Interferon regulatory factor 4 plays a pivotal role in the development of aGVHD-associated colitis

Figures & data

Figure 1. Irf4^-/- CD4⁺ T cell induce less aGVHD and GVHD associated colitis.

Lethally irradiated BALB/c recipients that received 5 × 10⁶ CD45.2⁺ WT BL/6 BM depleted for CD90.2⁺ cells at day one and 1 × 10⁶ CD4⁺ CD45.1⁺ Luciferase⁺ T cells from WT, Irf4^-/- or Irf4^± donors with BL/6 background at day two (a) were monitored every 2–3 days for clinical GVHD score (b) and for colitis development at the end of the experiment (c). Data represent the mean ± standard deviation combined from 3 to 8 independent experiments (a-b: BM = 8, Irf4^-/- = 7, Irf4^± = 4, WT = 5 experiments; c: all groups = 3 experiments). (d) The colons of BM and CD4⁺ T cell recipients were analyzed at day 14 post irradiation. Histological sections were stained with Hematoxylin/Eosin (HE) and scored for histopathological changes. One representative picture is shown per condition. Data represent the mean ± standard deviation combined from 1 to 3 independent experiments (BM = 2, Irf4^-/- = 2, Irf4^± = 1, WT = 3 experiments).

If applicable, all data points each representing an individual are shown. **P < .01, ***P < .005, **** P < .0001. Analyses were performed with the two tailed Mann-Whitney test.

Figure 2. Irf4^-/- CD4⁺ T cells show decelerated proliferation but migrate to the same aGVHD target organs like WT and Irf4^± CD4⁺ T cells.

The migration of 1 × 10⁶ CD4⁺ CD45.1⁺ Luciferase⁺ T cells from WT (red), Irf4^−/− (blue) or Irf4^+/- (orange) donors with BL/6 background in the lethally irradiated WT BL/6 CD0.2-depleted BM transplanted recipients was traced by measuring the frequency of CD45.1⁺ CD4⁺ T cells in the spleen at day 15 after irradiation via flow cytometry (a) and by bioluminescent imaging of the Luc⁺ CD4⁺ T cells considering the total bioluminescent signal (b) and the signal distribution throughout the body (c) at different time points. At day 14/15 (d, e) and day 31 (d, e) bioluminescent signal was additionally measured in the spleen and colon after dissection. Representative BLI pictures for the respective groups are shown.

Data represent the mean ± standard deviation combined from independent experiments. If applicable, all data points each representing an individual are shown. *P < .05, **P < .01, ***P < .005, ****P < .0001. The legend of Figure 2 applies for all subfigures. Analyses were performed with the two tailed Mann-Whitney test.

Figure 3. RNA sequencing analysis of re-isolated CD45.1⁺ CD4⁺ T cells shows differences in the transcriptomic landscape between transplanted Irf4^+/- and Irf4^-/- cells and reflects the observations described in vivo.

Lethally irradiated BALB/c recipients received 5 × 10⁶ CD45.2⁺ WT C57BL/6 BM depleted for CD90.2⁺ cells at day one and 1 × 10⁶ CD4⁺ CD45.1⁺ Luciferase⁺ T cells from Irf4^+/- or Irf4^-/- donors with C57BL/6 background at day two. CD4⁺ T cells were re-isolated and sorted for CD45.1⁺ CD4⁺ T cells with high purity and RNA was extracted from these cells for RNA sequencing analysis (a). (b) T helper cell subset-regulating and -suppressing genes were plotted as mean of RNA sequencing reads between the two groups. (c) The up- and downregulation of Th-specific transcription factors between Irf4^+/- and Irf4^-/- CD4⁺ T cells was plotted by the mean of 2-fold change + standard deviation.

Data represent the mean combined from two independent experiments from n = 5 Irf4^−/− versus n = 8 Irf4^+/- CD4⁺ T cell transplanted mice.

Figure 4. Irf4^-/- CD4⁺ T cells exhibit an altered memory/effector and a compromised Th phenotype on comparison to Irf4^+/- and WT CD4⁺ T cells.

Lethally irradiated BALB/c recipients received 5 × 10⁶ CD45.2⁺ WT BL/6 BM cells depleted for CD90.2⁺ cells at day one and 1 × 10⁶ CD4⁺ CD45.1⁺ Luciferase⁺ T cells from WT, Irf4^−/− or Irf4^+/- donors with BL/6 background at day two. Re-isolated CD45.1⁺ CD4⁺ T cells were analyzed for CD62L and CD44 expression to determine effector/memory development (a) and for activation- (CD69), memory- (CD122) and maturation- (CD127) markers (b) at day 15 post irradiation by flow cytometry. Representative flow cytometry plots for the distribution of Irf4^+/- (orange) and WT (red) versus Irf4^-/- (blue) CD4⁺ T cells in the effector/memory compartment are shown.

(c) The differentiation to Th-1, Th-17 and Th-2 CD4⁺ T cells was determined by intracellular staining for IFN-γ, IL-17A and IL-4 respectively and flow cytometry analysis upon PMA/Ionomycin stimulation of re-isolated CD45.1⁺ CD4⁺ T cells at day 15 post irradiation. (d) Re-isolated CD45.1⁺ CD4⁺ T cells at day 15 after irradiation were analyzed for the Treg specific transcription factor FoxP3 and Helios via flow cytometry. Data represent the mean combined independent experiments from n = 4-10 CD4⁺ T cell transplanted mice.

Data represent the mean ± standard deviation combined from independent experiments. If applicable, all data points each representing an individual are shown. *P < .05, **P < .01, ****P < .0001. Analyses were performed with the two tailed Mann-Whitney test.

Figure 5. IRF4 but not BACH2 expression are altered by GVHD immunosuppressant therapy and influence inflammatory versus anti-inflammatory signatures in intestinal biopsies of GVHD patients.

(a) Lethally irradiated BALB/c recipients received 5 × 10⁶ CD45.2⁺ WT C57BL/6 BM depleted for CD90.2⁺ cells at day one and 1 × 10⁶ CD4⁺ CD45.1⁺ Luciferase⁺ T cells from WT, Irf4^-/- or Irf4^+/- donors with C57BL/6 background at day two. Volcano plot mapping differentially expressed genes (DEGs) between Irf4^-/- and Irf4^± CD4⁺ T cells described in Figure 3 satisfying the criteria of log2 fold change > 1 or >-1 and p < 0.05. Significantly different expressed genes are labeled blue, genes of interest (GOI) are labeled black. (b) Western Blot detecting BACH2 and IRF4 protein expression of WT and Irf4^-/- CD4⁺ T cells before and after 10 days of MLR. WEHI-231 cells were used as control for BACH2 and IRF4 detection. ß-Actin was used as loading control.

(c) IRF4 and BACH2 expression in intestinal biopsies of n = 76 patients who received HSCT during different stages of GVHD development analyzed by qPCR. (d) IRF4 and BACH2 expression were quantified in CD4⁺ T cells derived from healthy donors (n = 8) after 3 days under stimulated or unstimulated conditions and simultaneous treatment with Cyclosporine A (CyA), Methylprednisolone (MP), a combination of both or without treatment. Samples were analyzed with Kruskal-Wallis test.

Intestinal biopsies of patients (n = 76) who received HSCT were divided into samples expressing IRF4 (e) or BACH2 (f) transcripts above and below median and plotted against NLRP3 (e) or FoxP3 (f) expression, quantified via qPCR (e) or immunohistochemistry (f) in these samples. Samples were analyzed by two-tailed Mann-Whitney-test. Whisker-box plots indicate the mean, min and max value of individual sample sizes indicated in the respective plots. Only significant statistical test results are indicated. *P < .05, **P < .01, ***P < .001, ****P < .0001.