Division of Rheumatology, Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, 1 Department of Immunology, Mayo Clinic, Rochester, MN 55905 and 2 Cancer Research Laboratory, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA
Correspondence to: W. Stohl; E-mail: stohl{at}usc.edu
Transmitting editor: T. Tedder
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: CD4+ cells; CD8+ cells; IgG; IgM; Ig-secreting cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the critical downregulators of T cell activation is CD152 (CTLA-4). In resting T cells, CD152 is predominantly expressed intracellularly with only limited cell surface expression (1). Following TCR engagement, intracellular and the limited cell surface CD152 focus to the sites of TCR engagement (2), with greater strength of the TCR signal resulting in greater surface accumulation of CD152 (3). CD152 mRNA levels are upregulated within hours (4), and cell surface CD152 expression greatly increases within days (5,6), although intracellular CD152 still predominates (7). At least two independent intracellular signaling pathways mediate CD152 upregulation (8).
CD80 (B7.1) and CD86 (B7.2), expressed by cells capable of antigen presentation (e.g. monocytes, macrophages, dendritic cells, B cells), are the natural ligands for CD152 (912). Engagement of CD152 delivers a direct inhibitory signal and also sequesters CD80/CD86, thereby indirectly impeding CD28-mediated activation (13,14). At least part of the inhibition is due to increased indoleamine 2,3-deoxygenase activity by dendritic cells with consequent increased catabolism of tryptophan (15). The net result of CD152 engagement is inhibition of activation-induced upregulation of membrane lipid raft expression (16) and arrest of T cells in the G0/G1 phase of the cell cycle (17,18).
Blockade of CD152 engagement has a myriad of biologic consequences. On the one hand, treatment with soluble anti-CD152 mAb results in enhanced in vitro T cell proliferation and IL2 production (5, 6, 19). Similar blockade of CD152 in vivo enhances expansion of antigen-specific T cells, promotes protective immunity against parasitic infection, blocks immunostimulatory chronic graft-versus-host disease and augments development of anti-tumor immunity (2023). On the other hand, in vivo blockade of CD152, rather than being beneficial, may be deleterious to the host, in that it accelerates and exacerbates onset and severity of experimental autoimmune encephalomyelitis (EAE) and autoimmune diabetes (2426) and confers susceptibility to EAE in otherwise resistant mice (27). Indeed, engagement of CD152 is crucial to development and/or maintenance of tolerance in vivo (28, 29), and the potent suppressor effects of CD4+CD25+ regulatory T cells are substantially mediated via CD152 (30,31).
Mice genetically deficient in CD152 (cd152/ mice) spontaneously develop massive systemic T lymphoproliferation with infiltration of numerous vital organs by lymphocytes and inflammatory cells (3234). Thymocyte development, including negative and positive selection, is normal in cd152/ mice (35,36), indicating that the physiologic defect is in control of peripheral T cell activation rather than in central T cell development. Since the accelerated T cell activation, lymphoproliferation and mortality are markedly attenuated in TCR-transgenic cd152/ mice with highly limited T cell repertoires (3639), it is likely that the proliferating T cells in these mice respond to self antigens and/or to highly prevalent environmental antigens. The fact that the activated T cells in non-transgenic cd152/ mice manifest a diverse and unbiased TCR repertoire (40) indicates that no individual self or environmental antigen is uniquely driving the pathologic response.
The ramifications of CD152 deficiency for B cells in vivo are less well understood. Increased B cell numbers and circulating Ig levels, as well as upregulation of B cell surface CD86, have been reported in cd152/ mice (32,34). This B cell hyperactivity may be secondary to excessive CD4+ cell-mediated help, inasmuch as treatment of cd152/ mice with a depleting anti-CD4 mAb blocks the B lymphocytosis (34). Since CD4+ T cell-mediated help is predominantly MHC class II (MHCII)-restricted and antibody responses to T cell-dependent antigens are profoundly blunted in MHCII-deficient (mhcii/) mice (41,42), one might predict that numbers of Ig-secreting cells (IgSC) and circulating Ig levels would be (near-) normal in MHCII-deficient cd152/ mice. Moreover, the widespread activation and expansion of CD8+ cells in cd152/ mice is also greatly attenuated by depletion of CD4+ cells (34), suggesting that dysregulation of CD4+ cells is also vital to dysregulation of CD8+ cells. Accordingly, one might predict that CD8+ cell activation and expansion in MHCII-deficient cd152/ mice would be, at most, very limited.
