice. As expected, no endogenous CTLA-4 was detected on freshly isolated cells from BL/6 mice. In vitro activation of LN cells led to augmentation of CTLA-4 cell-surface expression. In all experiments, higher levels of CTLA-4 could be detected on the surface of activated T cells from Y201V/CTLA-4/ mice than from C57BL/6 or FL/CTLA-4/ mice. In contrast, there were greater quantities of intracellular CTLA-4 in T cells from C57BL/6 mice compared with T cells from Y201V/CTLA-4/ mice. The FL CTLA-4 receptor was consistently expressed at lower surface levels than the endogenous CTLA-4 receptor or the CTLA-4 Y201V receptor.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1. Increased cell-surface expression of the Y201V CTLA-4 receptor on T cells. LN cells harvested from C57BL/6, FL/CTLA-4/ or Y201V/CTLA-4/ mice were stained with anti-TCRCyChrome and either anti-CTLA-4PE (open histograms) or a control hamster IgGPE (shaded histograms). For day 2 panels, LN cells were activated for 48 h with anti-CD3 (0.1 µg/ml) and anti-CD28 (1 µg/ml) and CTLA-4 surface and intracellular expression assessed. CTLA-4 expression was determined on electronically gated TCR+ cells. The annotated values represent the CTLA-4 MFI. Note that the MFI of the IgG control (not annotated) is different for surface versus intracellular stainings.
|
|
Mutation of CTLA-4 Y201, by increasing CTLA-4 cell-surface expression, may lead to increased inhibitory function in vivo, unless the Y201 residue was necessary for transmitting CTLA-4 downstream signals. Hence, Y201V transgenic mice might be expected to display an immunodeficient phenotype that was similar to other mouse models in which B7/CD28 interactions are interrupted or reduced, such as mice deficient in B7-1 and B7-2 (17), mice deficient for CD28 (18) or mice transgenic for CTLA-4Ig (19). However, in contrast to these other models, and in contrast to CTLA-4/ mice reconstituted with the FL transgene, reconstitution of CTLA-4/ mice with the Y201V transgene yielded a phenotype of lymphoproliferation. Beginning at
3 months of age, Y201V/CTLA-4/ mice started to accumulate increased numbers of LN cells (Fig. 2). As mice continued to age, grossly enlarged mesenteric nodes could be seen and the increase in LN cell numbers became more striking. Older mice accumulated 25 times greater LN cells compared with age-matched controls (Fig. 2, Y201V #1 versus C57BL/6 or FL, P = 0.0001). The number of LN cells in mice derived from a second founder (Y201V #2) was also significantly increased (Y201V #2 versus FL, P = 0.0003; versus C57BL/6, P = 0.007). For subsequent experiments, mice derived from the first Y201V founder were used.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2. Accumulation of Y201V/CTLA-4/ LN cells in mice with increasing age. Brachial, axillary, inguinal and mesenteric nodes were harvested from 2- to 7-month-old C57BL/6, FL/CTLA-4/ (FL) and Y201V/CTLA-4/ (Y201V #1) mice and the number of cells per animal determined. The results for a second Y201V founder is also shown (Y201V #2). P values were obtained using Students t-test. The bar denotes the mean of each group.
|
|
Correlation of T cell activation and memory cell markers with lymphadenopathy revealed that T cells derived from younger Y201V/CTLA-4/ mice in which lymphadenopathy was absent (2 months) or mild (3 months) exhibited a naive phenotype, with fewer cells expressing CD69 and CD25, and more cells expressing CD62L, compared with T cells from age-matched controls (Fig. 3). With increasing age and prominence of lymphadenopathy (6 months), a greater percentage of cells was found that expressed CD69, and had down-regulated CD62L (Fig. 3) and CD45RB (data not shown). The percentage of CD4+ cells expressing the activation marker CD25 was initially decreased and was not increased even when lymphadenopathy was prominent. This may be due to a requirement for CD28 signals for the maintenance of CD4+CD25+ T cell homeostasis (20). Activation markers on FL/CTLA-4/ T cells were similar to those on wild-type T cells (data not shown). Thus, the lymphadenopathy in Y201V/CTLA-4/ mice was characterized by an initial accumulation of cells having a naive phenotype; accumulation of activated or memory cells occurred later in the disease process.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 3. Sequential accumulation of naive and activated/memory T cells in Y201V/CTLA-4/ mice. LN harvested from 2-, 3- or 6-month-old Y201V/CTLA-4/ mice or aged-matched C57BL/6 controls were stained for CD4 and either CD69, CD25 or CD62L. Cells were analyzed by flow cytometry. For comparison, activation markers on CD4+ T cells from a 1-month-old CTLA-4/ mouse is shown.
|
|
Activated T cells in CTLA/ mice were reported to preferentially differentiate along a Th2 pathway in vivo. (21,22) and we have previously demonstrated that T cells from mice that expressed a tailless CTLA-4 mutant also exhibited a Th2 phenotype (15). We therefore determined whether cytokine production by Y201V/CTLA-4/ T cells was similarly altered. As shown in Fig. 4(A), mixed LN cell cultures from Y201V/CTLA-4/ mice produced significantly greater amounts of IL-4 when compared with cells from FL/CTLA-4/ mice or from C57BL/6 controls when stimulated in vitro with anti-CD3 and anti-CD28. The increase in IL-4 production could be seen even prior to the onset of lymphadenopathy (data not shown), and became greater as mice grew older and developed progressive lymphadenopathy (Fig. 4B, 6 versus 3 months). In contrast, IFN-
production in mixed LN cell cultures from Y201V/CTLA-4/ mice tended to be less than controls, perhaps because of an inhibitory effect of IL-4 on IFN-
-producing cells, either in vivo and/or in vitro (23). Stimulation of purified CD4+ T cells confirmed that Y201V/CTLA-4/ CD4+ T cells produced increased quantities of IL-4 (Fig. 4C). Under these conditions, both wild-type and mutant T cells produced minimal amounts of IFN-
. Thus, consistent with other models in which CTLA-4 signaling was deficient, Y201V CTLA-4/ T cells were preferentially skewed in vivo toward a Th2 phenotype.
To examine if the in vivo accumulation of Y201V/CTLA-4/ LN cells was associated with their increased proliferation in vitro, purified CD4+ T cells from 3- to 4-month-old Y201V/CTLA-4/ and FL/CTLA-4/ mice were labeled with CFSE and stimulated with plate-bound anti-CD3 or with anti-CD3 plus anti-CD28. As shown in Fig. 5, the majority of FL/CTLA-4/ CD4+ T cells did not undergo cell division when stimulated with 2 µg/ml of anti-CD3. In contrast, a significant proportion of Y201V/CTLA-4/ T cells were induced to undergo cell division in response to the same stimulus. When provided with anti-CD3 plus anti-CD28 co-stimulation, both FL/CTLA-4/ and Y201V/CTLA-4/ CD4+ T cells were induced to divide equally robustly, and no differences were observed between the two. Thus, Y201V/CTLA-4/ T cells exhibit increased responsiveness to suboptimal mitogenic stimulation in vitro. These results were not simply due to an increased percentage of memory CD4+ T cells in Y201V/CTLA-4/ cultures, which would be expected to exhibit a lower threshold for activation compared with naïve T cells, as in these experiments the number of cells bearing memory markers was not increased (Fig. 5B).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5. Increased cell division in Y201V/CTLA-4/ CD4+ T cells in response to TCR stimulation. (A) Purified CD4+ T cells from Y201V/CTLA-4/ or FL/CTLA-4/ mice were labeled with CFSE and stimulated with plate-bound anti-CD3 (2 µg/ml) or with anti-CD3 (5 µg/ml) plus anti-CD28 (5 µg/ml). CFSE fluorescence was determined on day 5. (B) Equivalent expression of naive and memory T cell markers on CD4+ T cells used for in vitro stimulations in (A). The results are representative of three separate experiments.
|
|
One mechanism that could explain the increased proliferation and accumulation of lymphocytes in Y201V/CTLA-4/ mice was if there was impaired function of CD4+CD25+ regulatory T cells. This subset of T cells has been shown to regulate T cell homeostasis (24) and to be dependent upon CTLA-4 function (25,26). To explore the possibility that the regulation of CD4+CD25+ T cells by CTLA-4 was dependent upon the Y201 residue, we determined whether CD4+CD25+ T cells from Y201V/CTLA-4/ mice were capable of inhibiting the proliferation of wild-type CD4+ T cells in vitro. As shown in Fig. 6, CD4+CD25+ T cells from 3-month-old Y201V/CTLA-4/ mice inhibited the proliferation of wild-type CD4+CD25 T cells to a degree comparable to wild-type CD4+CD25+ T cells. Thus, the ability of CD4+CD25+ regulatory T cells to suppress proliferation was not critically dependent upon the Y201 residue of CTLA-4.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6. Inhibition of proliferation of wild-type CD4+CD25 T cells by Y201/CTLA-4/CD4+CD25+ T cells. CD25+CD4+ CD4+ T cells were isolated from 3-month-old Y201/CTLA-4/ mice (YV) or from C57BL/6 or +/+ littermate controls (WT). Cells enclosed within the rectangles were sorted as CD25+CD4+ T cells. The sorted populations were mixed at a ratio of 1:1 (CD25+:CD25) and incubated for 2 days with anti-CD3 (1µg/ml) and irradiated wild-type spleen cells as antigen-presenting cells. The results are representative of three independent experiments. The averages of triplicate cultures are shown.
