tnf receptor-associated factor 3 is required for t cell-mediated immunity and tcr/cd28 signaling

The Journal of Immunology

TNF Receptor-Associated Factor 3 Is Required forT Cell-Mediated Immunity and TCR/CD28 Signaling

Ping Xie,* Zachary J. Kraus,† Laura L. Stunz,‡ Yan Liu,* and Gail A. Bishop†,‡,x,{

We recently reported that TNFR-associated factor (TRAF)3, a ubiquitously expressed adaptor protein, promotes mature B cell

apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. In this article, we report the generation and

characterization of T cell-specific TRAF32/2 mice, in which the traf3 gene was deleted from thymocytes and T cells. Ablation of

TRAF3 in the T cell lineage did not affect CD4 or CD8 T cell populations in secondary lymphoid organs or the numbers or

proportions of CD4+,CD8+ or double-positive or double-negative thymocytes, except that the T cell-specific TRAF32/2 mice had

a 2-fold increase in FoxP3+ T cells. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3-deficient mice

exhibited defective IgG1 responses to a T-dependent Ag, as well as impaired T cell-mediated immunity to infection with Listeria

monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon costimulation and

that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide

insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity. The Journal of Immunology, 2011,

186: 143–155.

TNFR-associated factor (TRAF)3, a member of the TRAFfamily of cytoplasmic adaptor proteins, is used in signal-ing by the TNFR superfamily and TLRs (1–5). TRAF3

binds directly to almost all of the receptors of the TNFR super-family that do not contain death domains, including CD40,BAFFR, TACI, BCMA, LTbR, CD27, CD30, RANK, HVEM,EDAR, XEDAR, 4-1BB, OX-40, and GITR, as well as a viraloncogenic mimic of the TNFR family, latent membrane protein 1encoded by EBV (1, 4, 6–8). Recent evidence demonstrates thatTRAF3 also regulates signaling by TLR3, 4, 7/8, and 9 (5, 9, 10).The shared usage of TRAF3 by so many receptors predicts itsbroad functional roles. In support of this, mice made geneticallydeficient in TRAF3 die within 10 d of birth, demonstrating theubiquitous and critical developmental functions of TRAF3 (11).To circumvent the early lethality problem of TRAF32/2 mice

and to explore the in vivo functions of TRAF3 in various cell typesof adult mice, we recently generated conditional TRAF3-deficient(TRAF3flox/flox) mice by using a conditional gene-targeting strategy,

which allows deletion of the TRAF3 gene in specific cell typesor tissues (12). Such a mouse model is particularly useful, becauseit has become increasingly clear that specific TRAF functions canbe quite cell-type and receptor specific (3–5, 13, 14). Specific ab-lation of TRAF3 in B lymphocytes results in severe peripheralB cell hyperplasia, which culminates in hyperimmunoglobulinemia,splenomegaly, lymphadenopathy, and autoimmune reactivity. Re-sting splenic B cells from these mice exhibit remarkably prolongedsurvival ex vivo, independent of the B cell survival factor BAFF,and show increased levels of nuclear NF-kB2 but decreased levelsof protein kinase C-d in the nucleus (12). Furthermore, in vivoadministration of a soluble fusion protein that blocks BAFF andAPRIL from binding to their receptors does not reverse peripheralB cell hyperplasia in B-TRAF32/2 mice (12). Thus, our findingsindicate that a major homeostatic function of TRAF3 in peripheralB cells is to promote spontaneous apoptosis, a conclusion sub-sequently confirmed by Gardam et al. (15).TRAFs 2 and 3 are now thought to play distinct and comple-

mentary functions in assembling a regulatory complex of TRAF2,TRAF3, cellular inhibitors of apoptosis 1/2, and NF-kB–inducingkinase in resting B cells (16, 17). Consistent with the notion thatprolonged survival is a predisposing factor for oncogenic trans-formation, two recent studies simultaneously reported that ho-mozygous deletion and inactivating mutations of the TRAF3 geneoccur in ∼12–17% of human patients with multiple myeloma, amalignancy of terminally differentiated B cells (18, 19). Collec-tively, these findings demonstrate that TRAF3 is a critical regu-lator of peripheral B cell homeostasis.In addition to TRAF3’s multiple roles in B lymphocytes, early

evidence implicates it in the regulation of T cell function. Inadoptive-transfer experiments, fetal liver cells from day 14TRAF32/2 embryos reconstituted T cell, B cell, granulocytic, anderythroid lineages in lethally irradiated mice (11). Interestingly,the immune response to a T-dependent (TD) Ag is defective inTRAF32/2 reconstituted mice, although the immune response toa T-independent Ag is normal. These findings indicate a re-quirement for TRAF3 in TD immune responses in vivo, but it isunclear how TRAF3 deficiency might affect T cell functions (11).In the 12 y since this early report, little has been uncovered about

*Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ08854; †Graduate Program in Immunology, ‡Department of Microbiology, and xDepart-ment of Internal Medicine, University of Iowa; and {Veterans Affairs Medical Center,Iowa City, IA 52242

Received for publication January 27, 2010. Accepted for publication October 16,2010.

This work was supported by a National Scientist Development grant from the Amer-ican Heart Association (to P.X.), National Institutes of Health Grant AI28847, anda Veterans Affairs Career Award (both to G.B.). This work was supported in part withresources and the use of facilities at the Iowa Veterans Affairs Medical Center.

Address correspondence and reprint requests to Dr. Gail A. Bishop, 2193B MERF,Department of Microbiology, University of Iowa, Iowa City, IA 52242. E-mailaddress: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this paper: DP, double positive; LAT, linker of activatedT cells; LLO, listeriolysin O; LMC, littermate control; LM-OVA, Listeria monocy-togenes expressing secreted ovalbumin protein; LN, lymph node; N, purified splenicnon-T cells; NS, nonspecific band; PI, propidium iodide; p.i., postinfection; PLC,phospholipase-C; T, T cells; TD, T-dependent; TNP, trinitrophenol; TNP-KLH,trinitrophenol-keyhole limpet hemocyanin; TRAF, TNFR-associated factor; Treg,regulatory T cell.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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the specific roles of TRAF3 in T cell biology. However, TRAF3binds to many TNFR family receptors expressed by T cells, andthese receptors make important contributions to T cell activationand costimulation (reviewed in Ref. 20). In 2008, Gardam et al.(15) reported the generation of TRAF3flox/floxLck-Cre mice. Theinvestigators found that TRAF3 deletion from T cells did not re-sult in T cell expansion in the spleen and lymph nodes (LNs). Incontrast to TRAF32/2 B cells, TRAF32/2 T cells do not exhibitprolonged survival, although high constitutive levels of NF-kB2processing were observed in both cell types in the absence ofTRAF3 (15). However, no further study of TRAF3 in T cell-mediated immunity or T cell signaling was described. In thecurrent study, we generated T cell-specific TRAF32 /2 mice(TRAF3flox/floxCD4-Cre, T-TRAF32/2 mice) to discover the func-tions of TRAF3 in T cell-mediated immunity.CD4-Cre–mediated deletion of floxed genes occurs predom-

inantly from double-positive (DP) thymocytes onward (21, 22),hence T-TRAF32/2 mice have TRAF3 deleted in CD4 and CD8T cells. We report that deletion of TRAF3 in T lymphocytesresulted in defective TD IgG1 responses, as well as impairedAg-specific CD8 and CD4 T cell responses to infection with theintracellular pathogen Listeria monocytogenes. Furthermore, wefound that TRAF3 was unexpectedly recruited to the TCR andCD28 signaling complex upon costimulation and that TCR/CD28-mediated signaling events were affected by TRAF3 deficiency.Taken together, our findings demonstrate essential roles forTRAF3 in TCR/CD28 signaling and T cell-mediated immunity.

