The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rifkin, I. R.
Right arrow Articles by Seldin, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rifkin, I. R.
Right arrow Articles by Seldin, D. C.
The Journal of Immunology, 1998, 161: 5164-5170.
Copyright © 1998 by The American Association of Immunologists

Acceleration of lpr Lymphoproliferative and Autoimmune Disease by Transgenic Protein Kinase CK2{alpha}1

Ian R. Rifkin*, Padma L. Channavajhala*,{dagger}, Heather L.B. Kiefer{dagger}, Adrienne J. Carmack*, Esther Landesman-Bollag*,{ddagger}, Britte C. Beaudette{dagger}, Brian Jersky§, David J. Salant*, Shyr-Te Ju*,{ddagger}, Ann Marshak-Rothstein{dagger} and David C. Seldin2,*,{dagger}

Departments of * Medicine, {dagger} Microbiology, and {ddagger} Pathology, Boston University Medical Center, Boston, MA 02118; and § Department of Mathematics, Sonoma State University, Rohnert Park, CA 94928


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MRL-lpr/lpr mice have a Fas receptor mutation that leads to abnormalities of apoptosis, lymphoproliferation, and a lupus-like autoimmune disease associated with the production of autoantibodies. Other than Fas pathway defects, little is known about molecular abnormalities that predispose to autoimmunity. Protein kinase CK2 (also termed casein kinase II), a serine-threonine protein kinase whose targets include many critical regulators of cellular growth, is highly expressed in a lymphoproliferative disease of cattle and in many human cancers. Overexpression of the CK2{alpha} catalytic subunit in lymphocytes of transgenic mice leads to T cell lymphoma. We hypothesized that CK2 dysregulation and Fas mutation might cooperatively augment lymphocyte proliferation and transformation. We find that in MRL-lpr/lpr mice bearing the CK2{alpha} transgene, the lymphoproliferative process is dramatically exacerbated, as these mice develop massive splenomegaly and lymphadenopathy by 12 wk of age in association with increased autoantibody production and accelerated renal disease. The lymphoid organs are filled with the unusual B220+CD4-CD8- T cells typically seen in MRL-lpr/lpr mice, not the B220-CD4+CD8+ or B220-CD4-CD8+ T cells typically seen in CK2{alpha} transgenic lymphomas. The T cells do not fulfill the criteria for transformation, as they are polyclonal and not transplantable or immortal in cell culture. Thus, although the lpr lymphoproliferative and autoimmune syndrome is potentiated by the presence of the CK2{alpha} transgene, this combination of apoptotic and proliferative abnormalities appears to be insufficient to transform lymphoid cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MRL strain mice homozygous for the lpr or gld mutations (MRL-lpr/lpr or MRL-gld/gld) develop an autoimmune syndrome associated with lymphoid hyperplasia and autoantibody production (1). This autoimmune syndrome has many similarities to human systemic lupus erythematosus (SLE),3 in that the mice develop a severe systemic immune complex-mediated disease associated with a range of autoantibody specificities (including antinuclear, anti-dsDNA and anti-SM Abs) characteristic of the human disease. The mice die at approximately 6 mo of age, generally because of renal failure secondary to glomerulonephritis (2). Although the lpr mutation alone can induce autoantibody production in otherwise nonautoimmune strains of mice (3), expression of clinical disease is accentuated by the effect of background genes in strains such as MRL (4). MRL strain mice without the lpr or gld mutations are also autoimmune prone, although they develop variable levels of autoantibodies and milder clinical disease (2). The lpr and gld defects have been identified as mutations in the genes encoding the cell surface molecules Fas and Fas ligand, respectively (5, 6). Engagement of Fas by its ligand has been shown to induce apoptotic cell death in a variety of cell types, including activated lymphocytes (7). With regard to T cells, in vitro studies have demonstrated that coexpression of Fas and Fas ligand by activated cells can lead to activation-induced cell death (8, 9, 10). In vivo studies have further suggested that the inability of lpr and gld T cells to undergo activation-induced cell death is associated with defects in the maintenance of peripheral tolerance (11) that may somehow allow for the expansion and dysregulated activity of autoreactive T cells capable of activating autoreactive B cells.

While the MRL-lpr/lpr mouse exhibits many of the autoimmune features of human SLE, another clinical syndrome, the Canale-Smith syndrome, has recently been demonstrated to be due to mutations in the human Fas gene (12, 13, 14). Patients with this disorder, now termed the "autoimmune lymphoproliferative syndrome" or ALPS, have waxing and waning peripheral lymphadenopathy and splenomegaly that is frequently misdiagnosed as non-Hodgkins’ lymphoma. They develop a wide range of autoantibodies, of which the anti-platelet and anti-erythrocyte Abs are most often clinically significant. Analysis of the Fas locus in these patients indicates that in contrast to the mice, the human patients appear to be heterozygous for a mutant Fas allele, which acts in a dominant negative fashion, hindering signaling through the normal Fas allele. Interestingly, some family members carrying the same mutant Fas allele do not develop the clinical syndrome (15), perhaps due to other genetic or environmental factors. ALPS patients and their asymptomatic relatives do not seem to have an incidence of true lymphoid neoplasms that is higher than expected.

Somatic mutations of Fas in cancer have recently been reported (16). An analysis of 54 samples of bone marrow from patients with multiple myeloma showed that six had lost Fas mRNA expression completely and another five had acquired Fas gene mutations that were not present in their normal peripheral blood cells. Two of the five myeloma specimens with mutations exhibited the identical mutation that has been seen in the germline DNA of some ALPS patients, and an additional mutation is similar to the lpr allele in mice. Thus, alterations of a single gene may play a role in either cancer or autoimmunity.

