Individual Effector/Regulator T Cell Ratios Impact Bone Regeneration

Abstract

There is increasing evidence that T lymphocytes play a key role in controlling endogenous regeneration. Regeneration appears to be impaired in case of local accumulation of CD8+ effector T cells (T_EFF), impairing endogenous regeneration by increasing a primary "useful" inflammation toward a damaging level. Thus, rescuing regeneration by regulating the heightened pro-inflammatory reaction employing regulatory CD4+ T (T_Reg) cells could represent an immunomodulatory option to enhance healing. Hypothesis was that CD4+ T_Reg might counteract undesired effects of CD8+ T_EFF. Using adoptive T_Reg transfer, bone healing was consistently improved in mice possessing an inexperienced immune system with low amounts of CD8+ T_EFF. In contrast, mice with an experienced immune system (high amounts of CD8+ T_EFF) showed heterogeneous bone repair with regeneration being dependent upon the individual T_EFF/T_Reg ratio. Thus, the healing outcome can only be improved by an adoptive T_Reg therapy, if an unfavorable T_EFF/T_Reg ratio can be reshaped; if the individual CD8+ T_EFF percentage, which is dependent on the individual immune experience can be changed toward a favorable ratio by the T_Reg transfer. Remarkably, also in patients with impaired fracture healing the T_EFF/T_Reg ratio was higher compared to uneventful healers, validating our finding in the mouse osteotomy model. Our data demonstrate for the first time the key-role of a balanced T_EFF/T_Reg response following injury needed to reach successful regeneration using bone as a model system. Considering this strategy, novel opportunities for immunotherapy in patients, which are at risk for impaired healing by targeting T_EFF cells and supporting T_Reg cells to enhance healing are possible.

Keywords: bone healing; effector T cell; mouse model; regeneration; regulatory T (Treg) cell.

Figure 1

Enhancing bone healing by adoptive transfer of murine CD4+ T_Reg in SPF mice. (A) Demonstration of the functionality of isolated murine CD4+ T_Reg in a suppression assay, illustrated is the suppression of the proliferation of CD4+CD25- T_Responder cells by using freshly isolated CD4+ T_Reg (n = 6). (B) Status of CD4+ T_Reg in the peripheral blood of the experimental mice 1 d after adoptive transfer, control (n = 6) vs. CD4+ T_Reg enriched mice (n = 6). (C,D) Improved bone healing by adoptive CD4+ T_Reg transfer as demonstrated by μCT evaluation after 21 d, control (C, left, control SPF) vs. T_Reg enriched mice (C, right, +T_Reg SPF). Quantification of the μCT data revealed a significantly higher BV/TV in the mice that received a CD4+ T_Reg transfer (+T_Reg) (n = 6) compared to the control SPF mice (n = 5) (D). BV, bone volume; TV, total callus volume; BV/TV, ratio of BV to TV; Mann-Whitney U-test.

Figure 2

Heterogeneous impact of adoptive transfer of CD4+ T_Reg on bone healing outcome in mice kept under non-SPF housing conditions. (A–D) μCT evaluation of the healing outcome after adoptive CD4+ T_Reg transfer after 21 d. (A) Representative images of the analyzed volume of interest of the healing bones are shown. +T_Reg treated mice clustered into responder (+T_Reg left, n = 4) and non-responder (+T_Reg right, n = 4). Control mice showed no complete bridging after 21 d (left, control, n = 6). In contrast, +T_Reg responder showed complete bridging, whereas non-responder showed no bridging. (B–D) Quantification of the μCT analysis, + T_Reg responder vs. + T_Reg non-responder vs. control mice. (E–G) Evaluation of the immune cell composition of +T_Reg responder vs. non-responder 21 d post-surgery. (E) Confirmation of CD4+ T_Reg engraftment in both, +T_Reg responder and non-responder compared to the control. (F,G) +T_Reg non-responder showed an elevated percentage of CD8+ T_EFF (F) as well as a significantly higher ratio of CD8+ T_EFF /CD4+ T_Reg (G) in the peripheral blood in comparison to the +T_Reg responder. TV, total volume; BV, bone volume; BV/TV, ratio of BV to TV; Mann–Whitney U-test, Bonferroni correction (B–E).

Figure 3

Individual effector/regulatory T cell ratio pre-osteotomy determines the healing outcome in mice. (A) Mice having a lower pre-surgery CD8+ T_EFF to CD4+ T_Reg ratio (left y-axis) showed a higher BV/TV (right y-axis, axis is reversed for a clearer depiction). Dashed line indicates the cut off between the +T_Reg responder (below the dashed line) and the +T_Reg non-responder (above the dashed line). (B) Adoptive CD4+ T_Reg transfer did not reshape the unfavorable CD8+ T_EFF/CD4+ T_Reg ratio. (C) Engraftment of CD4+CD25++ T cells 1 d post-surgery is elevated in both, the +T_Reg responder (n = 4) and +T_Reg non-responder (n = 3) in comparison to the control group (n = 6). 45BV, total bone volume; TV, total callus volume; BV/TV, ratio of BV to TV; Mann–Whitney U-test.

Figure 4

Impaired bone healing correlates with an elevated CD8+ T_EMRA level in both the peripheral blood as well as in the local fracture hematoma of patients. (A) Results of the flow cytometry analyses of terminally differentiated CD8+ effector T cells (CD8+T_EMRA) in the peripheral blood of fracture patients at the time of operation are shown. Impaired fracture healing patients showed a significantly higher frequency of CD45+CD3+CD8+CD57+CD28- T cells (CD57+CD28-+ in % of CD45+CD3+CD8+ T cells) compared to the normal healing patients indicates a dramatic increase of CD8+ T_EMRA: n = 12 for the impaired healing group, n = 23 for the normal healing group. (B) Quantification of CD8+ T cells in the hematoma region of fracture healing patients by epigenetic qPCR analysis. Impaired fracture healing patients showed an elevated level of CD8+ T cells in the local fracture area: n = 5 for the impaired healing group, n = 3 for the normal healing group (C,D) Representative x-ray images from the study cohort over a period of 15 months including the pre-surgery as well as the 6 and 15 months post-surgery states of fracture healing of an impaired healing (C) and a normal healing (D) patient, respectively. Student's t-test.

Figure 5

Impaired fracture healing correlates with an elevated level of CD4+ T_Reg and a higher ratio of CD8+ T_EMRA/CD4+ T_Reg. (A) Results of the flow cytometry analyses of the CD4+ T_Reg and the ratio CD8+T_EMRA/CD4+T_Reg in the peripheral blood of fracture patients at the time of operation are shown. Impaired fracture healing patients showed slightly elevated levels of CD45+CD3+CD4+CD25highCD127low T_Reg (CD25highCD127low in % of CD45+CD3+CD4+) compared to the normal healing patients indicates a partial, but not sufficient compensatory effect. The ratio of CD8+T_EMRA/CD4+T_Reg was significantly higher in patients with a delayed healing: n = 12 for the impaired healing group, n = 23 for the normal healing group. (B) Impaired fracture healing patients showed a significantly higher ratio of CD8+T cells/CD4+ T_Reg in the hematoma region demonstrating an insufficient compensatory effect of the CD4+ T_Reg in the impaired fracture healing patients: n = 5 for the impaired healing group, n = 3 for the normal healing group. Student's t-test.

Figure 6

Interdependency of the immune status and the healing outcome after bone fracture. The ratio of CD8+ effector T cells (CD8+ T_EFF) to CD4+ regulatory T cells (CD4+ T_Reg) affects the healing outcome after bone fracture. Under normal healing conditions, the T cell ratio is balanced. An elevated amount of CD8+ T_EFF relative to CD4+ T_Reg is unfavorable for successful healing. A shifted balance in the CD4+ T_Regsb direction improves bone fracture healing.