Biologic Agents in Crohn's Patients Reduce CD4+ T Cells Activation and Are Inversely Related to Treg Cells

Abstract

Crohn's disease (CD) is a chronic inflammatory disease with a complex interface of broad factors. There are two main treatments for Chron's disease: biological therapy and nonbiological therapy. Biological agent therapy (e.g., anti-TNF) is the most frequently prescribed treatment; however, it is not universally accessible. In fact, in Brazil, many patients are only given the option of receiving nonbiological therapy. This approach prolongs the subsequent clinical relapse; however, this procedure could be implicated in the immune response and enhance disease severity. Our purpose was to assess the effects of different treatments on CD4⁺ T cells in a cohort of patients with Crohn's disease compared with healthy individuals. To examine the immune status in a Brazilian cohort, we analyzed CD4⁺ T cells, activation status, cytokine production, and Treg cells in blood of Crohn's patients. Patients that underwent biological therapy can recover the percentage of CD4⁺CD73⁺ T cells, decrease the CD4⁺ T cell activation/effector functions, and maintain the peripheral percentage of regulatory T cells. These results show that anti-TNF agents can improve CD4⁺ T cell subsets, thereby inducing Crohn's patients to relapse and remission rates.

Figure 1

Phenotypic characterization of CD4⁺ T cells. Green plots healthy individuals, red triangles represent Crohn's patients undergoing biological therapy, and blue triangles represent Crohn's patients undergoing nonbiological therapy. (a) The subject's PBMC showing the percentage of total CD4⁺ T cells. (b)–(d) Median fluorescence intensity (MFI) of CD4⁺ T cells expressing CD73⁺, HLA-DR⁺, and CD38⁺, respectively. Each dot represents an individual, and bars indicate medians in the graphs. Statistical analysis was performed using the Kruskal–Wallis test.

Figure 2

Characteristics of memory and effector markers on CD4⁺ T cells. Green plots healthy individuals and red triangles represent Crohn's patients undergoing biological therapy. (a) Percentage of CD4⁺ T cells expressing CD45RO⁺. (b) Median fluorescence intensity (MFI) of CD4⁺ T cells expressing CD45RO⁺. (c) Percentage of CD4⁺ T cells expressing RORγt⁺. (d) Median fluorescence intensity (MFI) of CD4⁺ T cells expressing RORgt+. Each dot represents an individual, and bars indicate medians in the graphs. Statistical analysis was performed using the Kruskal–Wallis test.

Figure 3

Biological therapy shows a lower percentage of CD4⁺ subsets of cells. Analysis was done previously on gating CD4⁺ T cells. (a) Frequencies of Treg suppressor cells on PBMCs. (b) Percentage of FOXP3⁺CD25⁺CD73⁺CD39⁺ in the groups. (c) The ratio of secretion of IL17 and expression of Treg in the groups of treated patients. Intracellular cytokine staining of PBMCs from a representative Crohn's patient after stimulation with PMA/ionomycin. (d)-(e) Correlation of percentage of natural Tregs and CD4⁺HLA-DR⁺ T cells and CD4⁺CD38⁺ T cells in Crohn's patients undergoing biological therapy, respectively. Each dot represents an individual, and bars indicate medians in the graphs. Statistical analysis was performed using the Kruskal–Wallis test and correlation Pearson test with alpha equal to 0.05.

Figure 4

Presence of Treg suppressor on the intestine. (a) Six immunostaining with the specific antibodies. From right to left, we have the merged images. The top right is an example of lesion tissue and bottom-right healthy margins. The six quadrants from the left show an example of a single stain. (b) Counting of suppressor Treg cells in the intestine. Each dot represents the median of the count. Statistical analysis was performed using the Mann–Whitney test.