Materials and MethodsGeneration of T-TRAF32/2 mice

TRAF3flox/flox mice were generated as previously described (12). CD4-Cretransgenic mice (21) were purchased from Taconic Farms (Germantown,NY). The Taconic CD4-Cre mice have been bred for nine generations ontoC57BL/6 mice. TRAF3flox/flox mice were crossed with CD4-Cre mice togenerate TRAF3+/floxCD4-Cre mice, which were subsequently backcrossedwith TRAF3flox/flox mice to generate TRAF3flox/floxCD4-Cre (T-TRAF32/2)mice. Mouse tails were screened by genomic PCR using primer sets FC3 +BT6 and Cre-F + Cre-R, as described (12). Deletion of exons 1 and 2 of theTRAF3 gene in thymocytes and splenic T cells was detected by genomicPCR using primers U7 and BT6, as previously described (12). T-TRAF32/2

mice analyzed had a mixed genetic background between 129/SvJ andC57BL/6, and littermates were used as controls for all experiments. All micewere kept in specific pathogen-free conditions in the Gene Targeting CoreFacility at the University of Iowa and were used in accordancewith NationalInstitutes of Health guidelines and under an animal protocol approved by theAnimal Care and Use Committee of the University of Iowa.

Abs and reagents

Polyclonal rabbit Abs to TRAF1 (N19), TRAF3 (H122), and TRAF6(H274) were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonalrabbit Ab to TRAF2 was from Medical and Biological Laboratories(Nagoya, Japan). FITC-, PE-, or Cy5-labeled mAbs against mouse CD3,TCRab, TCRgd, CD4, CD8, CD45R (B220), CD25, CD43, CD44,CD62L, CD127, and FoxP3 were purchased from eBioscience (San Diego,CA). Anti-mouse IFN-g, TNF-a, IL-4, and IL-2 ELISA Ab pairs were alsopurchased from eBioscience. FITC- or PE-labeled Abs against mouse TNF-a and IFN-g were purchased from BioLegend (San Diego, CA). Polyclonalrabbit Abs against total or phosphorylated ERK, PLCg1, Lck, and ZAP70were from Cell Signaling Technology (Beverly, MA). Anti-actin Ab wasfrom Chemicon International (Temecula, CA). HRP-labeled secondary Abswere from Jackson ImmunoResearch Laboratories (West Grove, PA). Al-kaline phosphatase-conjugated polyclonal goat Abs specific for mouse IgMand IgG1 were from Southern Biotechnology Associates (Birmingham, AL).Pan T cell, CD4, CD8, and regulatory T cell (Treg) purification kits werefrom Miltenyi Biotec (Auburn, CA). A mouse anti-hamster IgG Ab (cloneMAH1.12) was obtained from R&D Systems (Minneapolis, MN). ElongaseDNA polymerase, CFSE labeling kit, Dynabeads-protein G, and tissue cul-ture supplements, including stock solutions of sodium pyruvate, L-glutamine,nonessential amino acids, and HEPES (pH 7.55), were from Invitrogen

(Carlsbad, CA). DNA oligonucleotide primers were obtained from IntegratedDNATechnologies (Coralville, IA). Alkaline phosphatase substrates, Igepal,and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis,MO).

Flow cytometry

Single-cell suspensions were made from the thymus, spleen, and LN.Erythrocytes from spleen were depleted with ACK lysis buffer. Cells (0.53106) were then blocked with rat serum and FcR-blocking Ab (2.4G2) andincubated with various Abs conjugated to FITC, PE, PerCP, or Cy5 formultiple color fluorescence surface staining. For cellularity analysis, cell-surface markers examined included CD3, TCRab, TCRgd, CD4, CD8,CD45R (B220), CD25, CD43, CD44, CD62L, and CD127, as indicatedin the figures. For FoxP3 staining, cells were stained with Cy5-labeledanti-CD4 and PE-labeled anti-CD25 Abs, followed by fixation and per-meabilization using a BD Cytofix/Cytoperm kit (BD Biosciences, SanJose, CA) and intracellular staining with FITC-labeled anti-FoxP3 Abs.Listmode data were acquired on a FACSCalibur (BD Biosciences) usingCell Quest software. The results were analyzed using FlowJo software(Tree Star, Ashland, OR). Forward light scatter/side scatter gating forsingle lymphocytes, which excludes cell aggregates, small erythrocytes,and dead cell debris, was used to analyze flow-cytometric data.

Immunization and trinitrophenol-specific Ig ELISA

For TD Ab responses, mice were immunized i.p. with 100 mg tri-nitrophenol-keyhole limpet hemocyanin (TNP-KLH; Biosource Technol-ogies, Vacaville, CA) precipitated in alum and boosted with 100 mgTNP-KLH/Alum on day 21. Sera were collected on days 7, 14, and 28 afterthe first immunization. Serum levels of anti-trinitrophenol (TNP) IgM andIgG1 were measured by ELISA as described previously (12). Standardcurves were determined on each plate using serial dilutions of purifiedTNP-specific IgM or IgG1 standards (BD Pharmingen, San Diego, CA).Plates were read on a Versamax plate reader (Molecular Devices, Sunny-vale, CA), and results were analyzed using SoftMax Pro 4.0 software.Multiple 1:5 or 1:10 serial dilutions of each serum sample were examined.Each standard curve contained 11 dilution points; in all cases, the co-efficient of determination for the standard curve (r2) was .0.98. The di-lution factor that gave A405 (OD 405 nm) values within the linear range(0.1–1.5) of standard curves of ELISA was used to calculate the concen-trations of TNP-specific IgM and IgG1.

L. monocytogenes infection

Recombinant L. monocytogenes expressing secreted OVA protein (LM-OVA) (23) was provided by Dr. John Harty (The University of Iowa).Eight- to twelve-week-old mice were infected i.v. with 0.053 LD50 (5 3103 CFU) virulent LM-OVA. At days 3 and 7 (primary response) post-infection (p.i.), spleens and livers were collected to determine bacterialload, as detailed below. Livers and spleens were homogenized in 10 ml0.2% Igepal in H2O. Organ homogenates were serially diluted and platedon streptomycin agar plates to determine CFU of LM-OVA in liver andspleen. Splenocytes were also collected at the time points listed in thefigures for flow-cytometric analysis of OVA-specific CD8 and listeriolysinO (LLO)-specific CD4 T lymphocytes.

Enumerating OVA-specific or LLO-specific T lymphocytes byintracellular staining for IFN-g and TNF-a

Quantification of Ag-specific CD8 and CD4 T cell responses was de-termined by intracellular cytokine staining, as described (24). Briefly,spleens were harvested from infected mice, and erythrocytes were de-pleted. Splenocytes were washed and resuspended in fresh medium. Totalsplenocytes were counted on a hemacytometer, and 200 ml splenocytes(2 3 106 cells) was transferred to sterile plastic tubes. Two hundredmicroliters of medium plus 2 ml/ml GolgiPlug (brefeldin A; BD Phar-mingen), with or without 1 mM purified OVA peptide (SIINFEKL, forOVA-specific CD8 T cell analysis) or 5 mM LLO O Ag LLO190–201 (forLLO-specific CD4 T cell analysis), was added to the tubes. Splenocyteswere subsequently incubated at 37˚C for 6 h before staining. Splenocyteswere surface stained with allophycocyanin-labeled anti-Thy1.2 and PerCP-labeled anti-CD8a or anti-CD4 Abs before fixation and intracellular cy-tokine staining. For IFN-g–producing cell analysis, staining was com-pleted using PE-labeled anti–IFN-g Abs. For TNF-a–producing cell anal-ysis, cells were costained with FITC-labeled anti–IFN-g and PE-labeledanti–TNF-a Abs.

144 TRAF3 REGULATES T CELL-MEDIATED IMMUNITY

Liver lymphocyte analysis

Littermate control (LMC) and T-TRAF32/2 mice, 7–8 wk old, wereinfected i.v. with 0.3 LD50 virulent L. monocytogenes. On days 5 and 7 p.i.,mice were euthanized, and their livers were perfused with cold Hanksmedium, rinsed in PBS, and forced through a 70-mm screen, according tothe protocol of Schmidt et al. (25). After centrifugation at 500 3 g, 4˚C,the pellet was resuspended in 15 ml 35% Percoll in Hanks medium.The cells were pelleted again at 500 3 g at 25˚C with no brake. Theresulting pellet was treated with ACK to lyse RBCs and then suspended in10 ml RPMI 1640 + 10% FCS and washed three times by centrifugation toyield the mononuclear cell fraction. Total cell counts were determined, andthe cells were stained with allophycocyanin–anti-CD4 and FITC–anti-CD8and analyzed on a FACSCalibur.

Splenic total, CD4, and CD8 T cell purification

Splenic T cell subsets were prepared from naive 2–3-mo-old mice. SplenicT cells and non-T cells were separated using a mouse Pan T Cell IsolationKit (#130-090-861; Miltenyi Biotec) and a magnetic separator (MiltenyiBiotec), following the manufacturer’s protocols. CD4 Th cells were pu-rified using a mouse CD4 T Cell Isolation Kit by negative selection (#130-090-860, Miltenyi Biotec), and Tregs were depleted using a mouse CD4+

CD25+ Regulatory T Cell Isolation Kit (#130-091-041; Miltenyi Biotec),following the manufacturer’s protocols. CD8 T cells were purified usinga mouse CD8 T Cell Isolation Kit by negative selection (#130-090-859;Miltenyi Biotec), following the manufacturer’s protocols. The purity ofisolated populations was monitored by FACS analysis using B220-, CD3-,CD4-, CD8-, or CD25-specific Abs, and cell preparations with .90%purity were used for further experiments. Purified splenic T cells werecultured in mouse culture medium (RPMI 1640 medium supplementedwith 5% FCS, 10 mM 2-ME, 10 mM HEPES [pH 7.55], 1 mM sodiumpyruvate, 2 mM L-glutamine, and 0.1 mM nonessential amino acids). Fordetection of TRAF3 expression, thymocytes, splenic T cells, or non-T cellsand purified CD4+ or CD8+ splenic T cells were directly lysed as pre-viously described (26). Immunoblot analysis was performed as previouslydescribed (26).

Survival assay and cell cycle analysis

Purified splenic CD4 (Treg depleted) or CD8 T cells (13 106/ml/well) werecultured in 24-well plates in the absence or presence of 0.5 mg/ml plate-bound anti-CD3 mAb (clone 145–2C11; eBioscience) with or without 2mg/ml soluble anti-CD28 mAb (clone 37.51; eBioscience) at 37˚C. At eachtime point, an aliquot of cells was removed to determine the number ofviable cells by staining with trypan blue. PI staining and CFSE labelingwere performed as previously described (27), and DNA content and CFSEintensity were quantified using a benchtop FACScan flow cytometer (BDBiosciences).

Cytokine ELISA

Purified splenic CD4 (Treg depleted) or CD8 T cells were cultured at 1 3106 cells/well in 24-well plates in the absence or presence of 0.5 mg/mlplate-bound anti-CD3 mAb with or without 2 mg/ml soluble anti-CD28mAb at 37˚C. Culture supernatants were collected at various time points.Concentrations of IL-2, IL-4, IFN-g, and TNF-a in culture supernatantswere determined by ELISA using cytokine-specific coating Abs and bio-tinylated detection Abs (eBioscience), as previously described (28).

Th1 polarization

Naive CD4+ T cells were purified from LMC and T-TRAF32/2 mice bynegative selection (Miltenyi Biotec CD4+ T cell isolation kit, #130-090-860), followed by positive selection on anti-CD62L beads (#130-049-701;Miltenyi Biotec). Cells were all .90% CD4+ by flow cytometry. Cellswere suspended in mouse culture medium at 0.5 3 106/ml and culturedin 24-well plates that had been coated overnight with 5 mg/ml anti-CD3agonistic Ab (eBioscience) in PBS and subsequently washed twice withmedium. Anti-CD28 agonistic Ab (10 mg/ml) and 20 U/ml IL-2(PeproTech, Rocky Hill, NJ) was added to all cultures, and the Th1 con-ditions included 4 ng/ml IL-12 p70 (BioLegend) and 2 mg/ml anti-mouseIL-4–blocking Ab (clone 11B11; eBioscience). After 4 d, the cells wereharvested, washed three times in Mouse Media, and replated at 53 105/mlin a 48-well plate in PMA (50 ng/ml) and ionomycin (1 mg/ml) or withplate-bound anti-CD3 Ab (0.5 mg/ml) and soluble anti-CD28 Ab (10 mg/ml). Supernatants were harvested from the PMA + ionomycin culturesafter 24 h and from the CD3 + CD28-stimulated cultures after 48 h of thesecondary culture. TNF, IL-2, and IFN-g were measured by ELISA, asdescribed above.

Early TCR signaling measurements

Splenic T cells were purified from 2–3-mo-old naive mice using the PanT cell purification kit, and Tregs were depleted using the Treg-purificationkit (both from Miltenyi Biotec). Purified T cells (without Tregs) werestimulated as previously described (29). Briefly, cells were starved inserum-free medium at 37˚C in 5% CO2 for 1 h and incubated with 5 mg/mlanti-CD3 Ab alone or with 5 mg/ml anti-CD3 Ab plus 5 mg/ml anti-CD28Ab on ice for 30 min. Cells were washed once in serum-free medium toremove unbound Ab. TCRs were cross-linked with a secondary Ab (mouseanti-hamster IgG, clone MAH1.12; R&D Systems) for various time peri-ods at 37˚C in a water bath, as indicated in the figures. TCR signaling wasterminated by adding hot 53 SDS sample buffer and boiling for 10 min at95˚C. Proteins were separated by SDS-PAGE and blotted with Abs tophospho- or total linker of activated T cells (LAT), phospholipase-C(PLC)-g1, ZAP70, ERK, and IkBa. Immunoblot analysis was performedusing various Abs, as previously described (26). Bands of immunoblotswere quantitated using a low-light imaging system (LAS-4000, FUJIFILMMedical Systems USA, Stamford, CT).

Sorting of Thy1+CD252CD44low cells

Spleen and LN cells from LMC and T-TRAF32/2 mice were treated withACK to lyse RBCs and washed three times in BCM-10 without phenol red.A total of 5 3 108 cells of each genotype were stained with allophyco-cyanin–anti-Thy1 Ab, PE–anti-CD44 Ab, and FITC–anti-CD25 Ab.The cells were sorted at the University of Iowa flow-cytometry facility onthe BD FACS Aria II. The cells were scatter gated on the lymphocytepopulation and then gated on the Thy1highCD252 population. The resultingpopulation was gated on the CD44low population, excluding the brightestCD44-stained population. The collected population was 17.2% of the totalT-TRAF32/2 cells and 15.9% of the LMC. The cells were kept at 4˚Cduring staining and collection. The sorted cells were serum starved andthen stimulated, as described above, for early TCR signaling measure-ments.

