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. 2008 Aug 1;112(3):652-60.
doi: 10.1182/blood-2008-01-134486. Epub 2008 May 27.

c-Cbl Expression Levels Regulate the Functional Responses of Human Central and Effector Memory CD4 T Cells

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Free PMC article

c-Cbl Expression Levels Regulate the Functional Responses of Human Central and Effector Memory CD4 T Cells

Nicolò C Brembilla et al. Blood. .
Free PMC article

Abstract

The biochemical mechanisms controlling the diverse functional outcomes of human central memory (CM) and effector memory (EM) T-cell responses triggered through the T-cell receptor (TCR) remain poorly understood. We implemented reverse phase protein arrays to profile TCR signaling components in human CD8 and CD4 memory T-cell subsets isolated ex vivo. As compared with CD4 CM cells, EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c-Cbl, Syk, Fyn, and LAT. Moreover, in EM cells reduced expression of negative regulator c-Cbl correlates with expression of c-Cbl kinases (Syk and Fyn), PI3K, and LAT. Importantly, consistent with reduced expression of c-Cbl, EM cells display a lower functional threshold than CM cells. Increasing c-Cbl content of EM cells to the same level as that of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism depends primarily on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Our study reports c-Cbl as a critical regulator of the functional responses of memory T cell subsets and identifies for the first time in humans a mechanism controlling the functional heterogeneity of memory CD4 cells.

Figures

Figure 1
Figure 1
Characteristics of RPP microarrays and validation of β-actin as protein for normalization. (A) Mean fluorescence intensity (MFI) of SLP-76–specific signal detected in spots containing equal protein amount of PBMC or intestinal Caco-2 cell lysates. Alternatively, exogenous RNase-2 was added to PBMC lysate and specific RNase-2 signals were normalized to β-actin content (bar graphs). Immunodetection of Western blots (WB) with specific antibodies for SLP-76 and RNAse-2 in the corresponding lysates are shown in right panels (300 000 cells/lane). (B) Quantitative analysis of Lck detection in serially diluted PBMC lysates using specific antibodies. The bottom panel shows corresponding spots printed in triplicate. (C) β-Actin as protein for normalization. β-Actin and β2-microglobulin contents were measured in lysates of sorted N, CM, EM, and E cells following SDS-PAGE and immunoblotting with specific antibodies. Shown are representative immunodetection and quantitative measurements of β-actin signals from WB in 3 cumulated subjects.
Figure 2
Figure 2
Analysis of the signaling components in CD8 and CD4 T-cell subsets. Signaling components were quantified by RPP arrays and normalized to β-actin in the CD8 (A) and CD4 (B) T-cell subsets. The colors indicate the mean log2 ratios of signaling components relative to N cells from 8 to 13 subjects. Signaling components showing significant differences (2-tailed paired t test; P < .05, shown in red) between CM and EM cells are represented as row ratios on individual graphs. CM and EM compartments are shown in black. Each set of linked dots corresponds to a distinct subject; n indicates the total number of subjects examined. Lck was detected using specific antibodies recognizing epitopes located at the N- and C-terminal ends of the protein (Lck N and Lck C, respectively) to ensure fully denaturing conditions.
Figure 3
Figure 3
Subject heterogeneity in the expression of signaling components in CD4 and CD8 T-cell subsets. Subject heterogeneity as expressed by CV of the 15 signaling components (n = 15) under study in 8 to 13 cumulated subjects in CD4 (A) and CD8 (B) T-cell subsets. Box plots were automatically generated using GraphPad, the box represents values between 25th and 75th percentile with a line at the median (50th percentile). The whiskers extend above and below the box to show the highest and the lowest values. Shown are signaling components with highest subject heterogeneity in CM or EM cells.
Figure 4
Figure 4
c-Cbl, but not Cbl-b, expression is reduced in EM CD4 T cells. (A) Quantification of c-Cbl expression level in CM and EM cells by RPP arrays and immunodetection of Western blots (WB) with antibodies specific for c-Cbl and actin. Shown are c-Cbl to actin ratios in 10 (RPP arrays, 30 cells equivalent/spot) or 4 (WB, 300 000 cells equivalent/lane) distinct subjects. Examples of detected spots (B) and quantified immunodetections (C) are shown. (D) Quantification of Cbl-b by RPP arrays in 9 subjects as described for panel A.
Figure 5
Figure 5
In EM cells, SLP-76, LAT, PI3K, and c-Cbl expression are coregulated. (A) Correlation of the expression of SLP-76, LAT, PI3K with c-Cbl in 8 to 10 distinct subjects. The significance of their linear correlation is shown (Pearson r and P < .05). (B) Correlation of the expression of TCR/CD3 ϵ and ζ components as described for panel A.
Figure 6
Figure 6
Functional threshold in CM and EM CD4 T cells. (A) Detection by FACS of intracellular production of IFN-γ and IL-2 in memory cells exposed for 1 night to graduated concentrations of immobilized anti-CD3 and 1 μg/mL soluble anti-CD28 antibodies. Shown is 1 representative response of 4 (left). Cumulated data of EM cells from 4 subjects in response to 5 μg/mL immobilized anti-CD3 antibody supplemented with 1 μg/mL soluble anti-CD28 or -CD4 antibodies are shown (right). (B) Proliferative potential of memory T cells measured by CFSE dilution in sorted CM and EM cells stimulated for 6 days as described for panel A (left). Shown are cumulated data of 4 subjects in response to 5 μg/mL of immobilized anti-CD3 antibody supplemented with 1 μg/mL soluble anti-CD28 antibody in the presence or absence of IL-2 (right). (C) Intracellular calcium mobilization in CM and EM cells in response to graduated concentrations of soluble mouse anti-CD3 and 1 μg/mL soluble anti-CD28 antibodies (left). The percentage of cell showing changes in intracellular calcium concentration was assessed by measuring variations of FL5/FL4 ratio (right).
Figure 7
Figure 7
Transduction of CTP-c-Cbl in EM cells results in lower functional response. (A) Limited amounts of CTP-GFP were transduced in memory cells and the transduction efficiency in CM and EM cells assessed by FACS. Thin and bold lines depict non-transduced and transduced cells, respectively. (B) Detection of c-Cbl following SDS-PAGE and immunoblotting of lysates of memory CD4 T cells previously transduced with wt or C381A CTP-c-Cbls and activated overnight with anti-CD3 and -CD28 antibodies. Vertical lines have been inserted to indicate repositioned gel lanes. (C) Proliferative capacities of CM and EM cells as measured by H3 thymidine incorporation (left). Memory cells were transduced with increasing amount of wt and C381A CTP-c-Cbls fusion proteins (AU: arbitrary units), stimulated for 16 hours with anti-CD3 (5 μg/mL) and -CD28 (1 μg/mL) antibodies and their proliferation measured after 3 days. CTP-mock transduction was used as negative control (formula image). Shown are data from 4 cumulated subjects (right). (D) Effect of CTP-c-Cbls transduction on memory T-cell cytokine production in response to the stimulation described for panel C. Cytokine production was normalized to non-transduced cells and CTP-mock transduction used as negative control. Significant changes (P < .05) in the functions of transduced cells relative to control and mock-transduced cells are indicated (★) as well as P values for significant differences between wt and C381A mutant c-Cbls. (E) Effect of CTP-c-Cbl on the proliferation threshold of purified EM cells stimulated as described for panel C. One representative example out of 3 is shown. (F) Impact of CTP-c-Cbl transduction on the proliferation of EM cells in response to IL-2, anti-CD3 (3 μg/mL) and -CD28 (1 μg/mL) antibodies. Each set of linked dots correspond to a distinct subject and significant changes (P < .05) between IL-2–treated and untreated cells are shown. (G) Quantification of the amounts of CTP-c-Cbls transduced in memory T cells relative to endogenous expression of c-Cbl following 16 hours of activation.

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