A clinical link between cancer and autoimmune disease has been long recognized (reviewed in Ref. 17 and 18), with the association between Sjogren’s syndrome and lymphoma being a striking example (19, 20). Defects in pathways of cell death may contribute to this link. As discussed above, Fas pathway mutations abrogate lymphocyte death leading to a breakdown of tolerance and autoimmunity, and perhaps also malignant transformation. Furthermore, one of the major mechanisms of tumorigenesis is defective cell death or apoptosis, for example, due to overexpression of bcl-2 or loss of p53. t(14;18) translocations leading to overexpression of the anti-apoptotic gene bcl-2 are seen in almost all human patients with low grade lymphoma; of note, mice transgenic for bcl-2 develop not only lymphoma, but also systemic autoimmunity (21). The tumor suppressor gene p53 has multiple roles in cells, one of which is to induce cell cycle arrest and apoptosis via bax. Loss of p53 is one of the most common events in human cancer, and mice in which p53 is deleted via homologous recombination are predisposed to T cell lymphoma and other cancers (22, 23, 24). One of the insights from the mouse models has been that death defects alone are not sufficient to transform cells. Both the bcl-2 transgenic and the p53 knockout mice develop clonal lymphomas, indicating that additional "hits" are required for lymphocyte transformation. In the p53 knockout mice, we have identified overexpression of the regulatory serine-threonine kinase CK2 as one of the additional molecular defects that accelerates lymphoma (25).

Protein kinase CK2 is a ubiquitous heterotetrameric serine-threonine kinase made up of two {alpha} or {alpha}' catalytic subunits and two ß regulatory subunits. CK2 has been reported to phosphorylate a variety of protein substrates including enzymes involved in nucleic acid synthesis (RNA polymerase I and II, DNA ligase), nuclear transcription factors (nuclear factor-{kappa}B, c-Myc, c-Jun, PU.1), signal transduction proteins, protein synthesis factors, and cytoskeletal proteins (reviewed most recently in Ref. 26). CK2 is active in proliferating cells, and a variety of human cancers are associated with high levels of CK2 activity (27, 28, 29, 30, 31). In animals, CK2 overexpression leads to lymphocyte proliferation. For example, massively elevated levels of CK2 are seen in cattle with theileriosis, a fatal lymphoproliferative disease caused by the protozoan parasite Theileria parva (32). We have shown that overexpression of the catalytic {alpha} subunit of CK2 in transgenic mice leads to T cell lymphoma (33), and CK2{alpha} overexpression accelerates lymphoma caused by overexpression of the transcription factor oncogenes myc (33) or tal-1 (34) or by loss of p53 (25). We hypothesized that the increased proliferation caused by CK2 overexpression and the death defect due to the lpr mutation of Fas might cooperate to accelerate lymphocyte transformation. Interestingly, this does not appear to be the case, but coexpression of CK2{alpha} and lpr proves to dramatically accelerate benign lymphoproliferation and autoimmunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

FVB mice transgenic for CK2{alpha} (from line TG.CKA) (33) and MRL-lpr/lpr mice (The Jackson Laboratory, Bar Harbor, ME) were intercrossed to obtain F1 progeny that were 50% FVB and 50% MRL background. F1 mice inheriting the CK2{alpha} transgene were backcrossed with MRL-lpr/lpr mice to obtain F2 mice that averaged 75% MRL background. F2 mice with the CK2{alpha} transgene that were homozygous for the lpr mutation developed lymphadenopathy more rapidly than their nontransgenic littermates. We chose to assess autoimmunity in the F3 backcross generation, which would average 87.5% MRL genetic background. Progeny of the F3 generation segregated with the expected Mendelian frequency into CK+ lpr/lpr (CK2{alpha} transgenic, homozygous for the lpr mutation); CK- lpr/lpr (nontransgenic, homozygous for the lpr mutation); CK+ lpr/+ (CK2{alpha} transgenic, heterozygous for the lpr mutation); CK- lpr/+ (nontransgenic, heterozygous for the lpr mutation). These four groups formed the experimental F3 generation cohort with 6 animals in the CK+ lpr/lpr group, 10 animals in the CK- lpr/lpr group, 10 animals in the CK+ lpr/+ group and 4 animals in the CK- lpr/+ group. Mice in this cohort were phlebotomized serially for analysis of autoantibody production by tail bleed under methoxyflurane anesthesia and euthanized for analysis of adenopathy and renal pathology at 12 wk of age. Other F2 mice were mated two further generations with p53 knockout mice (The Jackson Laboratory) to obtain cohorts of mice that were CK transgenic, lpr homozygous, and p53 homozygous or heterozygous null. These mice were used for lymphomagenesis studies but not for the autoantibody studies because of their mixed FVB, 129, and MRL genetic background. Genotyping was performed by PCR amplification of tail DNA using CK2{alpha} transgene-specific primers (25) in one reaction and primers specific for wild-type or mutant Fas in a second reaction (35). The wildtype and null alleles of p53 were also distinguished by PCR (25). Mice were maintained in a specific pathogen-free environment inside a two-way barrier at the Boston University School of Medicine Core Transgenic Facility (Boston, MA) in accordance with the regulations of the American Association for the Accreditation of Laboratory Animal Care. Mice were observed biweekly for the development of adenopathy or other signs of illness. Mice who developed adenopathy but appeared well were observed as long as possible to determine whether or not malignant lymphoma would develop.

Histology

For histologic examination, tissues were fixed in Optimal fix (American Histology Reagent Company, Lodi, CA) and processed and stained with hematoxylin and eosin in the Transgenic Core Pathology Laboratory at the University of California, Davis, CA.

Lymph node cell transplantation and culture

To determine whether lymphocytes were transformed, cells were transplanted into mixed background FVB/MRL recipients. Single cell suspensions were prepared from enlarged lymph nodes and 106–107 cells were injected s.c. under the flank skin of recipients and the recipients were then observed for up to 12 wk for evidence of tumor development. In addition the lymph node cells were tested for evidence of autonomous proliferation in vitro by resuspending the cells in RPMI 1640 supplemented with 10% FBS, 4 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 50 µm 2-ME and incubating in a 5% CO2 incubator at 37°C. Cell viability was assessed over time by trypan blue staining.