In this report, we demonstrate that these predictions are each incorrect. CD8+ cell activation and expansion in MHCII-deficient cd152/ mice are as dramatic as they are in MHCII-intact cd152/ mice. Furthermore, B cell hyperactivity also develops in MHCII-deficient cd152/ mice over time, although in a manner less rapid and less intense than that in corresponding MHCII-intact mice. Thus, CD152 plays a vital role in downregulating spontaneous MHCII-independent CD8+ cell expansion and B cell hyperactivity. Moreover, introduction of human MHCII into mhcii/cd152/ mice restores the more rapid kinetics and greater intensity of B cell hyperactivity, demonstrating a vital role for CD152 in downregulating both spontaneous MHCII-dependent as well as spontaneous MHCII-independent B cell hyperactivity.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice
All mice were housed at USC in a single specific pathogen-free room. MHCII-intact (mhcii+/+) cd152+/ mice bearing a C57Bl/6 (B6) background (35) were propagated by mating with B6 (cd152+/+) mice and screening the progeny for cd152 heterozygosity (see below). MHCII-deficient (mhcii/) cd152+/ mice were generated by first crossing mhcii+/+ cd152+/ mice with Aß0 mice (mhcii/cd152+/+) (41,43) and selecting for cd152 heterozygosity. The resulting mhcii+/cd152+/ mice were backcrossed to Aß0 mice and selected for cd152 heterozygosity and the absence of MHCII (see below).
To reconstitute mhcii/cd152/ mice with human MHCII, mhcii/cd152+/ mice were crossed with Aß0-DQ8 mice (mhcii/dq8+/+cd152+/+; deficient for murine endogenous MHCII and homozygous for a transgenic fragment containing both DQA*0301 and DQB*0302 genes) (44). The progeny (all mhcii/dq8+/) were selected for cd152 heterozygosity and were backcrossed to Aß0-DQ8 mice. Progeny from this backcross that were cd152+/ were screened for dq8 homozygosity by mating to B6 mice and testing the progeny for dq8 by PCR (see below). At least 12 pups from a given cd152+/ parent were tested, and cd152+/ parents were deemed to be dq8+/+ if all 12 pups bore dq8.
Assignment of cd152 genotype
cd152 genotype was determined by PCR. Small clippings of mouse tails were digested for >4 h at 56°C by proteinase K in Tris/EDTA/SDS buffer followed by phenol/chloroform extraction of genomic DNA. Genomic DNA was PCR-amplified for 4245 cycles each at 95°C for 1 min, 60°C for 1 min and 72°Cfor 1 min. The primer sequences used were: 5'-CCAGAACCATGCCCGGATTCTGACTTC-3' (cd152-intact sense), 5'-CCAAGTGCCCAGAGGGGCTGCTAAA-3' (cd152-deficient sense), 5'-AAACAACCCCAAGCTAACTGCGACAAGG-3' (anti-sense). The PCR products were subjected to electrophoresis in 1.5% agarose gels containing ethidium bromide, and bands were visualized under UV light. Band sizes for cd152-intact and cd152-deficient were 90 and
180 bp respectively.
Assignment of mhcii genotype
Both copies of the gene coding for I-Aßb are disrupted by a neo insertion in parental mhcii/ Aß0 mice (41). Staining splenic B cells for surface I-Ab readily identified by visual inspection the MHCII-intact, MHCII-intermediate and MHCII-deficient phenotypes, which correspond to mhcii+/+, mhcii+/ and mhcii/ genotypes respectively (Fig. 1). Although this procedure was suitable for mice being sacrificed, staining spleen cells was not suitable for mice being used for breeding. Unfortunately, staining of PBMC for surface I-Ab often yielded visually ambiguous results that did not permit discrimination between MHCII-intermediate and MHCII-deficient phenotypes. This was likely due to the lower percentages of MHCII+ cells in blood than those in spleen which, in turn, precluded clear separation of positively staining cells from negative cells. Fortunately, MHCII-deficient mice harbor markedly reduced numbers (and percentages) of CD4+ T cells in the periphery (41, 42), and staining PBMC for surface CD4 readily discriminated visually between the CD4-low phenotype (mhcii/ genotype) and CD4-high phenotype (mhcii+/ or mhcii+/+ genotypes) (Fig. 1).
|
Detection of dq8
The presence of both DQ8 and DQ8ß were detected by PCR using the following primer sequences: 5'-GAAGACATTGTGGCTGACCATGTTGCC-3' (DQ8
sense), 5'-AGCACAGCGATGTTTGTCAGTGCAAATTGCGG-3' (DQ8
anti-sense), 5'-AGGATTTGGTGTWCCAGTTTAAGGGCAT-3' (DQ8ß sense), 5'-TGCAAGGTCGTGCGGAGCTCCAA-3' (DQ8ß anti-sense). Band sizes for dq8
and dq8b were
250 and
350 bp respectively.
