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


     
 

The Journal of Immunology, 2007, 179, 7233 -7243
Copyright © 2007 by The American Association of Immunologists, Inc.
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 Shamim, M.
Right arrow Articles by Suresh, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shamim, M.
Right arrow Articles by Suresh, M.

Cbl-b Regulates Antigen-Induced TCR Down-Regulation and IFN-{gamma} Production by Effector CD8 T Cells without Affecting Functional Avidity1

Mohammed Shamim2,*, Som G. Nanjappa2,*, Anju Singh2,*, Erin Hemmila Plisch*, Scott E. LeBlanc{dagger}, Jane Walent*, John Svaren{dagger}, Christine Seroogy{ddagger} and M. Suresh3,*

* Department of Pathobiological Sciences, {dagger} Department of Comparative Biosciences, and {ddagger} Department of Pediatrics, University of Wisconsin, Madison, WI 53706


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The E3 ubiquitin ligase Cbl-b is a negative regulator of TCR signaling that: 1) sets the activation threshold for T cells; 2) is induced in anergic T cells; and 3) protects against autoimmunity. However, the role of Cbl-b in regulating CD8 T cell activation and functions during physiological T cell responses has not been systematically examined. Using the lymphocytic choriomeningitis virus infection model, we show that Cbl-b deficiency did not significantly affect the clonal expansion of virus-specific CD8 T cells. However, Cbl-b deficiency not only increased the steady-state cell surface expression levels of TCR and CD8 but also reduced Ag-induced down-modulation of cell surface TCR expression by effector CD8 T cells. Diminished Ag-stimulated TCR down-modulation and sustained Ag receptor signaling induced by Cbl-b deficiency markedly augmented IFN-{gamma} production, which is known to require substantial TCR occupancy. By contrast, Cbl-b deficiency minimally affected cell-mediated cytotoxicity, which requires limited engagement of TCRs. Surprisingly, despite elevated expression of CD8 and reduced Ag-induced TCR down-modulation, the functional avidity of Cbl-b-deficient effector CD8 T cells was comparable to that of wild-type effectors. Collectively, these data not only show that Cbl-b-imposed constraint on TCR signaling has differential effects on various facets of CD8 T cell response but also suggest that Cbl-b might mitigate tissue injury induced by the overproduction of IFN-{gamma} by CD8 T cells. These findings have implications in the development of therapies to bolster CD8 T cell function during viral infections or suppress T cell-mediated immunopathology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell receptor intracellular signaling is triggered by the interaction of cognate peptide/MHC complexes on APCs with TCR complexes on T cells. Signaling by the TCR complex is short lived, as is characteristic of all cell surface receptor systems that mediate stringently regulated biochemical pathways downstream of receptor activation (1). Proteins that negatively regulate the amplitude and duration of TCR signaling would therefore be expected to play critical roles in determining the cellular response to TCR-triggered signaling. Members of the Cbl family of E3 ubiquitin ligases have emerged as key proteins that modulate the intensity and duration of Ag receptor signaling in lymphocytes by regulating the turnover of p85 (a regulatory subunit of PI3K), phospholipase C-{gamma}, and proximal tyrosine kinases such as Lck, Fyn, and ZAP70, which control the earliest committing steps of TCR signaling (2). The step-wise inactivation and removal of critical activities from the TCR signaling pathway by Cbl-specific enzymatic modification sets the activation threshold for TCR-mediated cell responses, and fine-tunes the signaling pathway (2).

To date, three members of the Cbl family have been identified in mammals: c-Cbl, Cbl-b, and Cbl-c (3). Correlating with their relative abundance in thymic vs peripheral T cells, the activities of c-Cbl and Cbl-b appear to be important in down-regulating TCR signaling in thymocytes and mature peripheral T cells, respectively (4, 5, 6, 7). Cbl-b-deficient (Cbl-b–/–) mice do not express overt developmental anomalies in the immune system but develop autoimmune disease either spontaneously or upon immunization with myelin basic protein, which suggested that Cbl-b plays an important role in the maintenance of T cell tolerance to self-Ags (5, 6). Moreover, it has been shown that Cbl-b expression is strongly induced in anergic T cells (8, 9), and T cells from Cbl-b–/– mice appear refractory to the induction of anergy in vitro (8). T cells from Cbl-b–/– mice also hyperproliferate and produce high levels of IL-2 in response to suboptimal stimulation of Ag receptor signaling in vitro (5). Perhaps most interesting, unlike naive T cells from wild-type mice, naive Cbl-b–/– T cells can be fully activated without CD28-mediated costimulation or Vav1-dependent TCR signaling events, which implies that Cbl-b deficiency could reduce the activation threshold in CD28-deficient T cells (10, 11). Therefore, it is conceivable that the antigenic stimulation of autoreactive T cells in Cbl-b–/– mice without costimulation would lead to activation in contrast to the functional inactivation of Cbl-b-sufficient T cells in wild-type mice. Collectively, there is a consensus that Cbl-b regulates T cell activation by setting the thresholds of signaling pathways downstream of Ag receptors. Despite its well-recognized role in the induction of T cell anergy and protection against autoimmunity, the role of Cbl-b in regulating various facets of the physiological Ag-specific CD8 T cell response is not well understood (11, 12, 13). Using the lymphocytic choriomeningitis virus (LCMV)4 infection model in mice, we have determined the role of Cbl-b in regulating: 1) the expansion of virus-specific CD8 T cells during the primary response; 2) the expression of cell surface molecules including CD8 and TCR; 3) Ag-stimulated down-regulation of cell surface TCR expression by LCMV-specific effector CD8 T cells; 4) MHC I-restricted cytotoxic activity and cytokine production; 5) the functional avidity of LCMV-specific effector CD8 T cells as measured by LCMV-specific peptide stimulation of cytokine production in vitro; and 6) CD8 T cell responses to immunodominant vs subdominant epitopes. In this article, we show that the loss of Cbl-b had no significant effect on the clonal expansion of Ag-specific CD8 T cells in vivo during an acute LCMV infection. Instead, we show not only that Cbl-b deficiency selectively effected increased cell surface expression of TCR and CD8 molecules, but we also demonstrate that Cbl-b-deficiency resulted in diminished Ag-specific down-modulation of TCR surface expression and a concomitant increase in IFN-{gamma} production without affecting the CTL activity or functional avidity of the LCMV-specific effector CD8 T cells responding, in vitro. Additionally, Cbl-b deficiency did not significantly affect CD8 T cell responses to the immunodominant and subdominant epitopes. These findings have implications in understanding the regulation of CD8 T cell responses during acute viral infections and the development of immunotherapeutic modalities to alter TCR signaling and control CD8 T cell effector function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The P14 TCR transgenic mice (P14/+/+) on the C57BL/6/Thy1.1 background and Cbl-b-deficient background (P14/Cbl-b–/–) were provided by Drs. J. Grayson (Wake Forest University, Winston-Salem, NC) and P. S. Ohashi (University of Toronto, Ontario, Canada), respectively (5, 14). P14/+/+ mice on the C57BL/6/Ly5.1 background were provided by Dr. K. Murali-Krishna (University of Washington, Seattle, WA). Cbl-b–/– mice were provided by Dr. H. Gu (Columbia University, NY) (6). The C57BL/6, C57BL/6/Thy1.1, and C57BL/6/Ly5.1 mice were purchased from The Jackson Laboratory. All mice were maintained under specific-pathogen-free conditions at University of Wisconsin (Madison, WI), and all animals were used in accordance with the strict guidelines of the institutional animal care committee.

Virus

Mice were infected with 2 x 105 PFU of the Armstrong strain of LCMV. Infectious LCMV in the tissues was quantitated by a plaque assay using Vero cells (15).