Immunophenotyping

For immunophenotyping, single cell suspensions were prepared from lymph nodes. Contaminating red cells were lysed in hypotonic buffer and the remaining cells were washed twice in ice-cold HBSS with 2% FCS (2% HBSS). A total of 106 cells were stained with Abs to murine B220, CD3, CD4, CD8, Vß2, Vß4, Vß6, Vß8.1/8.2, Vß8.3, Vß10b, and Vß14 (PharMingen, San Diego, CA) all used at 1:100 dilution in 2% HBSS with 2% heat-inactivated normal rabbit serum. Cells were incubated in 50 µl of the primary Abs for 60 min on ice, washed twice with 2% HBSS, resuspended in 50 µl of either phycoerythrin-conjugated streptavidin or FITC-conjugated secondary Ab and incubated on ice for a further 30 min. After washing twice in 2% HBSS, cells were analyzed by flow cytometry using CellQuest software (Becton Dickinson, Mountain View, CA). Cells stained with an isotype-matched irrelevant Ab or with medium alone were used as negative controls and cells obtained from an age-matched MRL-+/+ mouse were used as the positive control.

TCR gene rearrangements

In addition to analyzing Vß usage on the surface of lymphocytes, lymphocyte DNA was analyzed to detect clonal rearrangements of the TCR gene Jß segment. Portions of lymph nodes were snap frozen on dry ice, then homogenized in 4 M guanidium isothiocyanate to inactivate endonucleases. The extract was centrifuged on a CsCl cushion at 42,000 rpm overnight and the DNA layer was removed and precipitated in ethanol. Southern blotting was performed with lymph node or control genomic tail DNA digested with HindIII, electrophoresed on 1% agarose gels (FMC, Rockland, ME), and vacuum blotted with 0.4 N NaOH onto nylon membranes (GeneScreen Plus, NEN, Boston, MA). A 1.6-kb DNA probe for the Jß2B exon of the TCR (36) was radiolabeled and hybridized with the blots overnight at 42°C, washed at 65°C with 2x SSC, 0.1% SDS, then 0.2x SSC, 0.1% SDS, and subjected to autoradiography.

Serologic studies

Serum IgG2a levels were measured by competitive RIA as previously described (37). Serum antinuclear Abs (ANA) were detected by indirect immunofluorescence using HEp-2 cells as substrate. Serum samples were diluted 1:100 in PBS + 0.2% BSA, placed on the HEp-2 cell-plated slides, and incubated for 60 min in a humidified chamber at room temperature. The positive control was a monoclonal ANA with specificity for dsDNA (kindly provided by Dr. Jan Erikson, Wistar Institute, Philadelphia, PA) and the negative control was pooled normal sera from BALB/c mice. Following incubation, the slides were washed for 5 min in PBS and then for 5 min in water. A FITC-conjugated goat anti-mouse total Ig (H + L) (Southern Biotechnology, Birmingham, AL) was then added at 1:100 dilution in PBS + 0.2% BSA, and the slides were incubated in the dark for a further 60 min in a humidified chamber at room temperature. Slides were then washed for 5 min in PBS and 5 min in water before being mounted with Vectashield (Vector Laboratories, Burlingame, CA) and examined by fluorescence microscopy. Scoring of the intensity of ANA staining was done by comparison with the positive control (scored as 3+) and the negative control (scored as 0).

Statistical analysis

In the analysis of IgG2a titers and spleen and lymph node weights the Student’s t test was used to determine the statistical significance of differences between groups. For analysis of glomerular cellularity, analysis of variance (ANOVA) was used to determine the statistical significance of differences between and within groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the CK2{alpha} transgene causes a massive increase in lymphadenopathy and splenomegaly in lpr/lpr mice

In initial stages of our experiments to determine whether the combination of the CK2{alpha} transgene and the lpr Fas allele accelerates autoimmunity or lymphomagenesis, we observed that the first (F2) generation of mice that were homozygous for lpr and bore the CK2{alpha} transgene developed adenopathy much sooner than their nontransgenic lpr/lpr littermates, even in a 75% MRL background. Thus, we decided to analyze mice of the F3 generation in detail, rather than inbreeding the transgene further generations into MRL. In the F3 cohort of mice, visible adenopathy developed in the CK+ lpr/lpr mice by 8–9 wk of age. By 12 wk of age two of the CK+ lpr/lpr mice had died and the others all had massive lymphadenopathy, whereas none of the animals in the other three groups had any visible adenopathy. The mice were euthanized at the 12-wk time point for analysis. Cervical, axillary, inguinal, mesenteric, para-aortic, and inframammary lymph nodes were collected to obtain total lymph node weight. Total lymph node weight at 12 wk of age (Fig. 1Go, black bars) in the CK+ lpr/lpr group was 10.65 ± 1.06 g (mean ± SD, n = 4) as compared with 2.38 ± 2.16 g in the CK- lpr/lpr group (n = 10), (p < 0.005). No difference was observed between the CK+ lpr/+ group (0.16 ± 0.09 g, n = 10) and the CK- lpr/+ group (0.27 ± 0.43 g, n = 4). Thus, the adenopathy was about fivefold greater in the CK+ lpr/lpr mice compared with the CK- lpr/lpr mice.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. CK2{alpha} transgenic lpr/lpr homozygous mice have increased hyperplasia of lymphoid organs compared to their nontransgenic lpr/lpr homozygous littermates. Mice were euthanized at 12 wk of age and cervical, axillary, inguinal, mesenteric, paraaortic, and inframammary lymph nodes were collected to obtain total lymph node weight (black bars). The spleens were also removed and weighed (gray bars). Means and standard errors are illustrated. The differences between the spleen and lymph node weights in the CK+ lpr/lpr and CK- lpr/lpr groups were highly significant (p < 0.005) by two-tailed unpaired Student’s t test assuming unequal variances, as indicated with asterisks.