Cell surface staining
Murine spleen mononuclear cells were isolated by mechanically teasing the spleens followed by Ficoll density gradient centrifugation. The isolated mononuclear cells were single-, double- or triple-stained with appropriate combinations of FITC-, PE- or Cy-Chrome-conjugated mAb specific for murine CD3, CD4, CD8, CD44, CD45R (B220), CD62L or I-Ab (BD PharMingen, San Diego, CA) and analyzed by flow cytometry. Cell debris, as determined by forward- and side-scatter characteristics, was electronically excluded from the analysis. At least 10 000 events were analyzed for each sample.
Serum Ig and spleen IgSC determinations
Mice were bled and/or sacrificed at the indicated times. Serum was assayed for total IgG and total IgM by ELISA. To do so, 96-well flat-bottomed plates were coated overnight at 4°C with goat anti-mouse IgG+IgA+IgM antibodies (Zymed Laboratories, South San Francisco, CA) and blocked with 10% FCS for 90 min at 37°C. After washing with 0.05% Tween-20 in PBS, diluted sera were added to the plates for 90 min at 37°C, washed, and detected with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-mouse IgM (Zymed) for 90 min at 37°C. After washing, color was developed by addition of o-phenylenediamine (Sigma-Aldrich, St. Louis, MO). The reaction was terminated by addition of 6N H2SO4, and OD450 was measured. Purified mouse IgG or IgM (Zymed) were used as standards.
Spleen cells were assayed for total IgSC by the reverse hemolytic plaque assay (45,46). Each plaque-forming cell was taken as an IgSC.
Statistical analysis
All analyses were performed using SigmaStat software (SPSS, Chicago, IL). To achieve normality, the results (cell numbers, cell percentages, Ig levels, IgSC) were log-transformed. Parametric testing between two matched or unmatched groups was performed by the paired or unpaired t-test respectively. Parametric testing among three or more groups was performed by one-way ANOVA. When log-transformation failed to generate normally distributed data or the equal variance test was not satisfied, non-parametric testing was performed by the MannWhitney rank sum test between two groups and by KruskalWallis one-way ANOVA on ranks among three or more groups. Correlations were determined by Pearson product moment correlation for interval data and by Spearman rank order correlation for ordinal data or for interval data which did not follow a normal distribution.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To formally assess MHCII-dependence of T cell expansion in cd152/ mice, we mated MHCII-deficient (mhcii/) cd152+/ male and female mice with each other, and the resulting F2 progeny was genotypically +/+, +/ and / for cd152 at the expected 1:2:1 ratio. There were considerably (70%) fewer splenic CD4+ cells in 3-week-old mhcii/cd152+/+ and mhcii/cd152+/ mice compared to those in their mhcii+/+ counterparts (compare Fig. 2E with B; P < 0.001 for each comparison), likely due to the absence of MHCII-based positive selection of CD4+ cells (41,42). In mhcii/cd152/ mice, the number of CD4+ cells was modestly greater (<2-fold) than those in cd152+/+ or cd152+/ littermates (P = 0.012), but the relative expansion was much more limited than that observed in mhcii+/+cd152/ mice (P < 0.001). Of note, CD4+ cells from all MHCII-deficient mice (including those with cd152+/+ or cd152+/ genotype) displayed an activated phenotype (Fig. 3 and data not shown), rendering moot the question of whether CD152 deficiency promotes activation of MHCII-unrestricted CD4+ cells.
The attenuated CD4+ cell expansion in mhcii/cd152/ mice notwithstanding, expansion CD8+ cells in these mice was unimpeded (compare Fig. 2F with C). Moreover, whereas CD8+ cells from MHCII-deficient cd152+/+ or cd152+/ mice displayed a non-activated phenotype, CD8+ cells from MHCII-deficient cd152/ mice displayed an activated phenotype similar to that of CD8+ cells from MHCII-intact cd152/ mice (Fig. 3). This demonstrates that the MHCII-independent increase in CD8+ cells in cd152/ mice is due to persistent CD8+ cell activation rather than to increased survival of resting or quiescent CD8+ cells.