|
|
 |
Discussion
|
---|
Our results demonstrate that the Y201 residue of the CTLA-4 molecule not only regulates CTLA-4 cell-surface expression, but also transmits inhibitory signals that are necessary for complete CTLA-4 function in vivo. Despite higher levels of expression of the Y201V receptor compared with the FL receptor, the FL receptor, but not the Y201V receptor, was able to correct the lymphoproliferative disorder of CTLA-4/ mice. The phenotype of Y201V/CTLA-4/ mice was in some ways similar to the phenotype of mice expressing the tailless (
Tail) CTLA-4 receptor. The overlapping phenotypes suggest that the lymphoproliferation seen in mice lacking the entire CTLA-4 cytoplasmic domain may have been due to the absence of the Y201 residue. The differences that existed between the two mutant mouse strains (slower onset of lymphadenopathy in Y201V/CTLA-4/ mice; predominant involvement of the mesenteric LN in Y201V/CTLA-4/ mice compared with the more widespread lymphadenopathy in
Tail/CTLA-4/ mice; accumulation of naive and activated/memory phenotype T cells in Y201V/CTLA-4/ mice compared with the accumulation of activated/memory T cells in
Tail/CTLA-4/ mice) can potentially be explained by differences in the levels of expression of the two mutant receptors on the surface of T cells. Unlike the Y201V receptor, which was expressed at significantly higher levels compared with endogenous CTLA-4 (Fig. 1), the expression of the
Tail receptor was lower, occurring at levels that did not exceed that of endogenous CTLA-4 (15). Because B7/CD28-dependent interactions are necessary for the development of the lymphoproliferative disorder of CTLA/ animals (27,28), a greater capacity of the Y201 mutant to compete with CD28 for ligands in vivo compared with the tailless mutant would be expected to lead to a milder phenotype in the Y201V/CTLA-4/ mice compared with
Tail/CTLA-4/ mice, even if in both mutant mouse strains, the Y201 residue conferred the majority of CTLA-4 inhibitory function. Of course, our results do not exclude the possibility that other amino acid residues within the cytoplasmic domain in addition to Y201 are also important for mediating CTLA-4 inhibitory function.
The phenotype of Y201V/CTLA-4/ mice that we initially reported was not one of lymphoproliferation (15). The majority of mice studied in the earlier report were 68 weeks of age, although some mice that were 3 months of age were also evaluated. In the current study, at 3 months of age, there was still considerable overlap between the range of LN cell numbers in Y201V mice (50200 x 106) versus control mice (50100 x 106). Therefore, it may be that if larger numbers of 3-month-old mice had been evaluated in the previous report, lymphadenopathy would have been observed. In addition, due to the breeding strategy used, the mice used in the earlier report were likely to have been homozygous for the CTLA-4 transgene. In contrast, in the present study, mice heterozygous for the transgene were exclusively used. A doubling of the transgene copy number in the homozygotes used in the previous study may have led to a greater degree of rescue and a less penetrant phenotype. A correlation between transgene copy number and degree of rescue also existed for the two Y201V founders described in the present study, in that mice derived from the Y201V #2 founder, which had the higher transgene copy number (data not shown), had a less marked, though still statistically significant increase in LN cell numbers compared with the first founder, which had the lower transgene copy number. Because the effects from loss of inhibitory signaling as a result of the Y201V mutation are likely to be balanced by increased cell-surface expression, even minor differences in protein expression that resulted from differences in transgene copy number might be expected to lead to differences in the absolute degree of rescue of the CTLA-4/ phenotype.