 
Spleen weights (Fig. 1Go, gray bars) were also much greater in the CK+ lpr/lpr group (2.74 ± 0.78 g) as compared with the CK- lpr/lpr group (0.46 ± 0.54 g), (p < 0.005), a difference of about sixfold. No difference was observed between the CK+ lpr/+ group (0.13 ± 0.01 g) and the CK- lpr/+ group (0.11 ± 0.03 g). Summing the weight of the lymph nodes and spleen, total lymphoid tissue in the CK+ lpr/lpr mice was about 13 g, or almost one-third of their body mass.

The augmented lymphoproliferative disease in CK+ lpr/lpr mice consists primarily of benign polyclonal B220+ CD4-CD8- T cells

A key biologic issue in this experiment was to determine whether the massive increase in lymphocytes in the lymph nodes and spleen of the lpr/lpr mice bearing the CK2{alpha} transgene was due to benign lymphoproliferation or malignant transformation. The initial criteria for evaluating this was the immunophenotype of the cells: lpr disease leads to the accumulation of an unusual B220+ CD4-CD8- T cell, while the lymphomas induced by the CK2{alpha} transgene generally consist of B220- CD4+CD8+ double-positive or CD4+CD8- or CD4-CD8+ single-positive T cells (33). By flow cytometry, consistently more than 70% of the cells in the lymph nodes of CK+ lpr/lpr mice were B220+ CD4-CD8- T cells, 20% were B cells, and less than 10% of the cells were phenotypically normal B220- T cells of the single CD4+ or CD8+ phenotype.

To determine whether these cells fulfilled the biologic definition of transformation, they were transplanted into mixed background FVB/MRL recipients. Lymphoma cells transplanted in this fashion typically grow and form s.c. nodules in 4–6 wk. None of 10 such transplants of CK+ lpr/lpr cells formed tumors in recipients. Furthermore, these cells did not grow autonomously in culture under standard lymphoma cell culture conditions. No significant difference in growth behavior in vitro was observed between CK+ lpr/lpr cells and CK- lpr/lpr cells: cells of either genotype slowly declined in viability with >90% dead by 7–10 days. No autonomous clones of cells arose, even with two additional weeks of culture.

These biologic tests indicate that the CK+ lpr/lpr lymphocytes were not transformed. To determine whether they were polyclonal or monoclonal in origin, Vß usage was analyzed by flow cytometry. The T cells from lymph nodes from two CK+ lpr/lpr mice were stained with seven different Abs against distinct Vß chains. Each node showed heterogeneous Vß usage (although some nodes showed skewing as has been previously reported in MRL-lpr/lpr mice, (38)), and the percentage of cells accounted for with this group of Abs ranged from 44 to 77% of the total number of lymphocytes (Table IGo). There was no case in which a single Vß accounted for the majority of the T cells, which would have indicated that there was a single dominant clone of that particular Vß. The Vß usage in the CK- lpr/lpr, CK+ lpr/+ and CK- lpr/- mice was similarly heterogeneous. Thus, the lymph nodes do not appear to be made up of cells that are progeny of a single T cell with a unique TCR on the surface.


View this table:
[in this window]
[in a new window]
  		Table I. Vß staining of lymph node cells demonstrates a polyclonal T cell response1

 
To verify that no predominant clonal TCR gene rearrangement occurred in the cells in these lymph nodes, DNA from five enlarged lymph nodes from CK+ lpr/lpr mice was hybridized with a probe directed against the Jß2 segment (36). This probe recognizes a band of about 1.8 kb in genomic DNA from either MRL or FVB mice digested with EcoRI, and only this genomic band is visible in DNA prepared from the five lymph nodes (Fig. 2Go). Thus the lymph node T cell population is presumed to contain many rearrangements, none of which can be detected as a dominant novel band. This same finding was confirmed with an alternative digestion with BglII (not shown).



View larger version (144K):
[in this window]
[in a new window]
  		FIGURE 2. CK2{alpha} lpr/lpr lymph nodes lack evidence of clonal TCR rearrangements. DNA was prepared from massively enlarged lymph nodes from five different CK+ lpr/lpr mice, digested with EcoRI, blotted, and hybridized with a probe directed against the Jß2 region. A single band migrating identically with the 1.8-kb germline band in genomic tail DNA (as indicated by an arrow) was seen in all specimens, without any evidence of novel TCR rearrangements as would be seen in clonally derived lymphomas.

 
These assays documented the polyclonal nontransformed nature of the lymphoproliferative process in the CK+ lpr/lpr group of mice. In an attempt to further bring out a contribution of the lpr mutant Fas allele to lymphocyte transformation, mice were intercrossed with p53 knockout mice to produce CK+ lpr/+ or lpr/lpr mice that were partially or completely deficient in p53. In preliminary experiments, no evidence of accelerated lymphocyte transformation over and above the contributions of the combinations of the CK2{alpha} transgene and p53 deficiency (25) was seen and these experiments were not pursued further (data not shown).

CK2 accelerates autoimmunity in both lpr/lpr homozygotes and lpr heterozygotes

The autoimmune syndrome in MRL-lpr/lpr mice is typified by the presence of antinuclear Abs, as is SLE in humans. To determine whether the exacerbated adenopathy produced by the CK2{alpha} transgene was accompanied by augmented autoantibody production, Abs against nuclear Ags present in fixed HEp-2 cells were assessed by immunofluorescence. At 7 wk of age, all six mice in the CK+ lpr/lpr group were ANA positive (with ANA scores ranging from 1+ to 3+) whereas only 1 of 10 mice in the CK- lpr/lpr group was ANA positive (ANA score 1+) (Fig. 3GoA). By 12 wk of age, all lpr/lpr mice (CK+ and CK-) were ANA positive. In the lpr/+ heterozygous mice at 7 wk of age, 4 of 10 mice in the CK+ lpr/+ group exhibited positive ANA staining whereas none of the 4 mice in the CK- lpr/+ group was ANA positive (Fig. 3GoA). By 12 wk of age, 7 of 10 mice in the CK+ lpr/+ group were ANA positive (with ANA scores ranging from 1+ to 3+), whereas all 4 mice in the CK- lpr/+ group remained ANA negative (Fig 3Go B).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. CK2{alpha} transgenic mice have higher levels of serum ANA compared with their nontransgenic (lpr/lpr or lpr/+) littermates. ANAs in serum were measured at 7 wk (A) and 12 wk (B) of age by immunofluorescence using fixed HEp-2 cells as substrate. Scores were assigned by comparison with the staining intensity of a monoclonal ANA with specificity for dsDNA.