Magnetic immunoprecipitation of the TCR/CD28 signalingcomplex

Splenic T cells were purified from 2–3-mo-old naive mice using the PanT cell purification kit (Miltenyi Biotec). Cells (3 3 107 cells for eachcondition) were rinsed with serum-free media and incubated with mAbs: 5mg/ml anti-CD3 alone, 5 mg/ml anti-CD28 alone, or 5 mg/ml anti-CD3plus 5 mg/ml anti-CD28 on ice for 30 min. Cells were washed once inserum-free medium to remove unbound Ab. Cells were subsequently in-cubated with protein G-magnetic beads (Dynabeads; Invitrogen) prearmedwith mouse anti-hamster IgG (cross-linking Ab for anti-CD3 and anti-CD28) on ice for 30 min to allow the binding of beads with Absto T cells and then were stimulated at 37˚C for 3 or 7 min. Cells werechilled on ice, pelleted by centrifugation at 4˚C, and lysed in 400 ml ice-cold lysis buffer (0.5% Triton X-100, 100 mM NaCl, 40 mM Tris [pH 7.5],1 mM CaCl2, 1 mM MgSO4, complete EDTA-free protease inhibitormixture [Roche Applied Science, Madison, WI], 2 mM Na3VO4 and 50mg/ml DNase) for 30 min on ice with repeated mixing. Magnetic beadswere pelleted on a magnetic rack without centrifugation (Invitrogen), andunbound materials were removed following the manufacturer’s protocol.The magnetic beads were washed five times with ice-cold lysis buffer(without DNase), left in a final volume of 50 ml, and boiled in SDS samplebuffer. Aliquots of lysates and the immunoprecipitates were separated bySDS-PAGE and analyzed by immunoblot analysis.

Statistics

For direct comparison of TNP-specific Ig isotype levels, OVA-specific CD8T cells, or LLO-specific CD4 T cells between LMC and T-TRAF32/2mice,statistical significance was determined using the unpaired t test for two-tailed data; p values, 0.05 are considered significant, and p values, 0.01are considered very significant.

ResultsGeneration of T-TRAF32/2 mice

We previously generated mice homozygous for the floxed TRAF3allele (TRAF3flox/flox) (12). To delete the loxP-flanked TRAF3alleles specifically in T lymphocytes, we used a transgenic mousestrain expressing Cre under the control of the CD4 promoter/enhancer/silencer, providing a T cell-specific source of Cre (21,30). It was shown that CD4-Cre mediates deletion of loxP-flanked

The Journal of Immunology 145

gene segments in most thymocytes at the CD4+CD8+ stage (21,

22). TRAF3flox/floxCD4-Cre mice were born at the expected Men-

delian frequencies and survive and breed normally. We verified

excision of the first two coding exons of the TRAF3 gene in thy-

mocytes by genomic PCR and the elimination of TRAF3 protein

expression in thymocytes, as well as in splenic CD4 and CD8

T cells of TRAF3flox/floxCD4-Cre mice (T-TRAF32/2 mice), by

Western blot analysis (Fig. 1A, 1B). Interestingly, we found that

TRAF1 and TRAF2 protein levels were modestly upregulated in

TRAF32/2 T cells. In contrast, the level of TRAF6 was not ob-

viously changed by TRAF3 deletion. Similarly, we also observed

modest upregulation of TRAF1 and TRAF2 in TRAF32/2 B cells

(12). This raises the possibility that upregulated TRAF1 and

TRAF2 may partially compensate for the loss of TRAF3 in both

cell types. Together, these results validated TRAF3flox/floxCD4-

Cre mice as T cell-specific TRAF32/2 (T-TRAF32/2) mice.

Thymocytes and peripheral T cell populations in T-TRAF32/2

mice

The thymus size of T-TRAF32/2 mice was comparable to that ofTRAF3flox/flox LMC mice. Flow-cytometric analyses revealed thatT-TRAF32/2 mice exhibited normal frequencies and numbers ofthymocyte populations, including double-negative (CD42CD82),DP (CD4+CD8+), and CD4 (CD4+CD82) and CD8 (CD42CD8+)single-positive cells (Fig. 1C, 1D). Thus, elimination of TRAF3from DP thymocytes did not affect CD4/CD8 lineage commitmentor survival in the thymus.In contrast to the severe splenomegaly and lymphadenopathy

observed in B-TRAF32/2 mice, adult T-TRAF32/2mice exhibitedslightly bigger spleens and normal LNs compared with those fromLMC mice or wild-type mice (Fig. 1E, P. Xie, unpublished obser-vations). We performed cellularity analysis by immunofluorescencestaining and flow cytometry to determine B and T cell populations

FIGURE 1. Normal thymocytes and pe-

ripheral B and T cell populations in T-

TRAF32/2 mice. A, Detection of the exci-

sion of exons 1 and 2 of the TRAF3 gene

(TRAF32 allele) in thymocytes by genomic

PCR analysis using primers U7 + BT6. A

2.6-kb PCR product was amplified from the

TRAF3flox allele, whereas a 645-bp PCR

product was amplified from the TRAF32

allele. B, Verification of TRAF3 deletion in

thymocytes and splenic T cells by Western

blot analysis. Splenic pan T, CD8+, and CD4+

T cells were purified from LMC and T-

TRAF32/2 (T-T32/2) mice by negative se-

lection using magnetic beads. Total cellu-

lar proteins were extracted from thymocytes,

purified splenic non-T cells (N), T cells (T),

or CD8+ or CD4+ T cells. Each protein

blot was immunoblotted for TRAF3 and then

stripped and reprobed for TRAF2, TRAF1,

TRAF6, and actin. NS, nonspecific band. C,

Representative FACS profiles of thymocytes

from LMC and T-TRAF32/2 mice examined

for CD4 and CD8 expression. D, Percentages

(upper panel) and numbers (lower panel) of

CD42CD82 (double negative), CD4+CD8+

(DP), CD4+, and CD8+ cells in the thymus

of LMC and T-TRAF32/2 mice. E, Slight

increase in spleen weight of T-TRAF32/2

mice. F, Percentages (upper panels) and

numbers (lower panels) of B and T cells in

spleens and LNs of LMC and T-TRAF32/2

mice. B cells and T cells were identified

by FACS analysis using B220 and CD3, re-

spectively. Data shown are results of five

independent experiments (mean 6 SEM).

Mice analyzed were 8–12 wk old.


in the spleen and LNs. We found that T-TRAF32/2 mice havenormal proportions and absolute numbers of B cells (B220+) andT cells (CD3+) in the spleen and LNs compared with LMC (Fig.1F). T-TRAF32/2 mice also display normal proportions of CD4(CD4+CD82CD252) and CD8 (CD42CD8+) T cells in the spleenand LNs (Fig. 2A). Interestingly, however, T-TRAF32/2 miceshow increased frequency and numbers of CD4+CD25+Foxp3+

Tregs in the spleen and LNs (Fig. 2A, 2B). These results dem-onstrate that deletion of TRAF3 from thymocytes at the DP stagedid not substantially affect CD4 and CD8 T cell development andhomeostasis, but it did alter the maturation or homeostasis ofTregs in secondary lymphoid organs.

Altered expression profile of CD44 and CD43 in T-TRAF32/2

mice

As the first step in examining the phenotype of TRAF32/2 T cellsin vivo, we determined the expression profile of a series of cellsurface markers on CD8 and CD4 T cells. We found that theexpression profiles of CD44, a marker of effector and memoryT cells, were considerably altered on CD8 and CD4 T cells in thespleen of T-TRAF32/2 mice (Fig. 2C). The CD44hi population ofCD8 T cells was absent in the spleen and LNs of T-TRAF32/2

mice, suggesting that they may have decreased proportions ofeffector and memory T cells. Interestingly, the expression level ofCD43 was drastically decreased on CD8, but not CD4, T cells inthe spleen and LNs of T-TRAF32/2 mice (Fig. 2D). In con-trast, the expression profiles of CD62L, CD69, and CD127 in T-TRAF32/2 mice were not different from those observed in LMC(P. Xie and L.L. Stunz, unpublished observations). Consideringthat CD44 and CD43 are involved in the regulation of T cellmigration, adhesion, and activation (31–34), altered expressionprofiles of these two molecules, together with the increased fre-quency of Tregs, predict that T-TRAF32/2 mice might exhibitaltered T cell-mediated immunity. Thus, we next sought to in-vestigate immune responses to TD Ag immunization and bacterialinfections.

Defective TD IgG1 response in T-TRAF32/2 mice

Following immunization with a TD Ag (TNP-KLH), T-TRAF32/2

mice showed a modestly decreased TNP-specific IgM response onday 7 but almost normal TNP-specific IgM responses on days 14and 28 compared with LMCmice (Fig. 3). In contrast, TNP-specificIgG1 responses were defective in T-TRAF32/2 mice at all timepoints examined in this study (Fig. 3). Our data extended the initialfindings using cells from the neonatally lethal TRAF32/2 mousethat suggested TRAF3 is required for TD Ab responses (11) andshowed that TRAF3 in T cells is especially critical for the pro-duction of IgG1. Importantly, our data also suggest that this re-quirement is intrinsic to T cell function.

Impaired primary T cell responses to L. monocytogenesinfections in T-TRAF32/2 mice

L. monocytogenes, a facultative intracellular bacterium, was usedas a model for T cell-mediated immune responses to bacterialinfections. LM-OVA (23) allowed the detection of Ag-specificCD8 T cell responses. Naive T-TRAF32/2 and LMC mice wereinfected i.v. with 0.05 3 LD50 (5 3 103 CFU) of virulentLM-OVA. Six of 24 (25%) T-TRAF32/2 mice succumbed to thislow, normally sublethal dose of LM-OVA infection between days7 and 16 p.i. (Fig. 4A). In contrast, all LMC mice (n = 28) sur-vived. Bacterial loads in the spleen on day 3 p.i. were comparablebetween LMC and T-TRAF32/2 mice, and both groups were ableto clear bacterial infection in the spleen on day 7 (P. Xie, un-published observations). However, two of six (33%) T-TRAF32/2

mice, but none of the LMC mice, had high bacterial loads in the

liver on day 7 p.i. (Fig. 4B). The slower clearance of bacteria inthe liver, but not in the spleen, suggests that TRAF32/2 T cellsmay have defects in migrating to target organs or in traffickingwithin the target tissues. To test this possibility, we analyzed CD4and CD8 T cells in the liver on days 5 and 7 p.i. We found thatT-TRAF32/2 mice had decreased numbers of CD4 and CD8 Tcells in the liver on day 5 but slightly increased numbers on day 7p.i. (Supplemental Fig. 1). Thus, TRAF32/2 CD4 and CD8 T cellsexhibited delayed responses in migrating and trafficking to theliver following primary L. monocytogenes infection. This is con-sistent with our observation that peripheral TRAF32/2 T cellsexhibited altered expression profiles of CD44 and CD43, twomolecules known to participate in the regulation of T cell mi-gration and trafficking (31–34). Together, these data demonstratethat TRAF3 deficiency in T cells compromised the ability of miceto resist L. monocytogenes infection.To investigate the mechanisms contributing to defective function

of TRAF3-deficient T cells, we enumerated Ag-specific T cells inLMC and T-TRAF32/2 mice using intracellular staining of IFN-gand TNF-a following in vitro stimulation with purified peptidesOVA257–264 (for CD8 T cell detection) or LLO190–201 (for CD4T cell detection). On day 7 postprimary infection, there weremarkedly fewer OVA-specific IFN-g– or TNF-a–producing CD8T cells in T-TRAF32/2 mice, both in frequency and total numbers(Fig. 4C, 4D). Reductions in the frequency and total numbers ofLLO-specific IFN-g– or TNF-a–producing CD4 T cells were evenmore striking in T-TRAF32/2 mice (Fig. 4C, 4D). However, onday 10 postprimary infection, the frequency and number of OVA-specific CD8 T cells were nearly normal in T-TRAF32/2 mice,whereas LLO-specific CD4 T-TRAF32/2 T cells were still mod-erately decreased compared with LMC (Fig. 4E). Thus, TRAF3deficiency seemed to delay the expansion of Ag-specific T cells,with CD4 T cells affected to a greater degree.

Decreased proliferation of TRAF32/2 CD4 and CD8 T cells inresponse to TCR and CD28 stimulation

Defective TD IgG1 responses and impaired T cell-mediated im-munity to bacterial infection observed in T-TRAF32/2 mice maybe due to multiple mechanisms, including decreased intrinsicfunction of CD4 helper and/or CD8 effector T cells or increasedsuppressive function of Tregs. Thus, we tested whether TRAF3deficiency affects the intrinsic function of CD4 and CD8 T cells.Splenic CD4 and CD8 T cells were purified from naive mice bynegative selection using magnetic beads, and Tregs were depletedfrom CD4 T cells as detailed inMaterials and Methods. Followingstimulation with anti-CD3 or anti-CD3 plus anti-CD28 mAbs,proliferation of CD4 and CD8 T cells was reduced in the absenceof TRAF3 (Fig. 5A). Analysis of cell-cycle distribution by PIstaining and flow cytometry showed that fewer TRAF32/2 CD4and CD8 T cells entered the cell cycle (S/G2/M phase), and moreTRAF32/2 T cells underwent activation-induced apoptosis (Fig.5B). This was in sharp contrast to B cells lacking TRAF3, whichshow marked resistance to apoptosis, possibly due to the lack ofTRAF3–NF-kB–inducing kinase interactions (12). CFSE-dilutionexperiments verified the decreased proliferation response inTRAF32/2 T cells following stimulation with anti-CD3 and anti-CD28 mAbs (Supplemental Fig. 2). In contrast, a normal pro-liferation response was seen in TRAF32/2 CD4 and CD8 T cellsfollowing stimulation with anti-CD3 Ab and ConA, thereby ex-cluding the possibility that TRAF3 deficiency globally impairscellular proliferation in T cells (Supplemental Fig. 2). Further,CD3 and CD28 were expressed on TRAF32/2 T cells at a levelequivalent to that observed in LMC T cells (P. Xie, unpublishedobservations), suggesting a direct role for TRAF3 in CD3- and


FIGURE 2. Increased Tregs and altered CD44 and CD43 expression profiles on peripheral CD8 and CD4 T cells in T-TRAF32/2 mice. A, Representative

FACS profiles of splenocytes from LMC and T-TRAF32/2 mice stained for CD4, CD25, and Foxp3 expression. B, Percentages (upper panels) and numbers

(lower panels) of CD4 (CD4+CD82CD252), CD8 (CD42CD8+), and Treg (CD4+CD25+Foxp3+) cells in spleens and LN of LMC and T-TRAF32/2 mice.

Data shown are results of four independent experiments (mean 6 SEM). C, Representative FACS profiles of CD44 expression on CD8+ or CD4+ T cells in

spleens, LN, and thymus of LMC and T-TRAF32/2 mice. D, Representative FACS profiles of CD43 expression on CD8+ or CD4+ T cells in spleens, LN,

and thymus of LMC and T-TRAF32/2 mice. Mice analyzed were 8–12 wk old.


CD28-induced proliferative responses. Taken together, these datademonstrate that TRAF3 promotes TCR- and CD28-mediated pro-liferation and protection from spontaneous apoptosis in T cells,which is in sharp contrast to the proapoptotic function ofTRAF3 in B cells.The increased apoptosis observed in TRAF32/2 T cells after

stimulation with anti-CD3Ab alone and anti-CD3 + anti-CD28 Abs(Fig. 5) could be due to a decrease in prosurvival factors, such asBcl-xL and Bcl-2, or an increase in Bim. Thus, we examined theprotein levels of Bcl-xL, Bcl-2, and Bim in purified TRAF32/2 andLMC T cells; they were equivalent between TRAF32/2 and LMCT cells in the absence and presence of CD3 + CD28 stimulation(Fig. 5C). We previously found that TRAF3 deficiency results inconstitutive processing of NF-kB2 from inactive p100 to the activep52, leading to prolonged B cell survival. We next examined NF-kB2 processing in purified TRAF32/2 and LMCT cells. Consistentwith the study by Gardam et al. (15), we observed markedly in-creased NF-kB2 processing in purified TRAF32/2 T cells com-pared with that observed in LMC cells (Supplemental Fig. 3).However, in contrast to TRAF32/2 B cells, constitutive NF-kB2processing did not lead to prolonged survival or decreased apo-ptosis in TRAF32/2 T cells, indicating that this is a cell type-specific phenomenon. Together, these results indicate that the in-creased apoptosis observed in TRAF32/2 T cells is not mediatedby alterations in Bcl-2 family member expression or constitutiveNF-kB2 activation. The specific mechanism responsible for thiselevated apoptosis will require additional investigation.

Diminished cytokine production of TRAF32/2 CD8 and CD4T cells in response to TCR and CD28 stimulation

One potential mechanism contributing to the observed defectiveAg-specific IgG1 response is decreased IL-4 secretion by TRAF32/2

CD4 Th cells. Moreover, LLO-specific CD4 and OVA-specific CD8TRAF32/2 T cells produced less IFN-g and TNF-a on a per-cellbasis as determined by intracellular staining and flow cytometry(Fig. 4C). These observations prompted us to further analyze cy-tokine production in isolated TRAF32/2 CD4 or CD8 T cells, de-pleted of Tregs, in response to TCR/CD28 stimulation. We foundthat following stimulation with anti-CD3 and anti-CD28 mAbs,production of IL-4, IL-2, TNF-a, and IFN-g was drastically di-minished by TRAF3 deficiency in CD4 T cells (Treg depleted) (Fig.6A) and was modestly reduced in TRAF32/2 CD8 T cells (Fig. 6B).However, production of IL-2 and TNF-a induced by PMA andionomycin was not decreased in TRAF32/2 CD4 and CD8 T cells(P. Xie, unpublished observations), thereby excluding the possibility

that TRAF3 deficiency globally affects the cytokine-productionmachinery in T cells. Such decreases in cytokine production inthe absence of TRAF3 may contribute to the defective TD IgG1responses (Fig. 3) and impaired T cell immunity to L. mono-cytogenes infection in T-TRAF32/2 mice (Fig. 4). The decreasedIL-2 production may also contribute to the reduced proliferationof TRAF32/2 CD4 T cells following TCR and CD28 stimulation(Fig. 5). The T cell-proliferation response and cytokine productionindicate that the intrinsic function of CD4 helper and/or CD8 ef-fector T cells in response to TCR/CD28 engagement is inhibited byTRAF3 deficiency.Another potential mechanism contributing to the defects in

expansion of Ag-specific CD4 T cells observed in T-TRAF32/2

mice (Fig. 4) is defective development or differentiation of Thcells. To test this, we performed in vitro skewing experiments withnaive CD4 T cells purified from LMC and T-TRAF32/2 mice.Because LLO peptide-specific, IFN-g– and TNF-a–producingCD4 T cells were analyzed in the L. monocytogenes-infectionexperiments (Fig. 4), our skewing experiment primarily focusedon Th1 polarization. Naive CD4 T cells were purified from LMCand T-TRAF32/2 mice and cultured in the presence of 5 mg/mlplate-bound anti-CD3 Ab, 10 mg/ml anti-CD28 Ab, 20 U/ml IL-2,4 ng/ml IL-12 p70, and 2 mg/ml anti-mouse IL-4 Ab for 4 d toinduce Th1 differentiation. We found that in vitro polarized Th1cells had a partial reduction in IFN-g and TNF-a production, aswell as a marked decrease in IL-2 secretion following stimulationwith PMA + ionomycin or CD3 + CD28 (Supplemental Fig. 4).These data suggest that differentiation and maturation of CD4+

Th1 cells, which require stimulation signals from the TCR andcostimulatory molecules, may be affected, in part, by TRAF3 de-ficiency.

Impaired phosphorylation of proximal signaling components ofTCR/CD28 in the absence of TRAF3

To understand how TRAF3 deficiency impaired the distal effec-tor functions of TCR and CD28 signaling, including proliferation,cytokine production, and in vitro Th1 polarization, we investigatedproximal-signaling events following TCR and CD28 engagementin TRAF32/2 T cells. Splenic T cells were purified from naivemice by negative selection, and Tregs were depleted using mag-netic beads. After stimulation with anti-CD3 or anti-CD3 + anti-CD28 mAbs, phosphorylation of proximal-signaling components(ERK, LAT, PLCg1, and ZAP70) was measured by immunoblotanalysis. Phosphorylation of these TCR-signaling components in-duced by anti-CD3 mAb alone was normal in TRAF32/2 T cells.

FIGURE 3. Defective TD IgG1 response in T-

TRAF32/2 mice. LMC and T-TRAF32/2 mice (8–10

wk old, n = 10 for each group) were immunized with

the TD Ag TNP-KLH/Alum and boosted on day 21

after the first immunization. Sera were collected on

days 7, 14, and 28 after the first immunization. Serum

levels of anti-TNP IgM and IgG1 were measured by

ELISA. Multiple serial dilutions of each serum sample

were tested to ensure the readout was within the linear

range of the assay.


In contrast, the synergistic effects induced by anti-CD3 + anti-CD28 mAbs were abolished by TRAF3 deficiency (Fig. 7A, Sup-plemental Fig. 5). Interestingly, activation of the classical NF-kB1pathway was not affected by TRAF3 deletion, as measured byphosphorylation and degradation of IkBa after stimulation withanti-CD3 Ab alone or anti-CD3 + anti-CD28 Abs (Fig. 7A, Sup-plemental Fig. 5). Considering that T-TRAF32/2 mice had a de-creased proportion of CD44hi memory T cells (Fig. 2), it ispossible that the decrease in CD44hi memory T cells contributes tothe observed decrease in phosphorylation of ERK, LAT, PLCg1,and ZAP70 following stimulation with TCR and CD28. We nextsorted Thy1+CD252CD44low naive T cells and repeated the ex-periments of stimulation with anti-CD3 + anti-CD28 Abs. We

found that sorted TRAF32/2 naive T cells also displayed partialimpairment in phosphorylation of ERK, LAT, PLCg1, and ZAP70in response to stimulation through CD3 and CD28 (Fig. 7B, Sup-plemental Fig. 5). Together, our results indicate that TRAF3 de-ficiency specifically impairs TCR and CD28 synergy at an earlyreceptor-proximal step, including phosphorylation of ERK, LAT,PLCg1, and ZAP70.

Recruitment of TRAF3 to the TCR/CD28-signaling complex

Previous studies in B lymphocytes demonstrated that ligation ofCD40 recruits TRAF3 to CD40 signaling rafts (35). This, togetherwith the receptor-proximal nature of TRAF3 effects in TCR/CD28signaling, prompted us to assess the possibility that TRAF3 asso-

FIGURE 4. Impaired primary T cell responses to L. monocytogenes infections in T-TRAF32/2 mice. A, Survival rate of LMC and T-TRAF32/2 mice

after primary infection with 53 103 CFU of virulent L. monocytogenes. B, Bacterial numbers in livers on day 7 p.i. C, Representative FACS profiles of Ag-

specific splenic CD8 and CD4 T cells on day 7 p.i., determined in the absence or presence of peptide stimulation. D, Percentages (among total splenocytes)

and numbers of Ag-specific (IFN-g– or TNF-a–producing) CD8 and CD4 T cells in spleens of LMC and T-TRAF32/2 mice on day 7 postprimary infection.

E, Percentages and numbers of Ag-specific T cells in spleens of LMC and T-TRAF32/2 mice on day 10 postprimary infection. The data are the results of

three independent experiments (mean 6 SEM). Mice infected were 2–3 mo old.


ciates with the TCR and/or CD28-signaling complex. Splenic totalT cells were purified from naive mice by negative selection. Wefound that upon stimulation with anti-CD3 or anti-CD28 mAbsalone, TRAF3 was not detected in the TCR or CD28-signalingcomplex in splenic T cells purified from wild-type or LMC mice(Fig. 7C). However, upon coligation of TCR and CD28 by anti-CD3 and anti-CD28 mAbs, TRAF3 was coimmunoprecipitatedwith the TCR/CD28-signaling complex in splenic T cells purifiedfrom LMC mice (Fig. 7C, 7D). It was shown that TRAF6 andMalt1 participate in TCR/CD28 signaling in T cells (36) and thatTRAF3 interacts with Malt1 in B cells (37). Thus, we examinedthe possible involvement of TRAF6 and Malt1 in the TRAF3association. However, coimmunoprecipitation of TRAF6 or Malt1

with CD3 + CD28 stimulation was not detected in T cells underour experimental conditions (Fig. 7C). Recruitment of ZAP70 tothe TCR/CD28-signaling complex was not affected in the absenceof TRAF3 (Fig. 7D). Thus, TRAF3 is a component of the TCR/CD28-signaling complex, but the absence/presence of TRAF3does not affect the association between TCR and ZAP70. To thebest of our knowledge, this is the first demonstration of the as-sociation of TRAF3 with an Ag-receptor complex.

DiscussionSubstantial progress has recently been made in understanding thefunction of TRAF3 in B lymphocytes (1, 12, 13, 15–17, 26, 38).Although initial studies of chimeric mice reconstituted with

FIGURE 5. Diminished proliferation and enhanced apoptosis of splenic CD4 T cells following stimulation through CD3 and CD28. Splenic CD4+ T cells

were purified from 2–3-mo-old LMC and T-TRAF32/2 mice, and Tregs were depleted using anti-CD25 magnetic beads. Cells were cultured ex vivo in the

absence or presence of stimulation with 0.5 mg/ml of plate-bound anti-CD3 mAb, alone or in combination with 2 mg/ml of soluble anti-CD28 mAb. A,

T cell survival ex vivo. The numbers of viable cells at each time point were determined by staining with trypan blue. Data shown are results of three

independent experiments (mean 6 SEM). B, Cell-cycle analysis by PI staining and FACS. Representative graphs of PI staining are shown, and the per-

centages of apoptotic cells (DNA content , 2n) and proliferating cells (2n , DNA content # 4n) are indicated. C, Western blot analysis of Bcl-xL, Bcl2,

and Bim. Splenic pan T cells were purified from 2–3-mo-old LMC and T-TRAF32/2 mice by negative selection, and Tregs were depleted using anti-CD25

Ab magnetic beads. Cells were stimulated with anti-CD3 + anti-CD28 Abs at 37˚C for the indicated times. Total cellular lysates were immunoblotted for

Bcl-xL, Bcl2, and Bim, followed by actin.


TRAF32/2 fetal liver cells implicated TRAF3 in the TD Ab re-sponse (11), no further information about the specific roles ofTRAF3 in T cell biology has been forthcoming, including whetherTRAF3 plays direct or indirect roles in T cell function. To addressthis important gap in knowledge, we generated and characterizedT cell-specific TRAF3-deficient mice. In addition to leading todefective TD IgG1 responses, ablation of TRAF3 in T cells im-paired T cell-mediated immunity to infection with L. mono-cytogenes, indicating that CD8 and CD4 T cells require TRAF3for optimal function. Thus, TRAF3 plays unique and indispen-sable roles in T cell immunity, which are not readily compensatedfor by other members of the TRAF family. Our subsequent evi-dence revealed that the defective T cell-mediated immunity ob-served in T-TRAF32/2 mice resulted from decreased intrinsicfunction of CD4 helper and CD8 effector T cells. Furthermore,TCR/CD28-mediated signaling events were affected by TRAF3deficiency in CD4 and CD8 T cells, including proliferation, cy-tokine production, and phosphorylation of ERK, LAT, PLCg1,and ZAP70. Strikingly, we found that TRAF3 was recruitedto the TCR/CD28-signaling complex and coimmunoprecipitatedwith TCR and CD28 upon costimulation. Together, our findingsidentify TRAF3 as an essential participant in TCR/CD28 sig-

naling and, thus, as a critical regulator of T cell-mediated im-munity.TRAF3 directly binds to almost all receptors of the TNFR su-

perfamily that do not contain death domains, as well as the EBV-encoded oncogenic protein latent membrane protein 1 (1, 4, 6–8).TRAF3 also regulates signaling by TLRs through interaction withadaptor proteins (5, 9, 10). Our present study expands TRAF3-interacting receptors to include the TCR/CD28 complex. It is in-triguing that TRAF3 was not detectably coimmunoprecipitatedwith CD3 or CD28 alone, although the cytoplasmic tail of mouseCD28 contains a putative TRAF3-binding site PYQPYA (resi-dues 203–208). One possibility is that association between CD28(or CD3) and TRAF3 is weak and susceptible to disruption by thedetergent contained in the lysis buffer but is strengthened bycoligation of CD3 and CD28. Alternatively, TRAF3 may berecruited by another signaling component that is present onlywhen CD3 and CD28 are engaged. Consistent with the lack ofdemonstrable direct binding between CD3 and TRAF3, CD3-induced phosphorylation of ERK, LAT, PLCg1, and ZAP70 wasnormal in TRAF32/2 T cells. However, CD3-induced prolifera-tion and cytokine production were also affected, in part, in theabsence of TRAF3, suggesting that TRAF3 also participates in

FIGURE 6. Decreased cytokine production by splenic CD4 and CD8 T cells following stimulation through CD3 and CD28. A, Splenic CD4 T cells were

purified from 2–3-mo-old LMC and T-TRAF32/2 mice, and Tregs were depleted using anti-CD25 magnetic beads. B, Splenic CD8 T cells were purified

from 2–3-mo-old LMC and T-TRAF32/2 mice by negative selection. Cells were cultured ex vivo in the absence or presence of stimulation with 0.5 mg/ml

of plate-bound anti-CD3 mAb, alone or in combination with 2 mg/ml of soluble anti-CD28 mAb. Levels of cytokines in the culture supernatants were

measured by ELISA. Results shown are representative of at least two independent experiments.


distal CD3-signaling events through indirect mechanisms. Uponcoligation of CD3 and CD28, TRAF3 recruitment to the TCR/CD28-signaling complex seems to be required for the synergybetween these two receptors, as evidenced by the abolished syn-ergistic effects on phosphorylation of ERK, LAT, PLCg1, andZAP70 observed in TRAF32 /2 T cells. It was shown thatengagement of CD40 or BAFFR induces rapid proteasome-dependent degradation of TRAF3 in B lymphocytes (16, 17, 38–40). Interestingly, a previous study showed that CD3 triggeringinduced TRAF3 cleavage by caspases in Jurkat T cells (41).However, we did not detect TRAF3 degradation or cleavage uponstimulation through CD3 or CD3 and CD28 in freshly isolatedmouse splenic T cells (P. Xie, unpublished observations). Recentevidence suggests that TRAF3 is an E3 ubiquitin ligase thatpreferentially assembles lysine 63-linked polyubiquitin chains

(42). Whether and how TRAF3 exerts its E3 ubiquitin ligase ac-tivity in TCR and CD28 signaling await further investigation.Although our evidence unequivocally demonstrated the direct

involvement of TRAF3 in CD3 and CD28-mediated T cell acti-vation, it remains possible that potential roles of TRAF3 in sig-naling by receptors of the TNFR superfamily also contribute to thedefective T cell immunity observed in T-TRAF32/2 mice. In thisregard, in vitro studies showed that TRAF3 can directly bind to4-1BB, CD27, TNFR2, GITR, OX-40, and CD30 expressed onT cells (1). Among these, GITR, CD27, OX-40, and 4-1BB weredemonstrated to make substantial contributions to T cell survival,proliferation, and cytokine production in response to stimulationwith TCR or TCR/CD28 (43–52). Thus, alterations in signaling bythese TNFR family members may also contribute to the defectiveTD IgG1 response and decreased T cell responses to L. mono-

FIGURE 7. TRAF3 is a proximal signaling component of TCR and CD28. A, Splenic pan T cells were purified from 2–3-mo-old LMC and T-TRAF32/2

mice by negative selection, and Tregs were depleted using anti-CD25 magnetic beads. Cells were stimulated with anti-CD3 or anti-CD3 + anti-CD28 Abs,

followed by a cross-linking Ab, as described in the Materials and Methods, at 37˚C for the indicated times. Total cellular lysates were immunoblotted for

phosphorylated (P-) or total ERK, LAT, PLCg1, ZAP70, and IkBa, followed by TRAF3. B, Splenic naive Thy1+CD252CD44low T cells were sorted from

2–3-mo-old LMC and T-TRAF32/2 mice using a BD FACS Aria II. Cells were stimulated with anti-CD3 + anti-CD28 Abs, followed by a cross-linking Ab,

as described in theMaterials and Methods, at 37˚C for the indicated times. Total cellular lysates were immunoblotted for phosphorylated (P-) or total ERK,

LAT, PLCg1, and ZAP70, followed by TRAF3. C, Splenic pan T cells were purified from 2–3-mo-old LMC mice by negative selection. Cells were

stimulated with anti-CD3, anti-CD28, or anti-CD3 + anti-CD28 mAbs, followed by Dynabeads coated with a cross-linking Ab, as described in the

Materials and Methods, at 37˚C for 3 min. The immunoprecipitates were analyzed by immunoblotting for TRAF3, Malt1, TRAF6, and ZAP70. D, Splenic

pan T cells were purified from 2–3-mo-old LMC and T-TRAF32/2 mice by negative selection. Cells were stimulated with anti-CD3 + anti-CD28 mAbs,

followed by Dynabeads coated with a cross-linking Ab, as described in Materials and Methods, at 37˚C for the indicated times. The immunoprecipitates

and residual lysates after immunoprecipitation were analyzed by immunoblotting for TRAF3 and ZAP70, followed by actin.


cytogenes infections observed in T-TRAF32/2 mice. Further stud-ies are needed to delineate specific roles of TRAF3 in signaling byGITR, CD27, OX-40, 4-1BB, TNFR2, or CD30. Our T-TRAF32/2

mice provide a valuable model system and useful tools for suchfuture studies.We found that T-TRAF32/2 mice displayed normal thymocyte

populations, as well as normal peripheral B, CD4, and CD8 T cellpopulations, but an increased frequency of CD4+CD25+FoxP3+

Tregs in secondary lymphoid organs. This suggests that TRAF3may regulate Treg maturation or homeostasis. It is very possiblethat the increased frequency of Tregs also contributes to the de-fects in T cell immunity observed in T-TRAF32/2 mice. An in-teresting study recently demonstrated that BAFF-transgenicmice also exhibit increased numbers of Tregs in the spleen (53).In B lymphocytes, BAFFR signals to stimulate NF-kB2 activationand promote peripheral B cell survival by overriding negativeregulatory functions of TRAF3 (4). However, BAFF stimulationdid not induce NF-kB2 activation in Tregs, and the increasedfrequency of Treg requires B cells in BAFF-transgenic mice, sug-gesting an indirect effect of BAFF on Tregs (53). We observedthat Tregs purified from T-TRAF32/2 mice did not show pro-longed survival in the absence or presence of BAFF (P. Xie, un-published observations). Thus, although BAFF and TRAF3 reg-ulate B cell survival through the same pathway, they may regulateTregs through distinct mechanisms. It is noteworthy that TNFR2,GITR, and TLR2 have been implicated in the regulation of Tregproliferation. In resting and activated states, mouse peripheralTregs express remarkably higher surface levels of TNFR2 than doCD4+CD252 T effector cells (54). TNF-a, in synergy with TCRand even more so with IL-2, markedly promotes the expansionand function of the Treg population (54). Similarly, although GITRis upregulated following activation of CD4+ and CD8+ T cells, asubstantially higher level of GITR is constitutively expressed onTregs (20, 55). GITR costimulates with TCR or IL-2 to induce theproliferation and cytokine production of Tregs, and GITR2/2 micedisplay a modest reduction in Tregs (55–57). In addition, TLR2 alsocostimulates with the TCR to control the expansion and function ofTregs (58). Thus, it remains to be determined whether TRAF3participates in signaling by TNFR2, GITR, and TLR2 in Tregs. Itwould also be interesting to further investigate whether the TRAF3-deficient Tregs have an increased suppressive function on a per-cellbasis and whether Tregs express less TRAF3.In the current study, we found that T-TRAF32/2 mice show

higher lethality and increased bacterial loads in the liver followinginfection with L. monocytogenes. In this context, it would be in-teresting to investigate whether deletion or inactivating mutationsof the TRAF3 genes or decreased expression of TRAF3 proteinsoccurs in T cells of human patients with immunodeficiencies orrecurrent infections that result from impaired T cell function.Regulation of lymphocyte homeostasis and functionality is

central to the proper functioning of the adaptive immune system invertebrates. Our findings obtained from B cell-specific TRAF32/2

mice (12) and T cell-specific TRAF32/2 mice presented in thisstudy provide definitive genetic and molecular evidence for thecrucial, but distinct, functions of TRAF3 in regulating B cellhomeostasis and T cell activation, respectively. These findingsdefine an essential role for TRAF3 in the adaptive-immune systemand provide a basis for rational approaches for the development ofTRAF3-specific therapeutic drugs to treat autoimmune diseases,immunodeficient disorders, and tumors.

AcknowledgmentsWe thank Drs. Vladimir Badovinac and Noah Butler for expert advice on L.

monocytogenes infection and liver-migration studies; Dr. John Colgan for

advice on T cell-polarization experiments; and Drs. Jon Houtman, Yufang

Shi, Arthur Weiss, and Gary Koretzky for advice on T cell-signaling stud-

ies, as well as for critical review of the manuscript. We also thank Thomas

Kinney for expert animal husbandry and Kyp Oxley and Sonja Smith for

excellent technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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tnf receptor-associated factor 3 is required for t cell-mediated immunity and tcr/cd28 signaling

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