Differential response of regulatory and conventional CD4⁺ lymphocytes to CD3 engagement: clues to a possible mechanism of anti-CD3 action? - PubMed (original) (raw)
Differential response of regulatory and conventional CD4⁺ lymphocytes to CD3 engagement: clues to a possible mechanism of anti-CD3 action?
Li Li et al. J Immunol. 2013.
Abstract
Several clinical trials have shown anti-CD3 treatment to be a promising therapy for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3(+) regulatory T cells (Tregs) are likely to be involved, but through unknown mechanistic pathways. We profiled the transcriptional consequences in CD4(+) Tregs and conventional T cells (Tconvs) in the first hours and days after anti-CD3 treatment of NOD mice. Anti-CD3 treatment led to a transient transcriptional response, terminating faster than most Ag-induced responses. Most transcripts were similarly induced in Tregs and Tconvs, but several were differential, in particular, those encoding the IL-7R and transcription factors Id2/3 and Gfi1, upregulated in Tregs but repressed in Tconvs. Because IL-7R was a plausible candidate for driving the homeostatic response of Tregs to anti-CD3, we tested its relevance by supplementation of anti-CD3 treatment with IL-7/anti-IL-7 complexes. Although ineffective alone, IL-7 significantly improved the rate of remission induced by anti-CD3. Four anti-human CD3 mAbs exhibited the same differential effect on IL-7R expression in human as in mouse cells, suggesting that the mechanism also underlies therapeutic effect in human cells, and perhaps a rationale for testing a combination of anti-CD3 and IL-7 for the treatment of recent-onset human type 1 diabetes. Thus, systems-level analysis of the response to anti-CD3 in the early phase of the treatment demonstrates different responses in Tregs and Tconvs, and provides new leads to a mechanistic understanding of its mechanism of action in reverting recent-onset diabetes.
Conflict of interest statement
Competing interests: This work was performed at Harvard Medical School, and the Harvard authors have no personal conflicts of interest.
Figures
Figure 1. Tconv and Treg cell respond differently to anti-CD3
Transcriptional profiles were generated from Tconv and Treg CD4+ splenocytes from pooled mice (n=3) at 2 to 72 hrs after treatment with anti-CD3. (A) Changes in activation markers that reflect TCR signalling. (B) Comparison of the changes elicited in Treg and Tconv cells (visualized as the ratio of expression at indicated times relative to untreated (t0)); numbers indicate the number of transcrips that change by 2.5-fold or more (B) A Tconv-Treg differential index was computed for each gene to reflect these differential changes (genes towards the top of the graph preferentially respond in Tconv, those at the bottom in Treg), and is plotted against the maximum change observed
Figure 2. Elements of the differential response of Tconv and Treg to anti-CD3
(A) The Tconv-Treg differential response, computed as in Fig1B, at 8 hrs (top panel) and 72 hrs (bottom) is plotted against the difference in expression between Tconv and Treg cells at baseline (y-axis), and the most extreme transcrips are shown by name. (B) Gene expression values (arbitrary units) for selected transcripts that are distinctively induced by anti-CD3 in Tconv and Treg cells. Values shown are average from two independent experiments.
Figure 3. IL7R expression on Tconv and Treg cells in response to anti-CD3
(A) Representative flow cytometric profile of IL7R and FoxP3 expression on TCRβ+CD4+ splenocytes of NOD mice, 72 hrs after treatment with anti-CD3 mAb (profiles representative of more than 5 different experiments. (B) Temporal analysis of IL7R expression on Tconv and Treg CD4+ splenocytes at different times after treatment with graded doses of anti-CD3 mAb i.p. *P ≤ 0.01 (Student’s t-test). Data are combined from 3 independent experiments, with 6 mice per dose.
Figure 4. Inverse relationship between IL7R expression and remaining surface CD3 after anti-CD3 mAb treatment
(A) Occupancy and clearance of CD3 molecules, and changes in IL7R expression, over a range of doses of anti-CD3. The amount of total, antibody-bound and free CD3 molecules, and of IL7R (as a FoldChange from untreated controls), on the surface of CD4+ Treg or Tconv cells was determined by flow cytometry 72 hrs after treatment with a range of doses of i.p. anti-CD3. Each dot is an individual mouse (B) CD3 molecules are similarly cleared in Treg and Tconv cells. Percent of remaining surface CD3 on Tconv and Treg cells from the same mouse, over a range of anti-CD3 doses (same experiments as Fig. 4A). (C) IL7R surface expression correlates with CD3 clearance in vivo. Changes in IL7R expression (as a FoldChange from untreated controls) plotted versus the percent of remaining surface CD3 on Tconv and Treg cells 72 hrs after treatment with a range of doses (same experiments as Fig. 4A). (D) In vitro effects of anti-CD3. Whole splenocytes were cultured with soluble anti-CD3 mAb at the concentrations shown, and remaining CD3 and IL7R expression on Tconv and Treg cells were determined at different times of culture. (E) IL7R surface expression correlates with CD3 clearance in culture. Change of IL7R expression plotted against the remaining CD3 expression on Tconv and Treg cell surface analyzed at 72 hrs of culture.
Figure 5. IL7 synergizes with anti-CD3 to induce expansion of Treg cells in vivo
(A) Percentage and absolute numbers (bottom panel) of FoxP3+CD4+ cells in the spleen, PLN, and pancreas of anti-CD3 treated (solid symbols, doses as shown) or anti-CD3 plus IL7/anti-IL7 (0.75μg/15μg) complexes treated (open symbols); grey symbol: IL7/anti-IL7 complexes alone. *P ≤ 0.0001. Data are combined from two independent experiments with 3 mice for each dose. (B) Proliferative response. CD4+ splenocytes from mice treated as indicated untreated, anti-CD3, anti-CD3 plus IL7, or control IgG and IL7 treated were analyzed by flow cytometry, 6 hrs after administration of with. EdU; top: profiles representative of 3 independent experiments. Bottom: time course analysis in response to graded doses of anti-CD3 alone or in combination with IL7/anti-IL7 complexes. *P ≤ 0.01. Data are combined with two independent experiments with 3 mice for each dose.
Figure 6. Synergistic effect of combined anti-CD3 and IL7 treatment in newly diabetic NOD mice
(A) Monitoring of blood glucose in recent-onset diabetic NOD females treated with anti-CD3 alone (50 μg daily, n=9), anti-CD3 together with IL7/anti-IL7 complexes (n=14), Il7 complexes alone, or none for five consecutive days; all mice received an insulin pellet to maintain glycemic control for 15-20 days. (B) Compilation of disease incidence in experiments as in A for several doses of anti-CD3 (IL7/anti-IL7 complexes at a constant dose of 0.75μg/15μg). (C) Insulitis in the pancreata of previously diabetic NOD female mice treated as in A (anti-CD3 n =5; anti-CD3 + IL7 complexes n =5), evaluated histologically after 100 days. Non-insulitic Ea16/NOD mice were included as a negative control. A minimum of 30 islets was examined for each mouse. (D) Representative islets from mice in long-term remission after treatment with anti-CD3 + IL7 complexes.
Figure 7. Differential IL7R expression on Tconv and Treg cells from human blood in response to anti-CD3
PBMCs from healthy volunteers were treated in culture with soluble anti-CD3 (4 different mAbs), and IL7R expression assessed in Treg and Tconv cells after 72 hrs by flow cytometry (A) Top: Scatter plots of FoxP3 and IL7R staining, values are MFI in FoxP3hi and FoxP3- cells; bottom: corresponding overlaid histograms. (B) Similar responses in Treg and Tconv cells from several independent donors. Changes in IL7R expression at 72 hrs (as FoldChange relative to t=0 control) induced by the different anti-CD3 mAbs on blood Tconv (green) and Treg (blue) from seven Caucasian donors.
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