 
In MRL-lpr/lpr mice, much of the autoantibody is of the IgG2a isotype, and total IgG2a levels are elevated (39). We measured serum IgG2a levels in the F3 cohort of mice, and found that they followed a pattern similar to the ANA. At 7–8 wk of age, IgG2a levels were 2.35 ± 0.6 mg/ml in the CK+ lpr/lpr group compared with 1.14 ± 0.2 mg/ml in the CK- group (p < 0.03). In the CK+ lpr/+ group IgG2a levels were 0.49 ± 0.1 mg/ml compared with 0.26 ± 0.1 mg/ml the CK- lpr/+ group (Fig. 4Go) (p < 0.03). The correlation of ANA positive with serum IgG2a levels was further reinforced by analysis of the CK+ lpr/+ group at 12 wk of age, which revealed an increase in IgG2a in the seven mice who were ANA positive (4.4 ± 0.82 mg/ml) as compared with the three who were ANA negative (0.75 ± 0.1 mg/ml).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. CK2{alpha} transgenic mice have higher levels of serum IgG2a compared with their nontransgenic (lpr/lpr or lpr/+) littermates. Serum levels of IgG2a were measured at 7–8 wk of age by competitive RIA. Means and SEs are shown. The increases in IgG2a levels by the presence of CK2{alpha} transgene for both the lpr/lpr and lpr/+ mice achieved statistical significance (p < 0.03) using a one-tailed unpaired Student’s t test assuming unequal variances, as indicated by asterisks.

 
One of the major consequences of autoantibody production in MRL-lpr/lpr mice is the development of glomerulonephritis, which generally progresses into clinically significant renal disease by 6 mo of age and contributes to the premature mortality of the mice. In the F3 cohort, which we analyzed at 12 wk of age, the histology of the kidneys was normal in the CK- lpr/lpr, CK+ lpr/+ and CK- lpr/+ animals (Fig. 5GoA). However, the CK+ lpr/lpr mice demonstrated severe renal disease with evidence of glomerular hypercellularity, mesangial matrix increase, cellular and fibrous glomerular crescent formation, tubular vacuolation, and cast formation (Fig. 5Go, B–D). Occasional foci of interstitial mononuclear cell infiltrates were seen. The glomerular hypercellularity was quantified by counting the number of nuclei (cells) per glomerulus in each of the groups. For 30 glomeruli in each of a pair of CK+ lpr/lpr mice, there were 93.4 ± 3.3 (mean ± SD) cells per glomerulus. The CK- lpr/lpr mice, the CK+ lpr/+ mice, and the CK- lpr/+ mice were nearly identical, at 41.6 ± 1.5, 43.7 ± 2.0, and 43.3 ± 1.6, respectively (Fig. 6Go). Interestingly, no differences were seen on indirect immunofluorescence staining for IgG (data not shown), consistent with reports that renal disease does not necessarily correlate with the degree of immune complex deposition (40, 41).



View larger version (154K):
[in this window]
[in a new window]
  		FIGURE 5. CK2{alpha} transgenic mice develop renal disease earlier than their nontransgenic littermates. Representative renal histology of CK-lpr/lpr mice (A) and CK+ lpr/lpr mice (B, C, and D) at 12 wk of age. A, A typically normal-appearing glomerulus (g) and tubules (t) are seen in a kidney section from a CK- lpr/lpr mouse. Note the cuboidal appearance of parietal epithelial cells (arrow), a normal feature of mouse glomeruli. B, An enlarged, hypercellular glomerulus in a CK+ lpr/lpr mouse indicative of proliferative glomerulonephritis. The tubular vacuolation (arrows) is suggestive of severe proteinuria. C, Crescentic glomerulonephritis in another CK+ lpr/lpr mouse. A large, circumferential fibrocellular crescent is seen (arrow) compressing the glomerular tuft, together with periglomerular fibrosis and an infiltrate of inflammatory cells. D, Large proteinaceous casts (arrows) within the lumen of renal tubules of a CK+ lpr/lpr mouse.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 6. CK2{alpha} transgenic lpr-lpr homozygous mice have glomerular hypercellularity at 3 mo of age. Two mice from each of the four experimental groups (CK+ lpr/lpr; CK- lpr/lpr; CK+ lpr/+; CK- lpr/+) were randomly selected at 12 wk of age and hematoxylin and eosin-stained sections of kidney were prepared. For each animal, the number of cells in each of 30 glomeruli was counted. Statistical analysis was performed by analysis of variance. A statistically significant difference (p < 0.0001) was found between the cell number in the CK+ lpr/lpr group and the other three groups (asterisk), indicating the presence of a proliferative glomerulonephritis in the CK+ lpr/lpr group. No differences were found between the other three groups or between the animals within each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that overexpression of the {alpha} catalytic subunit of the serine-threonine protein kinase CK2 markedly exacerbates the T cell lymphoproliferative disorder and autoimmune manifestations associated with defective Fas expression in lpr/lpr mice. The CK2{alpha} transgene-positive lpr/lpr mice examined at 12 wk of age had on average about fivefold greater total lymph node weight, sixfold greater spleen weight, and became ANA positive much earlier than their nontransgenic lpr/lpr littermates. The transgene-positive mice had histologic evidence of renal disease while the nontransgenic controls had not developed histologic abnormalities by the termination of the experiment.