In contrast to the early death of mhcii+/+cd152/ mice, mhcii/cd152/ mice live into adulthood and can reproduce (unpublished observations). This permitted us to mate mhcii/cd152+/ mice to mhcii/cd152/ mice, and the resulting pups (50% being cd152+/ and 50% being cd152/) were evaluated at 5 weeks of age. Although CD4+ cells were modestly increased (2-fold) in mhcii/cd152/ mice compared to those in cd152+/ littermates (P < 0.001, Fig. 2H), this increase was overwhelmed by the massive expansion (13-fold) of CD8+ cells in the cd152/ mice (P < 0.001, Fig. 2I). Thus, CD8+ cell activation and expansion is unfettered in the absence of CD152 in MHCII-deficient hosts, demonstrating a vital downregulatory role for CD152 in MHCII-independent CD8+ cell expansion.
Development of B cell hyperactivity in mhcii/cd152/ mice
Concurrent with the exuberant CD4+ and CD8+ cell expansion in 3-week-old mhcii+/+cd152/ mice, these mice, despite no increase in splenic B (B220+) cells, displayed markedly increased numbers of splenic IgSC (15-fold; P < 0.001) and circulating levels of IgG (
9-fold; P < 0.001) compared to cd152+/+ or cd152+/ littermates (Fig. 4AC). Circulating IgM levels were also increased
5-fold (P < 0.001, data not shown). In contrast, splenic IgSC and circulating IgG and IgM levels in 3-week-old mhcii/cd152/ mice were not appreciably different from those in their cd152+/+ or cd152+/ littermates (Fig. 4E and F and data not shown). However, by 5 weeks of age, B cell hyperactivity in mhcii/cd152/ was apparent, albeit not as dramatic as that appreciated in mhcii+/+cd152/ mice. Although no expansion of splenic B (B220+) cells in mhcii/cd152/ mice was detected, splenic IgSC numbers had increased
3-fold (P = 0.001), and serum IgG levels were clearly elevated in five of the nine cd152/ mice relative to their cd152+/ littermates (Fig. 4GI). Thus, there is an underlying MHCII-independent diathesis to B cell hyperactivity which CD152 normally controls. Of note, spleen CD8+ cell numbers correlated strongly with serum IgG levels in mhcii/cd152/ mice (Fig. 4J), suggesting that the increase in each was being driven by a common trigger (such as the MHCII-unrestricted CD4+ cells) and/or that the B cell hyperactivity was actually promoted (at least in part) by activated CD8+ cells.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD28-mediated signaling is vital to development of lymphoproliferation in MHCII-intact cd152/ mice. Treatment of such mice with CTLA4-Ig (which blocks CD28/CD80 and CD28/CD86 interactions) prevents their abnormal T cell activation and expansion for the duration of treatment (49). Moreover, MHCII-intact cd152/ mice that are also deficient in CD80 and CD86 (cd80/cd86/ genotype) are protected from the lymphoproliferative phenotype, but lymphoproliferation can be induced in these mice by artificial ligation of CD28 with an anti-CD28 mAb (50).
CD4+ cells also play a vital role in the lymphoproliferation that develops in cd152/ mice. In contrast to the MHCII-restriction exhibited by most CD4+ T cells, the great majority of CD8+ T cells are MHC class I-restricted. Nevertheless, their antigen-driven activation usually depends critically upon CD4+ cells (5153). The fact that the massive expansion of CD8+ cells in MHCII-intact cd152/ mice is largely prevented by treatment with a depleting anti-CD4 mAb (34) is consistent with this principle. Therefore, it was unexpected that despite substantially reduced numbers of CD4+ cells, CD8+ cell activation and expansion remained unabated in MHCII-deficient cd152/ mice. The degree of CD8+ cell activation and expansion in these mice was similar to that in their MHCII-intact counterparts and in their counterparts expressing human MHCII rather than murine MHCII (Figs 2 and 3). Taken together, these observations strongly suggest that the helper role played by CD4+ cells in CD8+ cell activation can be dissociated from the ability of CD4+ cells to recognize antigen in the context of MHCII. Whether CD28-mediated signaling plays a role in MHCII-independent activation of CD8+ cells remains to be formally established.
Our results of unabated CD8+ cell expansion in MHCII-deficient cd152/ mice can readily be reconciled with the prevention of CD8+ expansion in anti-CD4-treated cd152/ mice (34). In the latter study, repeated injections of a depleting anti-CD4 mAb, beginning within 2 days after birth, had to be administered to cd152/ mice over a 3-week period to effect continuous depletion of CD4+ cells. Without continuous CD4+ cell depletion, low numbers of activated CD4+ cells developed, and CD8+ cell expansion was not effectively blocked. Unfortunately, we could not similarly treat MHCII-deficient cd152/ mice with a depleting anti-CD4 mAb, since all MHCII-deficient mice in our colony, regardless of cd152 genotype, do not tolerate repeated i.p. injections and succumb prematurely. Whether this intolerance to repeated i.p. injections is due to undue susceptibility to occult infections in MHCII-deficient hosts or due to some other cause is uncertain.
Regardless, the great majority of the (MHCII-unrestricted) CD4+ cells in MHCII-deficient mice bear an activated phenotype (Fig. 3). Indeed, it may be that activated MHCII-unrestricted CD4+ cells are crucial helpers of CD8+ cell expansion even in MHCII-intact hosts. The vast majority of MHCII-unrestricted CD4+ cells are restricted by CD1d, so the requisite CD4+ cell-mediated help for antigen-driven CD8+ cell activation may arise from recognition by CD4+ cells of CD1d-restricted antigens. Future experiments utilizing CD1-deficient mice should help resolve this issue.
Alternatively, CD8+ cell activation and expansion in mhcii/cd152/ mice may be occurring by a pathway that altogether circumvents the requirement for antigen recognition by CD4+ cells. APC can be activated via engagement of their surface CD40 even in the absence of CD4+ cells (5153). CD154 (CD40 ligand) can be expressed by both CD4+ and double-negative (DN; CD4CD8) CD1d-restricted T cells (54, 55), so direct engagement of APC by CD154+ cells (even in an antigen-non-specific manner) could result in conditions permissive to activation and expansion of CD8+ cells. The reason that continuous depletion of CD4+ cells blocks CD8+ cell expansion in cd152/ hosts may have nothing to do with MHCII/CD4+ cell interactions but may be consequent to the elimination of most CD154+ cells. MHCII-deficient mice may have sufficient numbers of CD154+ cells to activate APC and permit unabated CD8+ cell activation and expansion. Experiments utilizing CD154-deficient mice should help clarify this point. In any case, our findings clearly point to a vital downregulatory role for CD152 in MHCII-independent activation and expansion of CD8+ cells.
In vivo deficiency of CD152 leads not only to marked activation and expansion of T cells, but it also leads to marked increases in IgSC and circulating Ig levels (Fig. 4). Of note, numbers of splenic B (B220+) cells are not increased in cd152/ mice despite the increased in vivo Ig production in such mice. This may reflect a preferential downregulatory effect of CD152 on differentiation, rather than on proliferation, of B cells. Alternatively, B cell numbers may truly increase in cd152/ mice, but the expanded B cells may preferentially home to extrasplenic sites. Increased numbers of lymph node B cells have been described in cd152/ mice (34), and other sites, such as the intestine or bone marrow, may also harbor increased numbers of B cells.
In either case, the observed B cell hyperactivity in cd152/ mice is effected via both MHCII-dependent and MHCII-independent pathways. In mhcii/cd152/ mice, B cell hyperactivity is not apparent at 3 weeks of age but is readily apparent at 5 weeks of age (Fig. 4), demonstrating MHCII-independent B cell hyperactivity in the absence of CD152. Introduction of a transgene for human DQ8 into cd152/ mice deficient for murine MHCII (but now expressing human MHCII) restores the vigorous B cell hyperactivity (Fig. 4), demonstrating superimposed MHCII-dependent B cell hyperactivity in the absence of CD152.
Of note, splenic IgSC numbers were essentially identical in MHCII-deficient cd152+/+ or cd152+/ mice compared to those in the corresponding MHC-intact mice (compare Fig. 4E with B). This strongly suggests that B cell maturation to the IgSC state can occur normally in the absence of MHCII. A component of this B cell maturation may be T cell-independent, but it is likely that there is also a T cell-dependent component. Although serum IgG levels were lower in MHCII-deficient cd152+/+ or cd152+/ mice compared to the respective levels in the corresponding MHC-intact mice (compare Fig. 4F with C), serum IgG levels had dramatically increased in the majority of MHCII-deficient cd152/ mice by 5 weeks of age (Fig. 4I). Importantly, serum IgG levels in these mice correlated strongly with the degree of CD8+ cell expansion (Fig. 4J), raising the possibility that a component of the MHCII-independent B cell hyperactivity in mhcii/cd152/ mice is driven by CD8+ cells. It may be that optimal treatment of autoimmune disorders associated with B cell hyperactivity may require targeting not just B cells and CD4+ cells but CD8+ cells as well.
![]() |
Acknowledgements |
---|
![]() |
Abbreviations |
---|
EAEexperimental autoimmune encephalomyelitis
IgSCIg-secreting cells
MHC IIMHC class II
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|