Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy

Abstract

Antigen-specific immunotherapy combats autoimmunity or allergy by reinstating immunological tolerance to target antigens without compromising immune function. Optimization of dosing strategy is critical for effective modulation of pathogenic CD4(+) T-cell activity. Here we report that dose escalation is imperative for safe, subcutaneous delivery of the high self-antigen doses required for effective tolerance induction and elicits anergic, interleukin (IL)-10-secreting regulatory CD4(+) T cells. Analysis of the CD4(+) T-cell transcriptome, at consecutive stages of escalating dose immunotherapy, reveals progressive suppression of transcripts positively regulating inflammatory effector function and repression of cell cycle pathways. We identify transcription factors, c-Maf and NFIL3, and negative co-stimulatory molecules, LAG-3, TIGIT, PD-1 and TIM-3, which characterize this regulatory CD4(+) T-cell population and whose expression correlates with the immunoregulatory cytokine IL-10. These results provide a rationale for dose escalation in T-cell-directed immunotherapy and reveal novel immunological and transcriptional signatures as surrogate markers of successful immunotherapy.

Figure 1. Peptide dose dictates regulatory CD4⁺ T-cell phenotype and protection from EAE.

CD4⁺ T cells from Tg4 mice treated 10 times s.c. with different doses of MBP Ac1-9[4Y] were re-stimulated in the presence of irradiated antigen-presenting cells (APCs) and a titration of MBP Ac1-9[4K]. (a) After 3 days, proliferative responses were measured by ³[H] thymidine incorporation. (b–d) Cytokines detected in cultures described in a, measured by an enzyme-linked immunosorbent assay (ELISA). (e) In vitro-expanded CD4⁺ T cells from peptide-treated Tg4 mice were cultured at a ratio of 1:1 with responder CD4⁺ T cells from untreated Tg4 mice, irradiated APCs and 0.1 μg ml⁻¹ MBP Ac1-9[4K]. After 3 days, proliferative responses were measured by ³[H] thymidine incorporation. Graph shows percentage suppression of responder CD4⁺ T-cell proliferation, relative to proliferation of responder cells cultured alone. Data (a–e) are representative of three similar experiments, error bars show s.e.m. of two independent biological replicates, each assayed in triplicate. (f) Onset and severity of EAE in Tg4 mice pre-treated with 10 doses of MBP Ac1-9[4Y] before immunization with spinal cord homogenate/Complete Freund's Adjuvant and Pertussis toxin. Results of two independent experiments are pooled, showing mean disease score±s.e.m. (n=7 PBS group, n=6 for each peptide-treated group).

Figure 2. High peptide doses administered subcutaneously induce inflammatory cytokines in TCR transgenic mice.

Tg4 mice were treated 10 times s.c. with 80 μg MBP Ac1-9[4Y]. (a) Shows the percentage of mice (n=15) free from adverse effects during the course of treatment. (b) Box-and-whisker plots show cytokine concentrations in serum (n=6), collected 2 h after successive 80 μg MBP Ac1-9[4Y] treatments as detected by a multiplex fluorescent bead immunoassay (MFBI). Box extends from 25th to 75th percentiles, horizontal line in boxes represents median value, error bars show minimum and maximum values. Data are representative of three independent experiments. (c) MFBI-detected serum cytokines from Tg4 Rag-1^−/− mice 2 h after a second s.c. injection with 80 μg MBP Ac1-9[4Y]. Mean cytokine concentration+s.e.m. is shown for four individual animals.

Figure 3. Peptide dose escalation desensitizes antigen-specific CD4⁺ T cells, allowing safe administration of high peptide doses subcutaneously.

(a) MBP Ac1-9[4Y] dose escalation strategy. (b) Mean serum cytokine concentrations, detected by MFBI, 2 h after treatment of Tg4 mice with either six 8-μg MBP Ac1-9[4Y] doses or EDI (illustrated in a). Representative of three experiments, error bars show±s.e.m. of three biological replicates. *P≤0.05, **P≤0.01, ***P≤0.001, two-way analysis of variance with Bonferroni post-test, comparing 8-μg MBP Ac1-9[4Y]- and EDI-treated mice. Tg4 mice were treated s.c. with high antigen doses, with (+) or without (−) prior dose escalation (dosing strategy illustrated in c). CD4⁺ T-cell expression of Ki67, CD69 and CD62L 2 h after peptide challenge in vivo was determined by flow cytometric analysis (d). Scatter plots show the percentage of CD4⁺ cells from individual mice, which are Ki67⁺, CD69⁺ or CD62L⁺, horizontal lines show means for each column. Results of two independent experiments are pooled (n=6). **P≤0.01, ***P≤0.001, unpaired t-test.

Figure 4. Dose escalation to higher peptide doses improves induction of a regulatory CD4⁺ T-cell phenotype.

(a) Treatment groups for EDI with increasingly higher final doses. (b) Proliferative responses of CD4⁺ T cells from EDI-treated Tg4 mice cultured with irradiated antigen-presenting cells (APCs) and MBP Ac1-9[4K] for 3 days, measured by ³[H] thymidine incorporation. Representative of three independent experiments each with two biological replicates assayed in triplicate. (c) Percentages of Vβ8⁺ T cells expressing IL-10 and IFN-γ after 6 days culture with 10 μg ml⁻¹ MBP Ac1-9[4K], detected by flow cytometric analysis. Representative of two similar experiments, error bars show+s.e.m. of two biological replicates. (d) For in vitro suppression assay, in vitro-expanded CD4⁺ T cells from peptide-treated Tg4 mice cultured 1:1 with carboxyfluorescein succinimidyl ester (CFSE)-labelled responder CD4⁺ T cells, APCs and 10 μg ml⁻¹ MBP Ac1-9[4K]. After 3 days, the proliferative response of CD4⁺CFSE⁺ responder cells was measured by flow cytometry. For in vivo suppression assay, 5 × 10⁶ Cell Trace Violet (CTV)-labelled CD45.1⁺ Tg4 CD4⁺ T cells were transferred i.v. into EDI-treated Tg4 CD45.2⁺ mice. After 24 h, mice were injected s.c. with 80 μg of MBP Ac1-9[4Y]. Three days after peptide challenge, Cell Trace Violet-labelled CD45.1⁺ CD4⁺ cells were recovered from spleens for flow cytometric analysis. Data in each plot are representative of two biological replicates, offset histograms show proliferation dye dilution and division indexes.

Figure 5. Escalating dose immunotherapy reduces EAE susceptibility.

(a) Tg4 mice were pre-treated s.c. with an escalating course of MBP Ac1-9[4Y], either 0.08 μg→0.8 μg→4 × 8 μg (esc. to 8 μg) or 0.08 μg→0.8 μg→8 μg→3 × 80 μg (esc. to 80 μg). In vitro-differentiated MBP Ac1-9-specific Th1 cells (10⁷) were transferred i.v. to treated animals. (b) Six-week-old Tg4 Rag-1^−/− mice, susceptible to spontaneous development of EAE, were EDI-treated (esc. to 80 μg, as above). (c) EAE was induced in Tg4 mice by s.c. injection of Complete Freund's Adjuvant (CFA)/spinal cord homogenate (SCH) with Pertussis toxin given on day 0 and day 2. Animals were then EDI-treated (arrows indicate day of treatment, dosing as above escalating to 80 μg). Animals were monitored daily for onset of EAE symptoms. Graphs show mean EAE score and s.e.m., summary panel shows incidence, mean maximum score and mortality for each experiment, n as shown in a–c.

Figure 6. Transcriptome analysis of CD4⁺ T cells at consecutive stages of escalating dose immunotherapy.

Tg4 Rag-1^−/−mice were EDI-treated s.c. with MBP Ac1-9[4Y]. CD4⁺ T-cell transcriptome analysis was undertaken at the indicated treatment stages (n=3 per time point, RNA pooled). (a) CPP-SOM of 1,893 regulated transcripts across six stages of EDI, illustrating treatment stage-specific transcriptional changes, relative to PBS-treated controls. Hexagons represent groups of co-clustered transcripts; colour changes show modulation of transcript expression (upregulation in red, downregulation in blue and moderate regulation in yellow and green). (b) Twelve gene clusters (colour-coded, labelled 1–12) obtained by two-phase SOM clustering. Bar graphs show the expression pattern of the seed unit used to derive each gene cluster. (c) Heatmaps showing expression patterns of clustered genes, and GO terms associated with each. Gene clusters are organized according to five dominant patterns; genes that are repressed from baseline, repressed following initial induction, incrementally induced, induced and then repressed and finally, induced and then maintained throughout the course of treatment. All GO terms are associated with at least three transcripts within a cluster, with a false-discovery rate (FDR) of <0.05.

Figure 7. Modulated expression of select genes associated with regulatory T-cell phenotype during escalating dose immunotherapy.

Tg4 Rag-1^−/− mice were EDI-treated s.c. with MBP Ac1-9[4Y]. CD4⁺ T-cell transcriptome analysis was undertaken at the indicated treatment stages (n=3 per time point, RNA pooled) and transcripts were grouped by two-phase SOM clustering (see Fig. 6). Heatmaps are used to illustrate the fold changes in mRNA expression of individual genes during EDI. The cluster from which individual genes are derived (illustrated in Fig. 6b) is indicated by a colour identifier. Genes that were not included in clustering analysis (did not demonstrate a two-fold or greater change at ≥;4 treatment points, compared with the PBS-treated control) are indicated by a hyphen.

Figure 8. CD4⁺ T-cell signature induced by dose escalation immunotherapy.

Tg4 mice were treated s.c. with an escalating dose of MBP Ac1-9[4Y] (0.08 μg→0.8 μg→8 μg→3 × 80 μg). (a) Real-time PCR analysis of mRNA of select genes expressed by CD4⁺ T cells, 2 h after peptide challenge in vivo, at the indicated stages of treatment. Graphs show mean expression values+s.e.m. for three replicate experiments, pooled (n=3 total). (b) Flow cytometric staining of IL-10, c-Maf, NFIL3, FoxP3, LAG-3, TIGIT, PD-1 and TIM-3 by CD4⁺ T cells at the indicated stages of treatment. Cells were harvested 2 h after peptide challenge in vivo. Data are representative of two to three independent experiments.

Figure 9. Correlation between IL-10 and expression of surface molecules associated with a regulatory T-cell phenotype.

Tg4^GFP/Il10 mice were treated with an escalating dose of MBP Ac1-9[4Y] (0.08 μg→0.8 μg→8 μg→3 × 80 μg), inducing GFP(IL-10) low (lo), intermediate (int) or high (hi) expression by CD4⁺ T cells. (a) Percentages of GFP(IL-10) lo, int and hi cells expressing LAG-3, TIGIT, PD-1,TIM-3 and CD49b at the cell surface. (b) Expression of GFP(IL-10) by CD4⁺ T cells expressing the indicated cell surface markers. Data are representative of nine mice, over three independent experiments.