Other studies have demonstrated that coexpression of transgenes such as bcl-2 (42) or the serine-threonine kinase pim-1 (43) or lack of TNF receptor type I (44) can accelerate the lymphoproliferative disease of lpr/lpr mice. These manipulations appear to target various arms of the apopototic cascades and were interpreted as demonstrating the cooperativity of multiple apoptosis abnormalities. In contrast, overexpression of the CK2{alpha} transgene enhances T cell cycling and activation responses but does not inhibit activation-induced cell death (S.-T. Ju and D. C. Seldin, unpublished observations). Thus, we attribute the dramatic lymphoproliferation seen in the CK+ lpr/lpr mice to the combination of a defect in cell death and cell cycling.

In spite of the acceleration of benign lymphoproliferative disease by the CK2{alpha} transgene, malignant lymphomas were not seen. This was also the case in the F2 generation of mice, some of whom lived as long as 18 wk, and in small numbers of mice bearing a p53 null allele. This contrasts with the ability of overexpression of CK2 to accelerate myc (33), tal-1 (34), or p53-deficiency (25) induced lymphomas. Thus, CK2 dysregulation can contribute to both a benign lymphoproliferative process and lymphocyte transformation, depending upon the context of other mutations. Fas appears to be an important regulator of the benign, but not the malignant, process in our mice; similar results have been found in the lpr/lpr mice transgenic for pim-1 kinase (43) where no malignant transformation was observed despite the documented oncogenic potential of pim-1. On the other hand, there was modest acceleration of the weak oncogenic effect of L-myc overexpression in an lpr/lpr background, suggesting a possible interaction of Fas mutation and myc dysregulation (45).

The dysregulation of lymphocyte growth in the CK2{alpha} transgenic MRL-lpr/lpr mice was accompanied by dysregulated lymphocyte function leading to accelerated autoimmune disease. The transgenic mice developed ANA, increased IgG2a, and renal disease earlier than their nontransgenic littermates. The renal disease seen in the CK+ lpr/lpr mice appeared histologically similar to that of older MRL-lpr/lpr mice, which has been described as glomerular hypercellularity due to mesangial and endothelial cell proliferation with occasional crescent formation (46). CK2 dysregulation also caused an acceleration of the development of ANA in lpr/+ heterozygous mice, although by the 12-wk termination of the experiment, no evidence of end organ disease had appeared. The development of autoantibodies in the lpr/+ heterozygote background is reminiscent of the autoimmune manifestations of the human ALPS disease, in which patients heterozygous for a Fas mutation develop a lymphoproliferative disease accompanied by a variety of autoantibodies. Interestingly, some of their clinically unaffected kinships may bear the identical mutant Fas allele (15), highlighting the role of other genetic or environmental determinants of autoimmune disease. This accelerated autoimmune disease in the CK2{alpha} transgenic lpr/lpr mice differs from the reports on the bcl-2 transgenic lpr/lpr mice (42) and the pim-1 transgenic lpr/lpr mice (43), where no enhancement of autoantibody production or increase in serum Ig was observed. Loss of the TNF receptor type I induced both accelerated lymphoproliferation and autoimmunity but not lymphoma (44), much as we see with overexpression of CK2{alpha}.

We have shown here that dysregulated expression of CK2 can contribute to autoimmunity, making it one of the few defined molecular abnormalities known to do so. Dysregulated expression of CK2 has been reported in a variety of human malignancies (27, 28, 29, 30, 31), and these experiments may provide a mechanistic link between lymphoid malignancies and autoimmune disease. However, another speculative interpretation of these experiments is that the pathways that cause benign lymphoproliferation and autoimmunity vs malignant lymphoma might in some circumstances diverge or even be exclusive of one another, as we did not see any cases of true lymphoma in the CK+ lpr/lpr mice. Perhaps lymphocytes at the stage of maturation necessary for the induction of autoimmunity are no longer as susceptible to transformation as their less differentiated precursors. Future studies examining the interaction of other oncogenes with Fas mutations will further clarify the biologic relationships between lymphoproliferation, autoimmunity, and lymphoma.


   Acknowledgments
 
We thank Judy Walls and Dr. Robert D. Cardiff in the Core Transgenic Pathology Laboratory at the University of California, Davis, CA, for assistance with the preparation and interpretation of histology. Dr. Barry Sleckman (Children’s Hospital, Boston MA) kindly provided the probe for the Jß2 region.


   Footnotes
 
1 This work was supported by Grant IRG-97U from the American Cancer Society (I.R.R.), National Institutes of Health Institutional Training Grant DK07053-22 (P.I. D.J.S.) (I.R.R.), National Institutes of Health Grant AI 32531 (A.M.R.), and a grant from the Massachusetts Division of the American Cancer Society (D.C.S.). Back

2 Address correspondence and reprint requests to Dr. David C. Seldin, Departments of Medicine and Microbiology, Boston Medical Center, 88 East Newton Street, Boston, MA 02118. E-mail address: Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; ALPS, autoimmune lymphoproliferative syndrome; ANA, anti-nuclear Ab(s). Back

Received for publication March 26, 1998. Accepted for publication July 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cohen, P., R. Eisenberg. 1991. lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol. 9:243.[Medline]
  2. Theofilopoulos, A., F. Dixon. 1985. Murine models of systemic lupus erythematosus. Adv. Immunol. 37:269.[Medline]
  3. Izui, S., V. Kelley, K. Masuda, H. Yoshida, J. Roths, E. Murphy. 1984. Induction of various autoantibodies by mutant gene lpr in several strains of mice. J. Immunol. 133:277.[Abstract]
  4. Kelley, V., J. Roths. 1985. Interaction of mutant lpr gene with background strain influences renal disease. Clin. Immunol. Immunopathol. 37:220.[Medline]
  5. Watanabe-Fukunaga, R., C. Brannan, N. Copeland, N. Jenkins, S. Nagata. 1992. Lymphoproliferative disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[Medline]
  6. Takahashi, T., M. Tanaka, C. Brannan, N. Jenkins, N. Copeland, T. Suda, S. Nagata. 1994. Generalized lymphoproliferative disease in mice caused by a point mutation in the Fas ligand. Cell 76:969.[Medline]
  7. Rouvier, E., M. Luciani, P. Golstein. 1993. Fas Involvement in Ca2+-independent T cell-mediated cytotoxicity. J. Exp. Med. 177:195.[Abstract/Free Full Text]
  8. Ju, S.-T., D. Panka, H. Cui, R. Ettinger, M. El-Khatib, D. Sherr, B. Stanger, A. Marshak-Rothstein. 1995. FasL interactions required for programmed cell death after T cell activation. Nature 373:444.[Medline]
  9. Brunner, T., R. Mogil, D. LaFace, N. Yoo, A. Mahboubi, F. Echeverri, S. Martin, W. Force, D. Lynch, C. Ware, D. Green. 1995. Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T cell hybridomas. Nature 373:441.[Medline]
  10. Dhein, J., H. Walczak, C. Baumler, K.-M. Debatin, P. Krammer. 1995. Autocrine T-cell suicide mediated by APO-1(Fas/CD95). Nature 373:438.[Medline]
  11. Singer, G., A. Abbas. 1994. The Fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1:365.[Medline]
  12. Drappa, J., A. Vaishnaw, K. Sullivan, J.-L. Chu, K. Elkon. 1996. Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity. N. Engl. J. Med. 335:1643.[Abstract/Free Full Text]
  13. Fisher, G., F. Rosenberg, S. Straus, J. Dale, L. Middelton, A. Lin, W. Strober, M. Lenardo, J. Puck. 1995. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935.[Medline]
  14. Rieux-Laucat, F., F. Le Deist, C. Hivroz, I. Roberts, K. Debatin, A. Fischer, J. de Villartay. 1995. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268:1347.[Abstract/Free Full Text]
  15. Sneller, M., J. Wang, J. Dale, W. Strober, L. Middelton, Y. Choi, T. Fleisher, M. Lim, E. Jaffe, J. Puck, M. Lenardo, S. Straus. 1997. Clinical, immunologic, and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis. Blood 89:1341.[Abstract/Free Full Text]
  16. Landowski, T., N. Qu, I. Buyuksal, J. Painter, W. Dalton. 1997. Mutations in the Fas antigen in patients with multiple myeloma. Blood 90:4266.[Abstract/Free Full Text]
  17. Fishman, P.. 1994. Autoimmunity and cancer. Isr. J. Med. Sci. 30:15.[Medline]
  18. Osterland, C.. 1996. Monoclonal gammopathies antigen driven in autoimmune disease. Clin. Exp. Rheumatol. 14:(Suppl.14):S37.
  19. Anaya, J., H. McGuff, P. Banks, N. Talal. 1996. Clinicopathological factors relating malignant lymphoma with Sjogren’s syndrome. Semin. Arthritis Rheum. 25:337.[Medline]
  20. Valesini, G., R. Priori, D. Bavoillot, J. Osborn, M. Danieli, N. D. Papa, R. Gerli, M. Pietrogrande, M. Sabbadini, F. Silvestris, L. Valsecchi. 1997. Differential risk of non-Hodgkin’s lymphoma in Italian patients with primary Sjogren’s syndrome. J. Rheumatol. 24:2376.[Medline]
  21. Strasser, A., S. Whittingham, D. Vaux, M. Bath, J. Adams, S. Cory, A. Harris. 1991. Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88:8661.[Abstract/Free Full Text]
  22. Donehower, L. A., M. Harvey, B. L. Slagle, M. J. McArthur, C. J. Montgomery, J. S. Butel, A. Bradley. 1992. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356:215.[Medline]
  23. Jacks, T., L. Remington, B. O. Williams, E. M. Schmitt, S. Halachmi, R. T. Bronson, R. A. Weinberg. 1994. Tumor spectrum analysis in p53-mutant mice. Curr Biol. 4:1.[Medline]
  24. Harvey, M., M. J. McArthur, C. J. Montgomery, A. Bradley, L. A. Donehower. 1993. Genetic background alters the spectrum of tumors that develop in p53-deficient mice. FASEB J. 7:938.[Abstract]
  25. Landesman-Bollag, E., P. L. Channavajhala, R. D. Cardiff, D. C. Seldin. 1998. p53 Deficiency and misexpression of protein kinase CK2{alpha} collaborate in the development of thymic lymphomas in mice. Oncogene. 16:2965.[Medline]
  26. Allende, J. E., C. C. Allende. 1995. Protein kinases. 4. Protein kinase CK2: an enzyme with multiple substrates and puzzling regulation [Review]. FASEB J. 9:313.[Abstract/Free Full Text]
  27. Faust, R. A., M. Gapany, P. Tristani, A. Davis, G. L. Adams, K. Ahmed. 1996. Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation. Cancer Lett. 101:31.[Medline]
  28. Yenice, S., A. T. Davis, S. A. Goueli, A. Akdas, C. Limas, K. Ahmed. 1994. Nuclear casein kinase 2 (CK-2) activity in human normal, benign hyperplastic, and cancerous prostate. Prostate 24:11.[Medline]
  29. Daya-Makin, M., J. S. Sanghera, T. L. Mogentale, M. Lipp, J. Parchomchuk, J. C. Hogg, S. L. Pelech. 1994. Activation of a tumor-associated protein kinase (p40TAK) and casein kinase 2 in human squamous cell carcinomas and adenocarcinomas of the lung. Cancer Res 54:2262.[Abstract/Free Full Text]
  30. Gapany, M., R. A. Faust, S. Tawfic, A. Davis, G. L. Adams, K. Ahmed. 1995. Association of elevated protein kinase CK2 activity with aggressive behavior of squamous cell carcinoma of the head and neck. Mol. Med. 1:659.[Medline]
  31. Munstermann, U., G. Fritz, G. Seitz, Y. P. Lu, H. R. Schneider, O. G. Issinger. 1990. Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue. Eur. J. Biochem. 189:251.[Medline]
  32. ole-MoiYoi, O. K., W. C. Brown, K. P. Iams, A. Nayer, T. Tsukamoto, M. D. Macklin. 1993. Evidence for the induction of casein kinase II in bovine lymphocytes transformed by the intracellular protozoan parasite Theileria parva. EMBO J. 12:1621.[Medline]
  33. Seldin, D. C., P. Leder. 1995. Casein kinase II {alpha} transgene-induced murine lymphoma: relation to theileriosis in cattle. Science 267:894.[Abstract/Free Full Text]
  34. Kelliher, M. A., D. C. Seldin, P. Leder. 1996. Tal-1 induces T cell acute lymphoblastic leukemia accelerated by casein kinase II{alpha}. EMBO J. 15:5160.[Medline]
  35. Wang, JKM, B. Zhu, S-T. Ju, J. Tschopp, A. Marshak-Rothstein. 1997. CD4+ T cells reactivated with superantigen are both more sensitive to FasL-mediated killing and express a higher level of FasL. Cell. Immunol. 179:153.[Medline]
  36. Siu, G., S. P. Clark, Y. Yoshikai, M. Malissen, Y. Yanagi, E. Strauss, T. W. Mak, L. Hood. 1984. The human T cell antigen receptor is encoded by variable, diversity, and joining gene segments that rearrange to generate a complete V gene. Cell 37:393.[Medline]
  37. Perkins, D., J. Michaelson, A. Marshak-Rothstein. 1987. The lpr gene is associated with resistance to engraftment by lymphoid but not erythroid stem cells from normal mice. J. Immunol. 138:466.[Abstract]
  38. Singer, P. A., A. N. Theofilopoulos. 1990. T-cell receptor Vß repertoire expression in murine models of SLE. Immunol. Rev. 188:103.
  39. Eisenberg, R., J. Winfield, P. Cohen. 1982. Subclass restriction of anti-Sm antibodies in MRL mice. J. Immunol. 129:2146.[Abstract]
  40. Clynes, R., C. Dumitru, J. Ravetch. 1998. Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science 279:1052.[Abstract/Free Full Text]
  41. Bullard, D., P. King, M. Hicks, B. Dupont, A. Beaudet, K. Elkon. 1997. Intercellular adhesion molecule-1 deficiency protects MRL/Mpj-Fas lpr mice from early lethality. J. Immunol. 159:2058.[Abstract]
  42. Reap, E., N. Felix, P. Wolthusen, B. Kotzin, P. Cohen, R. Eisenberg. 1995. bcl-2 transgenic lpr mice show profound enhancement of lymphadenopathy. J. Immunol. 155:5455.[Abstract]
  43. Moroy, T., A. Grzeschiczek, S. Petzold, K. Hartmann. 1993. Expression of a Pim-1 transgene accelerates lymphoproliferation and inhibits apoptosis in lpr/lpr mice. Proc. Natl. Acad. Sci. USA 90:10734.[Abstract/Free Full Text]
  44. Zhou, T., 3rd C. K. Edwards, P. Yang, Z. Wang, H. Bluethmann, J. D. Mountz. 1996. Greatly accelerated lymphadenopathy and autoimmune disease in lpr mice lacking tumor necrosis factor receptor I. J. Immunol. 156:2661.[Abstract]
  45. Zornig, M., A. Grzeschiczek, M. B. Kowalski, K. U. Hartmann, T. Moroy. 1995. Loss of Fas/Apo-1 receptor accelerates lymphomagenesis in E µ L-MYC transgenic mice but not in animals infected with MoMuLV. Oncogene 10:2397.[Medline]
  46. Andrews, B., R. Eisenberg, A. Theofilopoulos, S. Izui, C. Wilson, P. McConahey, E. Murphy, J. Roths, F. Dixon. 1978. Spontaneous murine lupus-like syndromes. J. Exp. Med. 148:1198.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
Z. Gurel, T. Ronni, S. Ho, J. Kuchar, K. J. Payne, C. W. Turk, and S. Dovat
Recruitment of Ikaros to Pericentromeric Heterochromatin Is Regulated by Phosphorylation
J. Biol. Chem., March 28, 2008; 283(13): 8291 - 8300.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. C. Beaudette-Zlatanova, T. Ling, M. J. Shlomchik, A. Marshak-Rothstein, and I. R. Rifkin
B Cells and Dendritic Cells from V{kappa}8 Light Chain Transgenic Mice Activate MRL-lpr/gld CD4+ T Cells
J. Immunol., July 1, 2006; 177(1): 45 - 52.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
P. Hilgard, M. J. Czaja, G. Gerken, and R. J. Stockert
Proapoptotic function of protein kinase CK2{alpha}" is mediated by a JNK signaling cascade
Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G192 - G201.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol PatholHome page
L. D. Koller, B. V. Stang, M. Posada de la Paz, and M. V. Ruiz Mendez
Pathology of "Toxic Oils" and Selected Metals in the MRL/lpr Mouse
Toxicol Pathol, October 1, 2001; 29(6): 630 - 638.
[Abstract] [PDF]

Home page
 J. Immunol.Home page
S. F. Greer, Y.-n. Wang, C. Raman, and L. B. Justement
CD45 Function Is Regulated by an Acidic 19-Amino Acid Insert in Domain II That Serves as a Binding and Phosphoacceptor Site for Casein Kinase 2
J. Immunol., June 15, 2001; 166(12): 7208 - 7218.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Pena-Rossi, L. A. Zuckerman, J. Strong, J. Kwan, W. Ferris, S. Chan, A. Tarakhovsky, A. D. Beyers, and N. Killeen
Negative Regulation of CD4 Lineage Development and Responses by CD5
J. Immunol., December 15, 1999; 163(12): 6494 - 6501.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rifkin, I. R.
Right arrow Articles by Seldin, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rifkin, I. R.
Right arrow Articles by Seldin, D. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS