Industrialized countries experience a population aging. Elderly patients, due to the experienced immunity, have a constant pro-inflammatory milieu. Little is known on how adaptive immunity impacts the tissue homeostasis and regeneration. The standardized housing of lab animals is specific pathogen free (SPF). However, this housing condition hinders antigen exposure and thus an aging of the adaptive immune system. We hypothesized that exposure to antigens and a developing adaptive immunity will impact tissue homeostasis and regeneration in mice. Mice kept under SPF housing or non-SPF were examined towards their immune status via flow cytometry, bone structure via microCT and bone competence via biomechanical torsional testing. MSCs from these mice were analyzed regarding their differentiation potential and ECM production under various immune cell signaling. Bone regeneration was analyzed in vivo in a mouse osteotomy model. The memory and effector compartment of the adaptive immunity was significantly increased in mice under non-SPF housing. This housing led to an increased femoral cortical thickness and torsional stiffness (p<0,05), whereas the tissue mineral density was not affected. The differentiation potential of stem cells under the influence of an aged immune milieu was significantly reduced. Bone formation was highly affected by the immune status and availed of a naïve immune cell milieu. Adaptive immunity directly impacts bone tissue formation, by exhibiting a constant stress, leading to structural differences in bone tissue organization as well as mechanical competence. For experimental settings, it appears highly relevant if mouse models have had the chance to develop an experienced immune system.

Recently, we could illustrate how tightly the bone and the immune system are interconnected during normal homeostasis but even stronger during bone regeneration. Specifically, the patient´s individual ratio of CD8+ effector T cells (TEFF, already identified as potential unfavorable cells for successful healing) to CD4+ regulatory T cells (TREG, one counterpart to CD8+ TEFF in controlling intratissue inflammation) prior to injury/ surgery appears to determine the healing outcome after fracture. We hypothesized that concentrating CD4+ TREG could serve as innovative therapeutic strategy to improve bone healing. We used an adoptive CD4+ TREG transfer in our well-established mouse osteotomy model. Before treatment, we identified the pre-surgery ratio of CD8+ TEFF/ CD4+ TREG by flow cytometry to characterize the healing potential of individual animals. Thereafter, we performed an adoptive CD4+ TREG transfer to reshape inflammation for supporting osteotomy healing. Across all groups, healing outcome was analyzed after 21 days post-surgery by µCT. Whereas TREG were highly supportive in SPF mice, we observed a heterogeneous clustered healing outcome in the non-SPF mice: TREG responder (improved healing outcome; p = 0.038) and TREG non-responder (impaired healing outcome; p = 0.024). Interestingly, the pre-/peri-surgery ratio of CD8+ TEFF/ CD4+ TREG was higher in the TREG non-responder (p=0.057). Thus, the amount of adoptively transferred CD4+ TREG was not sufficient to improve the healing outcome due to initial unfavorable high CD8+ TEFF/CD4+ TREG ratio. These results clearly show the importance of determining the individual immune status of each patient in the clinic before applying an immunotherapeutic approach.