We observed that LN homeostasis in Y201V/CTLA/ mutant mice was perturbed predominantly in the mesenteric LN. It may be that only in an environment of chronic antigenic stimulation such as exists in the intestinal lumen does inhibitory signaling through the CTLA-4 Y201 residue become critical for maintaining LN homeostasis. Furthermore, the natural bias toward Th2 responses that is often associated with gut-associated mucosal responses (29) may have provided an ideal environment for the generation of IL-4-secreting Y201V/CTLA/ T cells in the draining mesenteric LN. It was interesting that we could detect increased IL-4 production in vitro prior to any increase in LN cells in vivo. This raises the possibility that excessive IL-4 production may play a role in the initiation of lymphadenopathy in these mice.
The studies presented here provide evidence that in vivo, inhibitory signaling mediated by Y201 is an important mechanism for CTLA-4 function. However, consistent with the findings of other groups (1214), we were unable to show a defect in the ability of the CTLA-4 receptor bearing the Y201 mutation to inhibit cytokine secretion when CTLA-4 was co-ligated with the TCR and/or CD28 on T cells in vitro (data not shown). The disparate findings between the in vitro and in vivo results is likely due to an inability of the in vitro studies to adequately recapitulate the in vivo conditions of T cell activation under which the Y201 residue becomes necessary for CTLA-4 function. The idea that inhibitory signaling through Y201 may be important only under specific conditions of activation is consistent with our findings that Y201V/CTLA/ T cells exhibited increased cell division compared with cells expressing the FL CTLA-4 receptor in response to stimulation in vitro with low doses of anti-CD3, but not in response to stimulation with anti-CD3 plus anti-CD28 stimulation. Stimulation with higher doses of anti-CD3 (10 µg) similarly abolished the differences in cell division seen with Y201V/CTLA/ T cells compared with FL/CTLA/ T cells (data not shown). These results support a role for CTLA-4 in increasing the threshold for T cell activation (30) and, furthermore, suggest that the Y201 residue is important for this function. Accordingly, one setting where Y201 signaling may be physiologically important would be under conditions of TCR stimulation by weak ligands or self-ligands, where it would be desirable to raise the threshold for T cell activation.
The molecular mechanisms that underlie the defective function of the Y201V mutant remain unknown. A homologous motif (YMNM) is present within the cytoplasmic domain of the CD28 receptor and it has recently been reported that disruption of this motif in CD28 impairs the PI3K-dependent up-regulation of Bcl-xL that occurs with CD28 co-stimulation (31,32). Although the phosphorylated YVKM motif of CTLA-4 can also bind to PI3K, the contribution of loss of PI3K signaling to the lymphoproliferative phenotype we have observed is uncertain, as CTLA-4 engagement has been shown to have no effect on the up-regulation of Bcl-xL in T cells (33). Alternatively, it may be that the defective CTLA-4 function of the mutant receptor is due to disruption of protein interactions that are not phosphorylation dependent, such as the association between CTLA-4 with the clathrin-associated adaptor proteins AP-1 and AP-2 or with the serine/threonine phosphatase PP2A (34). The association of some cell-surface receptors with endocytic proteins has been shown to lead to the recruitment of signal transduction molecules (35). The inability of Y201V to associate with the endocytic machinery may prevent the activation of such signaling pathways which may be important for T cell inhibition.
Finally, we have demonstrated that the suppressor function of CD4+CD25+ regulatory T cells is not critically dependent upon the Y201 residue. It remains possible, however, that the decrease in the absolute number of CD4+CD25+ T cells present early on (Fig. 3) may have contributed to the loss of homeostasis that occurred later. However, in the absence of other contributing factors, this mechanism is unlikely to be solely responsible for the lymphadenopathy that develops in Y201V CTLA-4/ mice, as CD28/ cells also have diminished numbers of regulatory T cells, but do not develop adenopathy.
In summary, we have provided evidence that CTLA-4 inhibitory signals transmitted by Y201 contribute to the maintenance of LN homeostasis in vivo. Increased cell-surface expression of CTLA-4 that resulted from impaired endocytosis was unable to completely compensate for loss of inhibitory signals mediated by Y201.
 |
Acknowledgements
|
---|
We thank Dr Clara Abraham for critical reading of the manuscript and Dr Howard Petrie for valuable discussions. This work was supported by an NIH Clinical Investigator Award and by an American Society of Hematology Scholar Award (to E. C.).
 |
Abbreviations
|
---|
FLfull length
LNlymph node
PEphycoerythrin
PI3Kphosphatidylinositol 3-kinase
Y201tyrosine 201
 |
References
|
---|
- Alegre, M. L., Noel, P. J., Eisfelder, B. J., Chuang, E., Clark, M. R., Reiner, S. L. and Thompson, C. B. 1996. Regulation of surface and intracellular expression of CTLA4 on mouse T cells. J. Immunol. 157:4762.[Abstract]
- Linsley, P. S., Bradshaw, J., Greene, J., Peach, R., Bennett, K. L. and Mittler, R. S. 1996. Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity 4:535.[ISI][Medline]
- Shiratori, T., Miyatake, S., Ohno, H., Nakaseko, C., Isono, K., Bonifacino, J. S. and Saito, T. 1997. Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP- 2. Immunity 6:583.[ISI][Medline]
- Chuang, E., Alegre, M. L., Duckett, C. S., Noel, P. J., Vander Heiden, M. G. and Thompson, C. B. 1997. Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression. J. Immunol. 159:144.[Abstract]
- Bradshaw, J. D., Lu, P., Leytze, G., Rodgers, J., Schieven, G. L., Bennett, K. L., Linsley, P. S. and Kurtz, S. E. 1997. Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin-associated protein is negatively regulated by tyrosine phosphorylation. Biochemistry 36:15975.[CrossRef][ISI][Medline]
- Zhang, Y. and Allison, J. P. 1997. Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein. Proc. Natl Acad. Sci. USA 94:9273.[Abstract/Free Full Text]
- Follows, E. R., McPheat, J. C., Minshull, C., Moore, N. C., Pauptit, R. A., Rowsell, S., Stacey, C. L., Stanway, J. J., Taylor, I. W. and Abbott, W. M. 2001. Study of the interaction of the medium chain mu 2 subunit of the clathrin-associated adapter protein complex 2 with cytotoxic T-lymphocyte antigen 4 and CD28. Biochem. J. 359:427.[CrossRef][ISI][Medline]
- Schneider, H., Martin, M., Agarraberes, F. A., Yin, L., Rapoport, I., Kirchhausen, T. and Rudd, C. E. 1999. Cytolytic T lymphocyte-associated antigen-4 and the TCR
/CD3 complex, but not CD28, interact with clathrin adaptor complexes AP-1 and AP-2. J. Immunol. 163:1868.[Abstract/Free Full Text]
- Schneider, H., Prasad, K. V., Shoelson, S. E. and Rudd, C. E. 1995. CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells. J. Exp. Med. 181:351.[Abstract]
- Marengere, L. E., Waterhouse, P., Duncan, G. S., Mittrucker, H. W., Feng, G. S. and Mak, T. W. 1996. Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4. Science 272:1170.[Abstract]
- Schneider, H., da Rocha Dias, S., Hu, H. and Rudd, C. E. 2001. A regulatory role for cytoplasmic YVKM motif in CTLA-4 inhibition of TCR signaling. Eur. J. Immunol. 31:2042.[CrossRef][ISI][Medline]
- Nakaseko, C., Miyatake, S., Iida, T., Hara, S., Abe, R., Ohno, H., Saito, Y. and Saito, T. 1999. Cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement delivers an inhibitory signal through the membrane-proximal region in the absence of the tyrosine motif in the cytoplasmic tail. J. Exp. Med. 190:765.[Abstract/Free Full Text]
- Cinek, T., Sadra, A. and Imboden, J. B. 2000. Cutting edge: tyrosine-independent transmission of inhibitory signals by CTLA. J. Immunol. 164:5.[Abstract/Free Full Text]
- Baroja, M. L., Luxenberg, D., Chau, T., Ling, V., Strathdee, C. A., Carreno, B. M. and Madrenas, J. 2000. The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation. J. Immunol. 164:49.[Abstract/Free Full Text]
- Masteller, E. L., Chuang, E., Mullen, A. C., Reiner, S. L. and Thompson, C. B. 2000. Structural analysis of CTLA-4 function in vivo. J. Immunol. 164:5319.[Abstract/Free Full Text]
- Bird, J. J., Brown, D. R., Mullen, A. C., Moskowitz, N. H., Mahowald, M. A., Sider, J. R., Gajewski, T. F., Wang, C. R. and Reiner, S. L. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9:229.[ISI][Medline]
- Borriello, F., Sethna, M. P., Boyd, S. D., Schweitzer, A. N., Tivol, E. A., Jacoby, D., Strom, T. B., Simpson, E. M., Freeman, G. J. and Sharpe, A. H. 1997. B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 6:303.[ISI][Medline]
- Shahinian, A., Pfeffer, K., Lee, K. P., Kundig, T. M., Kishihara, K., Wakeham, A., Kawai, K., Ohashi, P. S., Thompson, C. B. and Mak, T. W. 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261:609.[ISI][Medline]
- Lane, P., Gerhard, W., Hubele, S., Lanzavecchia, A. and McConnell, F. 1993. Expression and functional properties of mouse B7/BB1 using a fusion protein between mouse CTLA4 and human gamma 1. Immunology 80:56.[ISI][Medline]
- Salomon, B., Lenschow, D. J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A. and Bluestone, J. A. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431.[ISI][Medline]
- Khattri, R., Auger, J. A., Griffin, M. D., Sharpe, A. H. and Bluestone, J. A. 1999. Lymphoproliferative disorder in CTLA-4 knockout mice is characterized by CD28-regulated activation of Th2 responses. J. Immunol. 162:5784.[Abstract/Free Full Text]
- Oosterwegel, M. A., Mandelbrot, D. A., Boyd, S. D., Lorsbach, R. B., Jarrett, D. Y., Abbas, A. K. and Sharpe, A. H. 1999. The role of CTLA-4 in regulating Th2 differentiation. J. Immunol. 163:26342639.[Abstract/Free Full Text]
- Smeltz, R. B., Chen, J., Ehrhardt, R. and Shevach, E. M. 2002. Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression. Nat. Rev. Immunol. 2:389.[ISI][Medline]
- Annacker, O., Pimenta-Araujo, R., Burlen-Defranoux, O., Barbosa, T. C., Cumano, A. and Bandeira, A. 2001. CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL. J. Immunol. 166:3008.[Abstract/Free Full Text]
- Takahashi, T., Tagami, T., Yamazaki, S., Uede, T., Shimizu, J., Sakaguchi, N., Mak, T. W. and Sakaguchi, S. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
- Read, S., Malmstrom, V. and Powrie, F. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
- Chambers, C. A., Sullivan, T. J. and Allison, J. P. 1997. Lymphoproliferation in CTLA-4-deficient mice is mediated by costimulation-dependent activation of CD4+ T cells. Immunity 7:885.[ISI][Medline]
- Mandelbrot, D. A., McAdam, A. J. and Sharpe, A. H. 1999. B7-1 or B7-2 is required to produce the lymphoproliferative phenotype in mice lacking cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). J. Exp. Med. 189:435.[Abstract/Free Full Text]
- Garside, P. and Mowat, A. M. 2001. Oral tolerance. Semin. Immunol. 13:177.[CrossRef][ISI][Medline]
- Chambers, C. A., Krummel, M. F., Boitel, B., Hurwitz, A., Sullivan, T. J., Fournier, S., Cassell, D., Brunner, M. and Allison, J. P. 1996. The role of CTLA-4 in the regulation and initiation of T-cell responses. Immunol. Rev. 153:27.[ISI][Medline]
- Okkenhaug, K., Wu, L., Garza, K. M., La Rose, J., Khoo, W., Odermatt, B., Mak, T. W., Ohashi, P. S. and Rottapel, R. 2001. A point mutation in CD28 distinguishes proliferative signals from survival signals. Nat. Immunol. 2:325.[CrossRef][ISI][Medline]
- Burr, J. S., Savage, N. D., Messah, G. E., Kimzey, S. L., Shaw, A. S., Arch, R. H. and Green, J. M. 2001. Cutting edge: distinct motifs within CD28 regulate T cell proliferation and induction of Bcl-XL. J. Immunol. 166:5331.[Abstract/Free Full Text]
- Blair, P. J., Riley, J. L., Levine, B. L., Lee, K. P., Craighead, N., Francomano, T., Perfetto, S. J., Gray, G. S., Carreno, B. M. and June, C. H. 1998. CTLA-4 ligation delivers a unique signal to resting human CD4 T cells that inhibits interleukin-2 secretion but allows Bcl-XL induction. J. Immunol. 160:12.[Abstract/Free Full Text]
- Chuang, E., Fisher, T. S., Morgan, R. W., Robbins, M. D., Duerr, J. M., Vander Heiden, M. G., Gardner, J. P., Hambor, J. E., Neveu, M. J. and Thompson, C. B. 2000. The CD28 and CTLA-4 receptors associate with the serine/threonine phosphatase PP2A. Immunity 13:313.[ISI][Medline]
- Di Fiore, P. P. and De Camilli, P. 2001. Endocytosis and signaling. an inseparable partnership. Cell 106:1.[ISI][Medline]