Adoptive transfer of P14 TCR transgenic CD8 T cells

P14/+/+ (Ly5.2+/Thy.1.1+) or P14/Cbl-b–/– (Ly5.2+/Thy.1.1–) CD8 T cells were adoptively transferred into C57BL/6/Ly5.1 mice by tail vein injection and infected with LCMV the next day. In some experiments, P14/+/+ (Ly5.1+/Thy.1.2+) or P14/Cbl-b–/– (Ly5.2+/Thy.1.2+) CD8 T cells were adoptively transferred into C57BL/6/Thy1.1 mice.

Flow cytometry

MHC class I tetramers were prepared and used as described previously (16). Briefly, single cell suspensions of splenocytes were stained with allophycocyanin-labeled MHC tetramers, PE-labeled anti-CD8 and FITC-labeled anti-CD44 Abs. In some experiments, anti-CD45.1 (Ly5.1), anti-CD45.2 (Ly5.2), anti-Thy1.1, anti-Thy1.2, anti-CD11a, and anti-CD43 Abs were used as costains with anti-CD8 and MHC I tetramers. To stain for intracellular granzyme B, splenocytes were stained for cell surface molecules and subsequently permeabilized and stained with anti-granzyme B Abs using the Cytofix/Cytoperm kit (BD Pharmingen). All Abs were purchased from BD-Pharmingen except the anti-granzyme and isotype control Abs, which were purchased from Caltag Laboratories.

Cbl-b induction and quantitation in vitro and in vivo

To assess Cbl-b induction in vitro, CD8 T cells were purified from spleens of uninfected +/+ C57BL/6 or Cbl-b–/– mice using CD8 T cell purification columns (R & D Systems) and stimulated with plastic-immobilized anti-CD3 Abs (1 µg/ml). Cell lysates were prepared by standard procedures and Cbl-b protein levels were quantitated by Western blotting. For Western blot analyses, goat anti-mouse anti-Cbl-b Abs (catalog no. sc-1435) and HRP-conjugated donkey anti-goat IgG were purchased from Santa Cruz Biotechnology. To verify that equal amounts of protein were loaded for gel electrophoresis, membranes were stripped and reprobed with anti-β actin Abs (provided by the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Iowa City, IA) and HRP-conjugated goat anti-mouse IgM (Jackson ImmunoResearch). Ab binding was visualized using the SuperSignal West Pico Chemiluminescence Substrate (Pierce Biotechnology) as per the manufacturer’s instructions.

To assess Cbl-b levels in vivo, naive P14/+/+/Thy1.2 or P14/Cbl-b–/–/Thy1.2 CD8 T cells were adoptively transferred into congenic C57BL/6/Thy1.1 mice and infected with LCMV. At different days after infection in vivo, splenocytes were stained for cell surface CD8/Thy1.2 and intracellular Cbl-b using the Cytofix/Cytoperm kit (BD Pharmingen). Goat anti-mouse anti-Cbl-b Abs (catalog no. sc-1435) and rabbit anti-goat Abs were purchased from Santa Cruz Biotechnology. Cbl-b-specific staining was assessed by comparing mean fluorescence intensities (MFI) between P14/+/+ and P14/Cbl-b–/– CD8 T cells using flow cytometry.

Assessment of ligand-induced TCR down-regulation

On day 8 after LCMV infection, single cell suspensions of splenocytes were cultured in vitro with or without the glycoprotein 33–41 peptide (GP33) for up to 5 h. After culture, cells were stained with anti-CD8, anti-Thy1.2/anti-Ly5.1/anti-Ly5.2, and Db/GP33 MHC I tetramers. As an index of cell surface TCR levels, MHC tetramer binding by P14 CD8 T cells was assessed by flow cytometry; the MFI of MHC I tetramer staining by P14 CD8 T cells was analyzed using FlowJo software (Tree Star). Peptide stimulation-induced TCR down-modulation was calculated using the following formula as described previously (7): percentage of TCR down-modulation (at time t) = 100 x [(MFI of unstimulated P14 CD8 T cells (at time t) – MFI of stimulated P14 CD8 T cells (at time t)) ÷ (MFI of unstimulated P14 CD8+ T cells (at time 0))].

CTL assay

The MHC I-restricted GP33-specific CTL activity in the spleens was assessed directly ex vivo by a standard 51Cr-release assay using GP33 peptide-coated and uncoated MC57 target cells (15).

Intracellular cytokine staining

Splenocytes were stimulated for 5 h with LCMV CTL epitope peptides in vitro, and the number of cytokine-producing CD8 T cells was quantitated by flow cytometry (16).

Staining for intracellular phosphorylated ERK1/2

Single cell suspensions of splenocytes were stimulated for 15 min with GP33 peptide (1 µg/ml) or phorbol myristate (80 µM; Fisher Biotech). Following stimulation, cells were fixed for 10 min at 37°C using freshly prepared paraformaldehyde at a final concentration of 2%. After fixation, cells were washed and subsequently permeabilized with ice-cold 90% methanol for 30 min on ice. Next, cells were stained with anti-Thy1.2 and anti-phospho ERK1/2 (T202/Y204) Abs (BD Biosciences). The percentages of Thy1.2+ P14 CD8 T cells showing phosphorylation of ERK1/2 were quantitated by flow cytometry as described previously (17, 18).

Assessment of in vivo proliferation of P14 CD8 T cells by measuring BrdU incorporation

Mice were pulsed with BrdU in drinking water (0.8 mg/ml) between days 0–4 or 4–8 after LCMV infection. At the end of each BrdU pulse, splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-BrdU using the BrdU staining kit (BD Pharmingen). The percentages of BrdU+ cells among Thy1.2+ P14 CD8 T cells were quantitated by flow cytometry as described elsewhere (19).

Quantitation of apoptotic P14 CD8 T cells directly ex vivo by annexin V staining

Splenocytes were stained with anti-CD8, anti-Thy1.2, and annexin V (BD-Pharmingen) directly ex vivo. The percentages of annexin Vhigh cells among Thy1.2+ P14 CD8 T cells were quantitated by flow cytometry as previously described (19, 20).

Statistical analyses

Statistical analyses of data were performed using the commercially available software (SYSTAT version 10.2).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Dynamic regulation of Cbl-b protein expression in Ag-specific CD8 T cells in vivo

Previous work has shown that Cbl-b mRNA and protein are up-regulated in anergic CD4 T cells and that Cbl-b–/– T cells are refractory to anergy induction (8, 9). In this study, we examined whether Cbl-b protein expression is altered in CD8 T cells following anti-CD3 stimulation in vitro. As shown in Fig. 1A, following stimulation with anti-CD3 there was a substantial up-regulation of Cbl-b protein expression in CD8 T cells as compared with unstimulated cells. Next, we examined the dynamics of Cbl-b expression in vivo in Ag-specific CD8 T cells during acute infection of mice with LCMV. Naive wild-type (P14/+/+) or Cbl-b-deficient (P14/Cbl-b–/–) TCR transgenic P14 CD8 T cells (specific to the Db-restricted GP33 epitope of LCMV) were adoptively transferred into congenic Thy1.1 C57BL/6 mice that were subsequently infected with the Armstrong strain of LCMV. On days 3, 5 and 8 postinfection (PI), Cbl-b protein expression in P14/+/+ CD8 T cells was assessed by flow cytometry. Data in Fig. 1B illustrate the dynamic alteration in Cbl-b protein levels in Ag-specific CD8 T cells during a primary T cell response to an acute LCMV infection. In vivo activation induced a substantial increase in Cbl-b protein levels in P14/+/+ CD8 T cells; Cbl-b protein levels on days 3 and 5 PI were ~5-fold higher, respectively, as compared with naive CD8 T cells. Notably, the highest Cbl-b protein levels were detected on days 3 and 5 PI, which coincided with high viral titers and the phase of intense Ag-driven proliferation of the anti-LCMV CD8 T cell response (16). Notably, concomitant with the diminishing viral load there were marked reductions in Cbl-b protein expression between days 5 and 8 PI. Thus, Cbl-b protein levels appear to be regulated by TCR signaling in Ag-stimulated CD8 T cells in vivo. Stringent regulation of CD8 T cell expansion and effector function is essential to mitigate undesirable cytokine and/or perforin-dependent immunopathology (21). Based on the established role for Cbl-b as an attenuator of TCR signaling (5, 6, 11, 22, 23, 24), the up-regulation of Cbl-b expression might be important in dampening Ag-triggered effector functions of CD8 T cells.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 1. Dynamics of Cbl-b expression in Ag-specific CD8 T cells. A, Cbl-b expression induced by anti-CD3 stimulation in vitro. Purified CD8 T cells from +/+ or Cbl-b–/– mice were cultured in vitro with or without plastic-immobilized anti-CD3 Abs. At 24 and 48 h after anti-CD3 stimulation, cells were harvested and cell lysates were analyzed for Cbl-b expression using Western blot analysis; unstimulated cells (US) were included as controls. B, Cbl-b expression in P14/+/+ CD8 T cells in vivo. P14/+/+/Thy1.2 or P14/Cbl-b–/–/Thy1.2 donor CD8 T cells were adoptively transferred into congenic Thy1.1/C57BL/6 mice and infected with LCMV. On days 0 (naive), 3, 5, and 8 after infection, Cbl-b expression in P14 CD8 T cells was assessed by flow cytometry. The histograms in B are gated on Thy1.2+ P14 CD8 T cells; light and bold lines represent Cbl-b staining of P14/Cbl-b–/– and P14/+/+ CD8 T cells respectively. {Delta}MFIs for specific Cbl-b staining were calculated by subtracting the MFI of Cbl-b staining for P14/Cbl-b–/– CD8 T cells from the MFI of Cbl-b staining for P14/+/+ CD8 T cells.

 
Activation and expansion of Cbl-b-deficient TCR transgenic P14 CD8 T cells during an acute LCMV infection

In this study, using an adoptive transfer model, we have examined the CD8 T cell-autonomous effects of Cbl-b deficiency on the expansion of Ag-specific CD8 T cells during an acute LCMV infection. Naive +/+/Thy1.1/Ly5.2 (P14/+/+) or Cbl-b–/–/Thy1.2/Ly5.2 (P14/Cbl-b–/–) P14 CD8 T cells were adoptively transferred into congenic C57BL/6/Ly5.1 mice and infected with LCMV. On the 8th day after LCMV infection the number of P14/+/+ and P14/Cbl-b–/– CD8 T cells in the spleen was quantitated by flow cytometry. As shown in Fig. 2, A and B, LCMV infection induced potent expansion of P14/+/+ CD8 T cells in adoptive transfer recipients. Surprisingly, the expansion of P14/Cbl-b–/– CD8 T cells was similar to that of P14/+/+ CD8 T cells in LCMV-infected mice. To probe whether Cbl-b regulated the dynamics of CD8 T cell expansion during the T cell response, we performed detailed analyses of the kinetics of P14/+/+ and P14/Cbl-b–/– CD8 T cell response following LCMV infection (Fig. 2C). Although the number of P14/Cbl-b–/– CD8 T cells was slightly higher than that of P14/+/+ CD8 T cells on day 3 PI, the overall kinetics of P14 CD8 T cell response to LCMV appeared to be minimally affected by Cbl-b deficiency. We also examined the kinetics of LCMV clearance in the spleens of mice that received P14/+/+ or P14/Cbl-b–/– CD8 T cells. The overall kinetics of LCMV clearance in mice that were recipients of P14/+/+ or P14/Cbl-b–/– CD8 T cells were similar, and infectious LCMV levels in both groups of mice on day 8 PI were barely above the limit of detection (Fig. 2D). The viral titers tended to be lower in the spleens of mice that were recipients of P14/Cbl-b–/– CD8 T cells on day 5 PI, but the differences between P14/+/+ and P14/Cbl-b–/– CD8 T cell recipients did not reach statistical significance (p < 0.05).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 2. Expansion of +/+ and Cbl-b-deficient P14 CD8 T Cells during an acute LCMV infection. Purified naive P14/+/+ or P14/cbl-b–/– CD8 T cells were adoptively transferred into congenic Ly5.1/C57BL/6 mice and infected with LCMV. A and B, On day 8 PI the expansion of donor P14/+/+ and P14/Cbl-b–/– CD8 T cells in the spleens of LCMV-infected recipient C57BL/6/Ly5.1 mice were enumerated by flow cytometry. FACS plots in A are gated on total CD8 T cells, and the numbers shown are the percentages of P14 effector CD8 T cells among CD8 T cells; each data point in B represents an individual mouse. C, total number of P14/+/+ and P14/Cbl-b–/– on the indicated days after LCMV infection. Data in C show mean ± SD from the analyses of 4 to 5 mice per group at each time point. D, Kinetics of LCMV clearance in spleen of P14/+/+ and P14/Cbl-b–/– CD8 T cell recipients; data are the mean ± SD of four mice per group at each time point.

 
Data in Fig. 2 showed that Cbl-b deficiency did not affect the primary expansion of P14 CD8 T cells during a primary response to LCMV infection. In this study, we assessed LCMV-induced proliferation of P14/+/+ and P14/Cbl-b–/– CD8 T cells by quantitating cellular BrdU incorporation in vivo. Following the adoptive transfer of P14 CD8 T cells, mice were treated with BrdU during two intervals: days 0–4 PI (early) or days 4–8 PI (late). Fig. 3A shows BrdU incorporation by P14/+/+ and P14/Cbl-b–/– CD8 T cells during the early and late phases of the primary CD8 T cell response to LCMV. Similar to P14/+/+ CD8 T cells, not only were all P14/Cbl-b–/– CD8 T cells BrdU+, the intensity of BrdU staining (MFI) was comparable between P14/+/+ and P14/Cbl-b–/– CD8 T cells. These data suggested that Cbl-b deficiency did not significantly affect the proliferation of P14 CD8 T cells during a primary response to LCMV. To verify these results, we compared the proliferation of naive P14/+/+ and P14/Cbl-b–/– CD8 T cells in vitro following stimulation with the antigenic peptide GP33. Consistent with in vivo data, Cbl-b deficiency did not significantly affect the Ag-induced proliferation of P14 CD8 T cells in vitro (Table I).


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 3. Proliferation and apoptosis of P14/+/+ and P14/Cbl-b CD8 T cells. Purified P14/+/+/Thy1.2 or P14/Cbl-b–/–/Thy1.2 CD8 T cells were adoptively transferred into congenic C57BL/6/Thy1.1 mice and infected with LCMV. A, LCMV-infected mice were pulsed with BrdU between days 0–4 or 4–8 PI. At the end of each BrdU pulse (days 5 and 8 PI), the percentages of BrdU+ P14 CD8 T cells were determined by flow cytometry. The histograms are gated on P14 CD8 T cells, and the numbers are the mean MFI of staining for BrdU ± SD from four mice per group at each time point. B, On day 8 PI, splenocytes were stained directly ex vivo with anti-Thy1.2, anti-CD8, and annexin V. The percentages of annexin Vhigh cells among P14/+/+ or P14/Cbl-b–/– CD8 T cells were determined by flow cytometry (n = 4).

 

View this table:
[in this window]
[in a new window]

  		Table I. Proliferative responses of +/+ and Cbl-b–/– P14 CD8 T cells to antigenic stimulation in vitroa

 
We and others have reported that "proapoptotic" LCMV-specific effector CD8 T cells could be detected directly ex vivo by annexin V staining (19, 20). Next, we determined whether Cbl-b deficiency affected the proapoptotic phenotype of P14 effector CD8 T cells on day 8 PI. As shown in Fig. 3B, the percentages of annexin Vhigh cells among P14/Cbl-b–/– CD8 T cells were slightly higher but not statistically significant (p < 0.05) as compared with P14/+/+ CD8 T cells. Collectively, data in Fig. 3 indicated that Cbl-b deficiency did not significantly (p < 0.05) affect either the proliferation or proapoptotic phenotype of P14 CD8 T cells during an acute LCMV infection. Responses of monoclonal TCR transgenic CD8 T cells might not always mimic polyclonal CD8 T cell responses. We addressed this concern by comparing the activation and expansion of polyclonal LCMV-specific CD8 T cells between +/+ and Cbl-b–/– mice; CD8 T responses to three LCMV epitopes and viral clearance in Cbl-b–/– mice was comparable to +/+ mice (data not shown).

Regulation of cell surface expression of TCR and CD8 on LCMV-specific effector CD8 T cells by Cbl-b

It is becoming increasingly evident that ubiquitylation by E3 ligases like Cbl-b regulates the abundance of cell surface receptors by effecting their ligand-activated internalization and trafficking to lysosomes (3, 22), which results in down-regulation and termination of receptor signaling (22). The effect of Cbl-b deficiency on the expression levels of cell surface proteins on effector CD8 T cells is yet to be studied. As described above, we adoptively transferred naive P14/+/+ or P14/Cbl-b–/– TCR transgenic CD8 T cells into congenic C57BL/6/Ly5.1 mice and infected them with LCMV. On day 8 PI, we determined the effect of Cbl-b deficiency on the expression of cell surface molecules on effector CD8 T cells. Fig. 4 shows that the MFIs of staining for CD8 and V{alpha}2 ({alpha}-chain of the P14 TCR) or P14/Cbl-b–/– effector CD8 T cells were significantly (p < 0.05) higher compared with P14/+/+ effector CD8 T cells. We confirmed the elevated levels of TCR expression on P14/Cbl-b–/– effector cells by measuring the MFI of staining with Db/GP33 tetramers (Fig. 4). The increased MFI for CD8 and V{alpha}2 by P14/Cbl-b CD8 T cells was not related to larger cell size, because analyses of forward scatter properties by flow cytometry showed no differences between P14/+/+ and P14/Cbl-b–/– effector CD8 T cells (data not shown). Moreover, Cbl-b deficiency selectively affected CD8 and V{alpha}2 expression but not that of CD11a, CD43, or CD44 (Fig. 4). Thus, Cbl-b negatively regulates the steady-state expression levels of TCR and CD8 on LCMV-specific effector CD8 T cells in vivo.


Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 4. Cbl-b regulates cell surface expression of TCR and CD8 on LCMV-specific effector CD8 T cells. Naive P14/+/+ and P14/Cbl-b–/– CD8 T cells were adoptively transferred into congenic C57BL/6/Ly5.1 mice and infected with LCMV. On day 8 PI, splenocytes were stained with anti-CD8, anti-V{alpha}2, anti-Ly5.2, anti-CD44, anti-CD43, or anti-CD11a Abs and Db/GP33 tetramers. Histograms are gated on Ly5.2+ CD8 T cells (light line, P14/+/+; bold line, P14/Cbl-b–/–) and numbers are the MFIs of staining ± SD (n = 4).

 
It has been reported that the combined deficiency of Cbl-b and c-Cbl markedly impaired the anti-CD3-induced down-regulation of TCRs in naive CD4 T cells in vitro; the deficiency of Cbl-b, but not that of c-Cbl alone, led to a measurable reduction in the ligand-induced down-modulation of TCR at 5 and 8 h after stimulation (7). In this study, we examined the role of Cbl-b in mediating Ag-stimulated down-modulation of TCR expression on P14 effector CD8 T cells ex vivo. As described above, following adoptive transfer of naive +/+ and Cbl-b–/– P14 CD8 T cells, recipient mice were infected with LCMV. On day 8 PI, splenocytes from LCMV-infected mice were cultured ex vivo with various concentrations of the GP33 peptide for 5 h to induce TCR down-regulation. Following antigenic stimulation, binding of the Db/GP33 tetramer to P14/+/+ or P14/Cbl-b–/– effector CD8 T cells was quantitated by flow cytometry. As shown in Fig. 5A, stimulation with the GP33 peptide induced substantial loss of tetramer binding by P14/+/+ effector CD8 T cells at all concentrations of the peptide; only 15–20% of P14/+/+ effector CD8 T cells bound the Db/GP33 tetramer in peptide-stimulated cultures. In striking contrast, ~60% of P14/Cbl-b–/– effector CD8 T cells still bound to Db/GP33 MHC I tetramers following stimulation with the GP33 peptide.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. Regulation of ligand-induced down-modulation of TCR by Cbl-b. Naive P14/+/+ and P14/Cbl-b–/– CD8 T cells were adoptively transferred into congenic C57BL/6/Ly5.1 mice and infected with LCMV. A, On day 8 PI, splenocytes were stimulated with the indicated concentrations of GP33 peptide for 5 h and Db/GP33 tetramer-binding P14 CD8 T cells were quantitated by flow cytometry. A, FACS plots are gated on total splenocytes and the numbers are the calculated percentages of tetramer-binding cells among total P14 CD8 T cells ± SD (n = 4); note that all tetramer-binding CD8 T cells were independently confirmed to be donor +/+ or Cbl-b–/– P14 CD8 T cells. B, Kinetics of ligand-induced TCR down-modulation. On day 8 PI, splenocytes were stimulated with 1 µg/ml GP33 peptide, and at the indicated times after stimulation the MFIs of tetramer staining for P14 CD8 T cells were assessed by flow cytometry; MFI values were used to calculate percentage of TCR down-modulation as described in Materials and Methods (n = 4).

 
To more precisely measure Ag-induced loss of tetramer binding to P14 CD8 T cells, we quantitated MFIs for the tetramer staining of P14/+/+ and P14/Cbl-b–/– effector CD8 T cells following peptide stimulation. In P14/Cbl-b–/– CD8 T cells, not only did peptide stimulation decreased the MFI of tetramer staining in a dose-dependent fashion, the MFIs of tetramer binding by P14/Cbl-b–/– CD8 T cells were higher at all peptide concentrations compared with P14/+/+ CD8 T cells (data not shown). Next, we examined the effect of Cbl-b deficiency on the dynamics of peptide-induced alterations in MHC I tetramer binding to P14 CD8 T cells. At different time points after peptide stimulation, we calculated TCR down-modulation by P14/+/+ and P14/Cbl-b–/– CD8 T cells using MFI values for tetramer staining (Fig. 5B). As shown in Fig. 5B, at 3 and 5 h after peptide stimulation TCR down-modulation by P14/Cbl-b–/– effector CD8 T cells was substantially lower compared with that by P14/+/+ CD8 T cells. Taken together, data in Fig. 5 show that Cbl-b plays an important role in Ag-induced down-modulation of TCRs in virus-specific effector CD8 T cells.

Role of Cbl-b in regulating cytotoxic activity, cytokine production, and functional avidity of LCMV-specific effector CD8 T cells

Data in Fig. 5 showed that P14/Cbl-b–/– effector CD8 T cells down-regulate their TCR less effectively compared with P14/+/+ CD8 T cells. In this study, we assessed whether sustained TCR expression on P14/Cbl-b–/– P14 effector CD8 T cells affected their MHC I-restricted cytolytic ability. As described above, naive P14/+/+ and P14/Cbl-b–/– naive CD8 T cells were adoptively transferred into congenic C57BL/6 hosts that were subsequently infected with LCMV. On day 8 PI we enumerated the number of donor P14/+/+ or P14/Cbl-b–/– effector CD8 T cells in the spleens of recipient mice by flow cytometry as in Fig. 1. Based on this information, we normalized the number of P14 CD8 T cells between the samples and compared their CTL activity directly ex vivo. Data in Fig. 6A indicated that the MHC I-restricted CTL activity of P14/Cbl-b–/– effector CD8 T cells was comparable to those of P14/+/+ effector cells. Likewise, intracellular levels of granzyme B in P14/+/+ and P14/Cbl-b–/– CD8 T cells were similar (Fig. 6B).


Figure 6
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 6. MHC I-restricted cytotoxic activity of wild-type and Cbl-b-deficient effector CD8 T cells. Purified naive P14/+/+ and P14/Cbl-b–/– CD8 T cells were adoptively transferred into congenic C57BL/6/Ly5.1 recipients and infected with LCMV. A, CTL activity of P14/+/+ and P14/Cbl-b–/– CD8 T cells. On day 8 PI, the percentages of P14 CD8 T cells were assessed by flow cytometry and the frequencies of P14 CD8 T cells were normalized in all samples; GP33-specific CTL activity was assessed at the indicated E:T ratios in a standard 51Cr release assay. Data show the calculated percentage of specific lysis from analyses of four mice per group. B, The levels of intracellular granzyme B in P14/+/+ and P14/Cbl-b–/– effector CD8 T cells on day 8 PI. The histograms are gated on Db/GP33 tetramer-binding P14 effector CD8 T cells and the numbers are the MFIs of staining for granzyme B ± SD (n = 4); lighter and bolder lines represents staining with isotype control and anti-granzyme Abs, respectively. Data are derived from two independent experiments.

 
Loss of Cbl-b-mediated negative regulation of TCR signaling lowers the threshold required for T cell activation in vitro (5, 6); Cbl-b–/– T cells showed strong responses to stimulation with suboptimal concentrations of anti-CD3 Abs in vitro. However, it is unknown whether diminished Ag-induced TCR down-regulation would affect the activation threshold of Cbl-b–/– effector CD8 T cells. The cytokine-producing ability of CD8 T cells, measured as a function of the concentration of the stimulating antigenic peptide, is termed "functional avidity" (25). Significant to our studies, the functional avidity of LCMV-specific effector CD8 T cells directly correlated with the levels of intracellular Lck (25), which is an important target molecule of Cbl-b in T cells (2, 22). Therefore, we theorized that Cbl-b might regulate the functional avidity of CD8 T cells by modulating the abundance of Lck, cell surface CD8, and TCR. In this study, we investigated whether Cbl-b deficiency affected the functional avidity of Ag-specific effector CD8 T cells during an acute LCMV infection. As described above, we adoptively transferred naive P14/+/+ or P14/Cbl-b–/– CD8 T cells into congenic C57BL/6/Ly5.1 mice and subsequently infected them with LCMV. On day 8 PI, splenocytes were stimulated with graded concentrations of the GP33 peptide and the percentages of IFN-{gamma}-producing P14 CD8 T cells were determined by intracellular cytokine staining. Data in Fig. 7A show that the percentage of IFN-{gamma}-producing cells among P14/+/+ effector CD8 T cells increased in a peptide dose-dependent fashion. Surprisingly, at all peptide concentrations the fractions of IFN-{gamma}-producing cells of total P14/Cbl-b–/– CD8 T cells were comparable to those of the P14/+/+ CD8 T cells; the half-max for both the P14/+/+ and the P14/Cbl-b–/– CD8 T cells was 0.0001–0.001 µg/ml. Thus, despite expressing higher levels of cell surface CD8 and TCR, the overall functional avidity of Cbl-b-deficient LCMV-specific effector CD8 T cells was comparable to that of the +/+ effectors.


Figure 7
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 7. Role of Cbl-b in regulating functional avidity and cytokine production of effector CD8 T cells. Naive P14/+/+ or P14/Cbl-b–/– CD8 T cells were adoptively transferred into C57BL/6/Ly5.1 mice, which were subsequently infected with LCMV. On day 8 PI, splenocytes were stimulated with the indicated concentrations of the GP33 peptide and IFN-{gamma} production by P14 CD8 T cells was assessed by intracellular cytokine staining. A, The data are expressed as a percentage of maximum response (percentages of IFN-{gamma}-producing cells of total P14 CD8 T cells at a saturating peptide concentration of 0.1 µg/ml GP33 peptide for each mouse) (n = 4). B, MFI of IFN-{gamma} staining in P14 CD8 T cells (n = 4). Data are representative of two independent experiments.

 
Next, we asked whether Cbl-b regulates the levels of IFN-{gamma} produced by P14 effector CD8 T cells in response to varying degrees of antigenic stimulation. We compared the MFIs for IFN-{gamma} staining between P14/+/+ and P14/Cbl-b–/– effector CD8 T cells following ex vivo stimulation with graded concentrations of the antigenic peptide GP33–41 (Fig. 7B). Both P14/+/+ and P14/Cbl-b–/– effector CD8 T cells achieved peak MFIs for IFN-{gamma} production at peptide concentrations of ≥0.01 µg/ml. However, the MFIs for IFN-{gamma} staining in P14/Cbl-b–/– CD8 T cells were substantially higher than in P14/+/+ CD8 T cells at peptide concentrations of 0.001 µg/ml or above. These findings strongly suggested that Cbl-b down-regulates Ag-induced IFN-{gamma} production by effector CD8 T cells. These data support the hypothesis that the up-regulation of Cbl-b expression in effector CD8 T cells (Fig. 1B) might be involved in dampening IFN-{gamma} production in response to antigenic stimulation. In contrast to the hyperinduction of IFN-{gamma}, Ag-induced IL-2 production by P14/Cbl-b–/– effector CD8 T cells was comparable to that by P14/+/+ effector CD8 T cells (data not shown).

Effect of Cbl-b deficiency on TCR signaling in LCMV-specific effector CD8 T cells

It has been shown that impaired down-modulation of TCR expression leads to sustained TCR signaling in Cbl-b-deficient T cells in vitro (7). It was of interest to determine whether the impaired down-regulation of TCR by P14/Cbl-b–/– effector CD8 T cells altered biochemical signaling induced by antigenic stimulation. Also, we were interested to test whether increased IFN-{gamma} production by P14/Cbl-b–/– effector CD8 T cells was associated with an alteration of TCR signaling. As above, naive P14/+/+ or P14/Cbl-b–/– CD8 T cells were adoptively transferred into congenic C57BL/6 hosts and infected with LCMV. Stimulation of the TCR induces transient phosphorylation of ERK1/2, and Cbl-b deficiency has been reported to sustain ERK1/2 phosphorylation in anti-CD3-stimulated T cells in vitro (7). In this study, on day 8 PI, we assessed ERK1/2 phosphorylation induced by PMA or antigenic stimulation of P14/+/+ and P14/Cbl-b–/– effector CD8 T cells ex vivo. As shown in Fig. 8, at 15 min after stimulation with either PMA or the GP33 peptide a larger proportion of P14/Cbl-b–/– CD8 T cells exhibit substantial ERK1/2 phosphorylation compared with P14/+/+ CD8 T cells. These data suggested that Cbl-b plays a role in regulating TCR signal transduction in effector CD8 T cells.


Figure 8
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 8. Activation of ERK1/2 in wild-type and Cbl-b-deficient effector CD8 T cells. Purified P14/+/+/Thy1.2 or P14/Cbl-b–/–/Thy1.2 CD8 T cells were adoptively transferred into congenic C57BL/6/Thy1.1 mice and infected with LCMV. On day 8 PI, splenocytes were stimulated with PMA or the GP33 peptide for 15 min. The levels of phosphorylated ERK1/2 in P14/+/+ and P14/Cbl-b–/– effector CD8 T cells were assessed by flow cytometry. The histograms are gated on Thy1.2+ P14 CD8 T cells; dotted lines and filled histograms show staining by anti-phosphorylated ERK1/2 Abs in unstimulated and stimulated cells, respectively. The numbers are the percentages of P14 CD8 T cells that express phosphorylated ERK1/2, and data are representative of analysis of four mice per group.

 
Role of Cbl-b in regulating CD8 T cell responses to dominant vs subdominant epitopes during an acute LCMV infection

Based on the magnitude of the CD8 T cell response to individual epitopes, the well-defined CD8 T cell epitopes present in the nucleoprotein (NP) and GP of LCMV follow a reproducible immunodominance hierarchy (16). Immunodominance might result from the interplay of multiple factors including: 1) abundance and half-life of peptide/MHC complexes on the APC; 2) precursor frequencies of epitope-specific CD8 T cells in the T cell repertoire; 3) avidity and/or affinity of the TCR to the peptide/MHC complex; and 4) ability of immunodominant-epitope-specific CD8 T cells to suppress CD8 T cell responses to subdominant epitopes (26, 27). In this study, we determined whether the Cbl-b-imposed threshold in TCR signaling regulated the immunodominance hierarchy of the CD8 T cell response to LCMV. Groups of +/+ and Cbl-b–/– mice were infected with LCMV and on day 8 PI we quantitated the percentages of CD8 T cells specific to immunodominant and subdominant LCMV epitopes by intracellular cytokine staining. As shown in Fig. 9A, CD8 T cells specific to immunodominant (NP396 and GP33) and subdominant (GP276, NP205, and GP118) epitopes were readily detected in the spleens of +/+ and Cbl-b–/– mice. However, the immunodominance hierarchy of the anti-LCMV CD8 T cell response in +/+ and Cbl-b–/– mice was similar (Fig. 9, A and B), which suggested that the negative reg-ulation of TCR signaling by Cbl-b might not play a role in determining the immunodominance hierarchy of the primary CD8 T cell response to LCMV.


Figure 9
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 9. Immunodominance hierarchy of CD8 T cell responses to LCMV in Cbl-b–/– mice. Groups of +/+ and Cbl-b–/– mice were infected with LCMV, and on day 8 PI CD8 T cells specific to the indicated epitopes were quantitated by intracellular cytokine staining. The dot plots in A are gated on total viable splenocytes and the numbers in A and the data in B (n = 4) are the percentages of epitope-specific CD8 T cells of total LCMV-specific CD8 T cells. Data are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Regulation of the duration and intensity of Ag receptor signaling is important from the standpoint of the induction of potent T cell responses against infections and tumors and protection against autoimmunity. Previous work has clearly demonstrated a nonredundant role for Cbl-b in the termination of TCR signaling and protection against T cell-dependent autoimmunity (5, 6). However, it remained to be determined whether Cbl-b regulates Ag-specific T cell responses to foreign proteins such as viral Ags. In this article, we have documented that TCR signaling in the absence of Cbl-b did not significantly affect the primary expansion of monoclonal or polyclonal CD8 T cells during an acute LCMV infection. We show that Cbl-b down-regulated the expression level of cell surface molecules, including TCR and CD8, on effector CD8 T cells, and we further demonstrate that Cbl-b plays an integral role as a negative regulator of TCR signaling in effector CD8 T cells by effecting Ag-induced down-regulation of Ag receptors and selectively limiting the production of IFN-{gamma}. Surprisingly, diminished Cbl-b-dependent Ag-induced TCR down-regulation did not affect either the functional avidity of effector CD8 T cells or the immunodominance hierarchy of the CD8 T cell response. Collectively, these findings demonstrate differential effects of Cbl-b deficiency on events downstream of TCR signaling in effector CD8 T cells in the context of an acute viral infection.

Using Western blot analysis we show that the expression of Cbl-b in anti-CD3-stimulated CD8 T cells in vitro was higher than in unstimulated cells; controls included CD8 T cells from Cbl-b-deficient mice. In contrast to our studies, Li et al. (28) have previously reported the reduced expression of Cbl-b in T cells stimulated with anti-CD3 in vitro. The discrepancy in findings might be related to differences in experimental conditions, including: 1) the concentrations of anti-CD3 used and/or the blotting procedures; and 2) the use of total T cells (CD4 and CD8) in their study vs purified CD8 T cells in the present study. Nevertheless, in our studies of Cbl-b regulation under physiological conditions in vivo, we observe that Cbl-b expression is increased at days 3 and 5 PI in LCMV-specific CD8 T cells at a time when viral titers reach high levels in the spleen. Notably, with a reducing viral load there was a concomitant reduction in the expression levels of Cbl-b in LCMV-specific CD8 T cells, which suggests that Cbl-b levels might be regulated by TCR signaling in vivo. It has been previously reported that the stimulation of T cells in vitro with anti-CD3 in the presence of CD28-dependent costimulation induces the ubiquitination and degradation of Cbl-b within 1–2 h, which is followed by a CTLA-4-dependent re-expression (28, 29). Therefore, it is possible that induction of Cbl-b in LCMV-specific CD8 T cells at days 3 and 5 PI might reflect CTLA-4-dependent re-expression.

Although it is clear that Cbl-b sets the threshold for TCR signaling and protects against autoimmunity, the role of Cbl-b in regulating Ag-specific CD8 T cell responses to foreign Ags or infections has not been examined. In this study, under physiological conditions, we have determined the CD8 T cell-autonomous effects of Cbl-b in regulating the primary expansion of virus-specific CD8 T cells during an acute viral infection. Our studies show that the net proliferative expansion of Cbl-b-deficient, TCR transgenic CD8 T cells was largely similar to those of wild-type cells during an acute LCMV infection. These results were surprising, because Cbl-b-deficient T (CD4 plus CD8) cells have been reported to exhibit enhanced proliferative responses to antigenic stimulation in vitro (5, 6). However, supporting our results, two previous in vivo studies involving injection of antigenic peptide or LCMV infection of P14 TCR transgenic mice have reported less than remarkable differences in expansion between P14/+/+ and P14/Cbl-b–/– CD8 T cells (9, 12). First, in the CD8 T cell anergy model, which involves the injection of antigenic peptide GP33 into TCR transgenic mice, the percentages of P14/+/+ and P14/Cbl-b–/– CD8 T cells among splenocytes on day 2 after peptide injections were ~30% and 40% respectively (9). Second, in a virus-induced diabetes model, following LCMV infection of P14 TCR transgenic mice, the percentages of P14/+/+ and P14/Cbl-b–/– cells among total CD8 T cells in the spleen on day 7 PI did not appear to be statistically significant (12). Additionally, the percentages of IFN-{gamma}- or IL-4- producing CD4 T cells were similar in wild-type and Cbl-b–/– mice infected with vesicular stomatitis virus (10). Collectively, these findings along with our studies suggest that Cbl-b deficiency has a minimal effect on the primary expansion of Ag-specific CD8 and CD4 T cells in several models in vivo. Why are Cbl-b-deficient CD8 T cells not hyperproliferative in vivo during an acute viral infection? Viral infections induce potent host innate immune/inflammatory responses, including the production of type I IFNs, which determine the nature and magnitude of primary anti-viral T cell responses (30, 31, 32). Therefore, it is conceivable that the potent stimulation of wild-type T cells by these innate/inflammatory factors during an acute viral infection could overcome Cbl-b-induced constraints on TCR signaling and T cell activation in vivo. By contrast, it is possible that Cbl-b might regulate the activation of T cells, especially under noninflammatory conditions, by reducing the TCR activation threshold to less potent antigenic stimulation with auto-Ags and tumor Ags. This notion is supported by the observed increase in the susceptibility of Cbl-b–/– mice to spontaneous or experimentally induced autoimmune diseases and enhanced CD8 T cell-dependent immunity against tumors (5, 6, 33, 34). In comparison with an acute infection induced by the Armstrong strain of LCMV, the infection of mice with rapidly replicating strains of LCMV such as LCMV-clone 13 establishes chronic infections by clonally exhausting CD8 T cells of certain specificities (35). Clonal exhaustion is directly related to the intensity and duration of antigenic stimulation in vivo during a chronic LCMV infection (36). Preliminary analyses of virus-specific CD8 T cell responses to LCMV-clone 13 in +/+ and Cbl-b–/– mice have indicated that Cbl-b deficiency accentuates the clonal exhaustion of virus-specific CD8 T cells (S. Nanjappa and M. Suresh, manuscript in preparation). Thus, Cbl-b appears to play a protective role against the clonal exhaustion of virus-specific CD8 T cells by dampening TCR signaling during a chronic LCMV infection.

Elegant work by Gu’s group (7) has demonstrated that c-Cbl and Cbl-b limit Ag receptor signaling in T cells by effecting ligand-induced down-modulation of cell surface TCR. In their studies, the deficiency of Cbl-b alone led to a measurable reduction in anti-CD3-induced TCR down-regulation in CD4 T cells, but the effects of the double deficiency of c-Cbl and Cbl-b on TCR levels were more profound, especially at 5 and 8 h after stimulation. In this article we show that Ag-induced down-modulation of TCR in Ag-specific effector CD8 T cells is markedly reduced in the absence of Cbl-b at 3 and 5 h after stimulation. Additionally, we demonstrate a nonredundant role for Cbl-b in suppressing steady-state expression of cell surface CD8 on LCMV-specific effector CD8 T cells. Reduced ligand-induced TCR down-regulation coupled with enhanced expression of cell surface CD8 would be expected to increase the overall avidity of interactions between APCs and Cbl-b-deficient effector CD8 T cells. We tested whether alterations in the levels of cell surface TCR and CD8 induced by Cbl-b deficiency affected the MHC I-restricted cytolytic and cytokine-producing functions of effector CD8 T cells directly ex vivo. Cbl-b deficiency did not significantly affect either the expression of effector molecule granzyme B or the ex vivo cytolytic activity of effector CD8 T cells. The process of cell-mediated cytotoxicity by effector CD8 T cells does not depend upon synapse formation and only requires the engagement of three TCRs by peptide/MHC complexes on the target cell (37). Hence, Cbl-b deficiency-induced enhancement of TCR signaling in effector CD8 T cells might be superfluous and is less likely to alter an already hypersensitive T cell activation process during target cell recognition and lysis.

By contrast to the supersensitive process of T cell activation during CTL killing (37), a stable mature immunological synapse induced by the engagement of higher number of TCRs might be required for the induction of cytokine production by effector CD8 T cells (38). In our studies, Cbl-b deficiency did not affect the functional avidity/activation threshold of effector CD8 T cells but effected substantially higher levels of Ag-induced IFN-{gamma} production of individual cells. Moreover, not only did Cbl-b-deficient CD8 T cells express higher levels of cell surface TCR than +/+ CD8 T cells following stimulation with the antigenic peptide, but Ag-induced ERK1/2 phosphorylation was sustained in a larger fraction of Cbl-b-deficient, LCMV-specific effector CD8 T cells compared with wild-type cells. These findings suggest that Cbl-b down-modulates Ag-induced IFN-{gamma} production by dampening TCR signaling, likely by limiting TCR occupancy. Thus, our findings demonstrate differential sensitivities of effector mechanisms (cytotoxicity vs IFN-{gamma} production) to regulation by Cbl-b. As discussed above, differences in the level of TCR occupancy required to trigger cytotoxicity and IFN-{gamma} production might explain why Cbl-b deficiency affected IFN-{gamma} production and not CTL activity of effector CD8 T cells. To reiterate, increased cell surface expression of TCR on Cbl-b–/– effector CD8 T cells is likely to augment cytokine production, a process that involves the triggering of a substantial proportion of TCRs, but is superfluous for cell-mediated cytotoxicity, which requires the engagement of only three TCRs (37, 38). Compared with the effects of CTL lysis, which are localized to a single cell, CD8 T cell-derived cytokines could have systemic ill effects if produced at supraphysiological levels (21). Therefore, stringent regulation of TCR signaling by Cbl-b to limit cytokine production might be essential to limit bystander tissue damage during an immune response.

Viral infections induce CD8 T cell responses to multiple epitopes, and the magnitude of response to each epitope in relationship to other epitopes exhibits a reproducible hierarchy, termed as immunodominant hierarchy (26, 27). The mechanisms underlying the immunodominant hierarchy among epitopes are multifactorial, as reviewed recently by Yewdell and colleagues (26, 27). Significant to the current study, the abundance of peptide/MHC I complexes on the surface of an APC and, therefore, the quality of TCR stimulation induced might vary among different epitopes. In this study we tested the hypothesis that enhanced TCR signaling induced by Cbl-b deficiency might alter the immunodominant hierarchy by bolstering CD8 T cell responses to "weaker" subdominant epitopes. Our studies show that Cbl-b did not significantly affect the immunodominant hierarchy of the CD8 T cell response to an acute LCMV infection. Nonetheless, these findings suggested that altering the TCR signaling thresholds might not modulate the magnitude of primary expansion to CD8 T cells to both dominant and subdominant epitopes. It is possible that factors that regulate Ag processing and/or presentation, and not the T cell precursor frequency or the activation threshold, are critical determinants of immunodominant hierarchy (39).

In summary, in this article we have done a systematic analysis of the role of Cbl-b in regulating Ag-specific CD8 T cell responses to an acute viral infection. Along with published work, our studies have clarified that constraints on TCR signaling induced by Cbl-b might be important for protection against autoimmunity and not essential for regulating the primary activation or expansion of virus-specific CD8 T cells during an acute viral infection (5, 6). In addition, our studies show that Cbl-b plays a critical role in limiting TCR occupancy and dampening Ag receptor signaling during the activation of effector CD8 T cells by peptide/MHC complexes. The loss of Cbl-b-dependent regulation of TCR signaling enhanced Ag-induced IFN-{gamma} production by effector CD8 T cells, which suggests that Cbl-b might mitigate systemic effects and/or local tissue injury induced by the overproduction of proinflammatory cytokines by effector CD8 T cells. These findings are expected to have implications in the development of therapeutic strategies to modulate TCR signaling to: 1) suppress cytokine production during T cell-dependent immunopathology; and 2) bolster cytokine production by T cells to promote viral clearance during chronic viral infections.


   Acknowledgments
 
We thank Drs. Ohashi and Gu for providing the P14/Cbl-b–/– and Cbl-b–/– mice, respectively. We also thank Katie Skell and Rajini Srinivasan for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Public Health Service Grants AI48785 and AI59804 from the National Institutes of Health (to M.S.). Back

2 M.S., S.N., and A.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. M. Suresh, Department of Pathobiological Sciences, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706. E-mail address: sureshm{at}svm.vetmed.wisc.edu Back

4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; GP33, glycoprotein 33–41 peptide; MFI, mean fluorescence intensity; NP, nucleoprotein; PI, postinfection. Back

Received for publication April 5, 2007. Accepted for publication September 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Lee, K. H., A. R. Dinner, C. Tu, G. Campi, S. Raychaudhuri, R. Varma, T. N. Sims, W. R. Burack, H. Wu, J. Wang, et al 2003. The immunological synapse balances T cell receptor signaling and degradation. Science 302: 1218-1222. [Abstract/Free Full Text]
  2. Schmidt, M. H., I. Dikic. 2005. The Cbl interactome and its functions. Nat. Rev. Mol. Cell Biol. 6: 907-919. [Medline]
  3. Thien, C. B., W. Y. Langdon. 2005. c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses. Biochem. J. 391: 153-166. [Medline]
  4. Murphy, M. A., R. G. Schnall, D. J. Venter, L. Barnett, I. Bertoncello, C. B. Thien, W. Y. Langdon, D. D. Bowtell. 1998. Tissue hyperplasia enhanced T-cell signaling via ZAP-70 in c-Cbl-deficient mice. Mol. Cell. Biol. 18: 4872-4882. [Abstract/Free Full Text]
  5. Bachmaier, K., C. Krawczyk, I. Kozieradzki, Y. Y. Kong, Y. Saski, A. Oliveira-dos-Santos, S. Maiathasan, D. Bouchard, A. Wakeham, A. Itie, et al 2000. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature 403: 211-216. [Medline]
  6. Chiang, Y. J., H. K. Kole, K. Brown, M. Naramura, S. Fukuhara, R. J. Hu, I. K. Jang, J. S. Gutking, E. Shevach, H. Gu. 2000. Cbl-b regulates the CD28 dependence of T-cell activation. Nature 403: 216-220. [Medline]
  7. Naramura, M., I. K. Jang, H. Kole, F. Huang, D. Haines, H. Gu. 2002. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-regulation. Nat. Immunol. 3: 1192-1199. [Medline]
  8. Heissmeyer, V., F. Macian, S. H. Im, R. Varma, S. Feske, K. Venuprasad, H. Gu, Y. C. Liu, M. L. Dustin, A. Rao. 2004. Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat. Immunol. 5: 255-265. [Medline]
  9. Jeon, M. S., A. Atfield, K. Venuprasad, C. Krawczyk, R. Sarao, C. Elly, C. Yang, S. Arya, K. Bachmaier, L. Su, et al 2004. Essential role of the E3 ubiquitin ligase Cbl-b in T cell anergy induction. Immunity 21: 167-177. [Medline]
  10. Krawczyk, C. M., R. G. Jones, A. Atfield, K. Bachmaier, S. Arya, B. Odermatt, P. S. Ohashi, J. M. Penninger. 2005. Differential control of CD28-regulated in vivo immunity by the E3 ligase Cbl-b. J. Immunol. 174: 1472-1478. [Abstract/Free Full Text]
  11. Krawczyk, C., K. Bachmaier, T. Sasaki, R. G. Jones, S. B. Snapper, D. Bouchard, I. Kozieradzki, P. S. Ohashi, F. W. Alt, J. M. Penninger. 2000. Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells. Immunity 13: 463-473. [Medline]
  12. Gronski, M. A., J. M. Boulter, D. Moskophidis, L. T. Nguyen, K. Holmberg, A. R. Elford, E. K. Deenick, H. O. Kim, J. M. Penninger, B. Odermatt, et al 2004. TCR affinity and negative regulation limit autoimmunity. Nat. Med. 10: 1234-1239. [Medline]
  13. Ohashi, P. S.. 2003. Negative selection and autoimmunity. Curr. Opin. Immunol. 15: 668-676. [Medline]
  14. Grayson, J. M., L. E. Harrington, J. G. Lanier, E. J. Wherry, R. Ahmed. 2002. Differential sensitivity of naive and memory CD8+ T cells to apoptosis in vivo. J. Immunol. 169: 3760-3770. [Abstract/Free Full Text]
  15. Ahmed, R., A. Salmi, L. D. Butler, J. M. Chiller, M. B. Oldstone. 1984. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice: role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160: 521-540. [Abstract/Free Full Text]
  16. Murali-Krishna, K., J. D Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. Miller, R. Ahmed. 1998. Counting antigen specific T cells: a re-evaluation of bystander activation during viral infection. Immunity 8: 177-187. [Medline]
  17. Chow, S., H. Patel, D. W. Hedley. 2001. Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 46: 72-78. [Medline]
  18. Kaech, S. M., S. Hemby, E. Kersh, R. Ahmed. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111: 837-851.
  19. Suresh, M., A. Singh, C. Fischer. 2005. Role of tumor necrosis factor receptors in regulating CD8 T-cell responses during acute lymphocytic choriomeningitis virus infection. J. Virol. 79: 202-213. [Abstract/Free Full Text]
  20. Wang, X. Z., S. E. Stepp, M. A. Brehm, H. D. Chen, L. K. Selin, R. M. Welsh. 2003. Virus-specific CD8 T cells in peripheral tissues are more resistant to apoptosis than those in lymphoid organs. Immunity 18: 631-642. [Medline]
  21. Slifka, M. K., J. L. Whitton. 2000. Antigen-specific regulation of T cell-mediated cytokine production. Immunity 12: 451-457. [Medline]
  22. Duan, L., A. L. Reddi, A. Ghosh, M. Dimri, H. Band. 2004. The Cbl family and other ubiquitin ligases: destructive forces in control of antigen receptor signaling. Immunity 21: 7-17. [Medline]
  23. Mueller, D. L.. 2004. E3 ubiquitin ligases as T cell anergy factors. Nat. Immunol. 5: 883-890. [Medline]
  24. Rangachari, M., J. M. Penninger. 2004. Negative regulation of T cell receptor signals. Curr. Opin. Pharmacol. 4: 415-422. [Medline]
  25. Slifka, M. K., J. L. Whitton. 2001. Functional avidity maturation of CD8+ T cells without selection of higher affinity TCR. Nat. Immunol. 2: 711-717. [Medline]
  26. Yewdell, J. W., J. R. Bennink. 1999. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu. Rev. Immunol. 17: 51-88. [Medline]
  27. Yewdell, J. W.. 2006. Confronting complexity: real-world immunodominance in antiviral CD8+ T cell responses. Immunity 25: 533-543. [Medline]
  28. Li, D., I. Gal, C. Vermes, M. L. Alegre, A. S. Chong, L. Chen, Q. Shao, V. Adarichev, X. Xu, T. Koreny, et al 2004. Cutting edge: Cbl-b, one of the key molecules tuning CD28- and CTLA-4-mediated T cell costimulation. J. Immunol. 173: 7135-7139. [Abstract/Free Full Text]
  29. Zhang, J., T. Bardos, D. Li, I. Gal, C. Vermes, J. Xu, K. Mikecz, A. Finnegan, S. Lipkowitz, T. T. Glant. 2002. Cutting edge: regulation of T cell activation threshold by CD28 costimulation through targeting Cbl-b for ubiquitination. J. Immunol. 169: 2236-2240. [Abstract/Free Full Text]
  30. Haring, J. S., V. P. Badovinac, J. T. Harty. 2006. Inflaming the CD8+ T cell response. Immunity 25: 19-29. [Medline]
  31. Kolumam, G. A., S. Thomas, L. J. Thompson, J. Sprent, K. Murali-Krishna. 2005. Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J. Exp. Med. 202: 637-650. [Abstract/Free Full Text]
  32. Thompson, L. J., G. A. Kolumam, S. Thomas, K. Murali-Krishna. 2006. Innate inflammatory signals induced by various pathogens differentially dictate the IFN-I dependence of CD8 T cells for clonal expansion and memory formation. J. Immunol. 177: 1746-1754. [Abstract/Free Full Text]
  33. Loeser, S., K. Loser, M. S. Bijker, M. Rangachari, S. H. van der Burg, T. Wada, S. Beissert, C. J. Melief, J. M. Penninger. 2007. Spontaneous tumor rejection by cbl-b-deficient CD8+ T cells. J. Exp. Med. 204: 879-891. [Abstract/Free Full Text]
  34. Chiang, J. Y., I. K. Jang, R. Hodes, H. Gu. 2007. Ablation of Cbl-b provides protection against transplanted and spontaneous tumors. J. Clin. Invest. 117: 1029-1036. [Medline]
  35. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, R. Ahmed. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188: 2205-2213. [Abstract/Free Full Text]
  36. Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, R. Ahmed. 2003. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77: 4911-4927. [Abstract/Free Full Text]
  37. Purbhoo, M. A., D. J. Irvine, J. B. Huppa, M. M. Davis. 2004. T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5: 524-530. [Medline]
  38. Valitutti, S., S. Muller, M. Dessing, A. Lanzavecchia. 1996. Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy. J. Exp. Med. 183: 1917-1921. [Abstract/Free Full Text]
  39. Rodriguez, F., S. Harkins, M. K. Slifka, J. L. Whitton. Immunodominance in virus-induced CD8+ T-cell responses is dramatically modified by DNA immunization and is regulated by {gamma} interferon. J. Virol. 76: 4251-4259.



This article has been cited by other articles:


Home page
 BloodHome page
I. Alcazar, I. Cortes, A. Zaballos, C. Hernandez, D. A. Fruman, D. F. Barber, and A. C. Carrera
p85{beta} phosphoinositide 3-kinase regulates CD28 coreceptor function
Blood, April 2, 2009; 113(14): 3198 - 3208.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Zhang, N. Zhang, and D. L. Mueller
Casitas B-Lineage Lymphoma b Inhibits Antigen Recognition and Slows Cell Cycle Progression at Late Times during CD4+ T Cell Clonal Expansion
J. Immunol., October 15, 2008; 181(8): 5331 - 5339.
[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 Shamim, M.
Right arrow Articles by Suresh, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shamim, M.
Right arrow Articles by Suresh, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS