Prognostic implications of a CD8+ TEMRA to CD4+Treg imbalance in mandibular fracture healing: a prospective analysis of immune profiles

Abstract

Introduction: Open reduction and fixation are the standard of care for treating mandibular fractures and usually lead to successful healing. However, complications such as delayed healing, non-union, and infection can compromise patient outcomes and increase healthcare costs. The initial inflammatory response, particularly the response involving specific CD8⁺ T cell subpopulations, is thought to play a critical role in healing long bone fractures. In this study, we investigated the role of these immune cell profiles in patients with impaired healing of mandibular fractures.

Materials and methods: In this prospective study, we included patients with mandibular fractures surgically treated at Charité - Universitätsmedizin Berlin, Germany, between September 2020 and December 2022. We used follow-up imaging and clinical assessment to evaluate bone healing. In addition, we analyzed immune cell profiles using flow cytometry and quantified cytokine levels using electrochemiluminescence-based multiplex immunoassays in preoperative blood samples.

Results: Out of the 55 patients enrolled, 38 met the inclusion criteria (30 men and 8 women; mean age 32.18 years). Radiographic evaluation revealed 31 cases of normal healing and 7 cases of incomplete consolidation, including 1 case of non-union. Patients with impaired healing exhibited increased levels of terminally differentiated effector memory CD8⁺ T cells (T_EMRA) and a higher T_EMRA to regulatory T cell (T_reg) ratio, compared with those with normal healing.

Conclusions: Our analysis of mandibular fracture cases confirms our initial hypothesis derived from long bone fracture healing: monitoring the T_EMRA to T_reg ratio in preoperative blood can be an early indicator of patients at risk of impaired bone healing. Radiologic follow-up enabled us to detect healing complications that might not be detected by clinical assessment only. This study highlights the potential of individual immune profiles to predict successful healing and may form the basis for future strategies to manage healing complications.

Keywords: TEMRA; bone healing; mandibular fractures; non-union; ossification; pseudarthrosis.

Figure 1

An illustration of the scoring system to evaluate bone healing based on follow-up imaging, with a score of 0 on the left side and a score of 2 on the right side.

Figure 2

Aheatmap of 10 metabolic and pro-inflammatory markers in patient serum. The data were divided into two groups based on the healing outcome: normal healing (n = 20) and impaired healing (n = 6). The mean cytokine levels (in pg/mL) are presented for each group.

Figure 3

Delayed bone healing is linked to a more experienced systemic adaptive immune profile. The distributions of different immune cell populations with regard to bone healing in patients with mandibular fractures are shown. (A) Lymphocytes, CD3⁺ T cells, CD4⁺ T cells, and CD8⁺ T cells. (B) CD4⁺ T cell subsets: central memory (CD4⁺ T_CM, CCR7⁺CD45RA^–), naive (CD4⁺ T_naïve, CCR7⁺CD45RA⁺), effector memory (CD4⁺ T_EM, CCR7^–CD45RA^–), and terminal differentiated effector memory (CD4⁺ T_EMRA, CCR7^–CD45RA⁺). (C) CD8⁺ T cell subsets: central memory (CD8⁺ T_CM, CCR7⁺CD45RA^–), naive (CD8⁺ T_naïve, CCR7⁺CD45RA⁺), effector memory (CD8⁺ T_EM, CCR7^–CD45RA^–), and terminal differentiated effector memory (CD8⁺ T_EMRA; CCR7^–CD45RA⁺). The data are presented as the mean ± standard deviation of the indicated group (n = 38). For A–C, an unpaired two-sided t-test was used for statistical analysis. *P < 0.05.

Figure 4

Elevated systemic levels of (CD28^–CD57^–) CD8⁺ T_EM-like and (CD28^–CD57⁺) CD8⁺ T_EMRA-like cells in patients with compromised bone healing. (A) CD8⁺CD28^– and CD4⁺CD28^– T cells. (B) CD8⁺CD57⁺ and CD4⁺CD57⁺ T cells. (C) Non-activated (CD28⁺CD57^–) CD8⁺ T cells, activated (CD28⁺CD57⁺) CD8⁺ T cells, effector memory-like (T_EM-like) (CD28^–CD57^–) CD8⁺ T cells, and terminal differentiated effector memory-like (T_EMRA-like) (CD28^–CD57⁺) CD8⁺ T cells. (D) Non-activated (CD28⁺CD57^–) CD4⁺ T cells, activated (CD28⁺CD57⁺) CD4⁺ T cells, (CD28^–CD57^–) CD4⁺ T_EM-like cells, and (CD28^–CD57⁺) CD4⁺ T_EMRA-like cells. The data are presented as the mean ± standard deviation of the indicated group (n = 38). For (A, B, D), an unpaired two-sided t-test was used for statistical analysis. For (C), the Mann–Whitney U test was employed for statistical analysis. *P < 0.05.

Figure 5

No significant differences in systemic CD4⁺ T_reg levels between normal and impaired healing. (A) CD4⁺CD25^hi T cells. (B) CD4⁺CD25^hiFoxP3⁺ T cells. (C) CD4⁺CD25^hiCD127^low/– T cells. (D) CD4⁺CD25^hiFoxP3⁺CD127^low/– T cells. The data are presented as the mean ± standard deviation of the indicated group (n = 38).

Figure 6

Significant association between the (CD28^–CD57^–) CD8⁺ T_EM-like or (CD28^–CD57⁺) CD8⁺ T_EMRA-like cell to (CD25^hiFoxP3⁺CD127^low/–) CD4⁺ T_reg ratios and impaired bone healing. (A) The CD8⁺ T_EM-like cell to CD4⁺ T_reg ratio. (B) The CD8⁺ T_EMRA cell to CD4⁺ T_reg ratio. The data are presented as the mean ± standard deviation for the indicated group (n = 38). For (A, B), an unpaired two-sided t-test was used for statistical analysis. *P < 0.05.

Figure 7

Correlation analysis, reveals there are no significant differences in marker stability across various CD8⁺ T cell subsets. (A) CD3⁺ T cells as a percent of CD45⁺ cells (Pearson r = 0.65, 95% CI 0.32–0.84, P = 0.001). (B) CD8+ T cells as a percent of CD3+ T cells (Pearson r = 0.86, 95% CI 0.68–0.94, P < 0.0001). (C) CD28^–CD57^– CD8⁺ T_EM-like cells as a percent of CD3⁺CD8⁺ T cells (Pearson r = 0.83, 95% CI 0.63–0.93, P < 0.0001). (D) CD28^–CD57⁺ CD8⁺ T_EMRA-like cells as a percent of CD3⁺CD8⁺ T cells (Pearson r = 0.86, 95% CI 0.69–0.94, P < 0.0001). (E) CCR7^–CD45RA^– CD8⁺ T_EM cells as a percent of CD3⁺CD8⁺ T cells (Pearson r = 0.87, 95% CI 0.70–0.95, P < 0.0001). (F) CCR7^–CD45RA⁺ CD8⁺ T_EMRA cells as a percent of CD3⁺CD8⁺ T cells (Pearson r = 0.91, 95% CI 0.79–0.96, P < 0.0001). The data are based on the pre- and postoperative blood samples from the same patients (n = 22).

Figure 8

The CD8⁺ T_EM-like and CD8⁺ T_EMRA-like cell to CD25^hiFoxP3⁺CD127^low/– CD4⁺ T_reg ratios predict impaired fracture healing. (A) The ROC curves for the (CD28^–CD57^–) CD8⁺ T_EM-like cell to CD25^hiFoxP3⁺CD127^low/– CD4⁺ T_reg ratio in preoperative blood samples (AUC = 0.81, standard error = 0.07, 95% CI 0.67–0.95, n = 37, P = 0.01). (B) The ROC curves for the (CD28^–CD57⁺) CD8⁺ T_EMRA-like cell to CD25^hiFoxP3⁺CD127^low/– CD4⁺ T_reg ratio in perioperative blood samples (AUC = 0.7714, standard error = 0.08742, 95% CI 0.60–0.94, n = 38, P = 0.027).