Alessandro Serra

Human memory T cells from the bone marrow are resting and maintain long-lasting systemic memory

Significance Memory T cells are essential components of immunological memory. In the apparent absence of antigen, numbers of recirculating antigen-specific memory T cells dwindle, provoking the question of whether there is immunological memory without memory T cells. Here we show that human memory T cells can reside in the bone marrow as resting cells in terms of proliferation, transcription, and mobility. The repertoire of bone marrow memory T cells is enriched for systemic pathogens representing persistent, recent, and childhood challenges. In terms of absolute numbers, memory T cells specific for systemic antigens are maintained predominantly in the bone marrow, in particular those representing historic encounters. In the bone marrow, a population of memory T cells has been described that promotes efficient secondary immune responses and has been considered to be preactivated, owing to its expression of CD69 and CD25. Here we show that human bone marrow professional memory T cells are not activated but are resting in terms of proliferation, transcription, and mobility. They are in the G0 phase of the cell cycle, and their transcriptome is that of resting T cells. The repertoire of CD4+ bone marrow memory T cells compared with CD4+ memory T cells from the blood is significantly enriched for T cells specific for cytomegalovirus-pp65 (immunodominant protein), tetanus toxoid, measles, mumps, and rubella. It is not enriched for vaccinia virus and Candida albicans-MP65 (immunodominant protein), typical pathogens of skin and/or mucosa. CD4+ memory T cells specific for measles are maintained nearly exclusively in the bone marrow. Thus, CD4+ memory T cells from the bone marrow provide long-term memory for systemic pathogens.

Macrophages in bone fracture healing: Their essential role in endochondral ossification

In fracture healing, skeletal and immune system are closely interacting through common cell precursors and molecular mediators. It is thought that the initial inflammatory reaction, which involves migration of macrophages into the fracture area, has a major impact on the long term outcome of bone repair. Interestingly, macrophages reside during all stages of fracture healing. Thus, we hypothesized a critical role for macrophages in the subsequent phases of bone regeneration. This study examined the impact of in vivo induced macrophage reduction, using clodronate liposomes, on the different healing phases of bone repair in a murine model of a standard closed femoral fracture. A reduction in macrophages had no obvious effect on the early fracture healing phase, but resulted in a delayed hard callus formation, thus severely altering endochondral ossification. Clodronate treated animals clearly showed delayed bony consolidation of cartilage and enhanced periosteal bone formation. Therefore, we decided to backtrack macrophage distribution during fracture healing in non-treated mice, focusing on the identification of the M1 and M2 subsets. We observed that M2 macrophages were clearly prevalent during the ossification phase. Therefore enhancement of M2 phenotype in macrophages was investigated as a way to further bone healing. Induction of M2 macrophages through interleukin 4 and 13 significantly enhanced bone formation during the 3week investigation period. These cumulative data illustrate their so far unreported highly important role in endochondral ossification and the necessity of a fine balance in M1/M2 macrophage function, which appears mandatory to fracture healing and successful regeneration.

T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion.

Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing.

T Lymphocytes Influence the Mineralization Process of Bone

Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells.

A4.01 T cells are critical regulators of soft callus mineralization and normal deposition of collagen I during bone repair

Background and objectives The critical interdependency between the skeletal and immune system has been demonstrated for the fracture healing process. However, the current state of the art is contradictory. On one hand, lymphopenic mice display alleged better healing after injury. On the other hand, several studies attribute either a positive or negative role to T and B cells in bone biology, autoimmunity and fracture healing. The objective of this study is to elucidate the specific role of T and B cells in fracture repair toward early callus mineralization, bone quality, migration of osteoblast precursors and Collagen I deposition. Materials and methods Unilateral closed femoral fractures were produced in RAG1-/-, TCR βδ chain -/-, JHT -/- and C57BL/6 wild-type mice. Bone healing was assessed by histology, biomechanical testing and fluorochrome deposition. Deposition and structural properties of CollagenI were studied by second harmonic and confocal microscopy. Callus gene expression was determined by microarray analysis. Osteoblast precursors, T and B cell migration in the callus were visualised by confocal microscopy. Results Fractured bones of RAG -/- mice mineralized faster, but were much less capable to withstand deformation. Similarly, RAG -/- mice deposited bone quicker during the earliest phases of healing, whereas at later stages underperformed relative to controls. RAG -/- mice also failed to deposit a cross gap layer of Collagen I which correlated with diminished immigration of osteoblast precursors in the peripheral callus. The remaining network of Collagen I fibres in these mice was found to be highly disordered. Interestingly, no alteration in osteoblast precursor migration was observed when only either T or B cells were absent. However, Collagen I deposition was strongly impaired in T cell but not in B cell deficient mice. There, Collagen I fibres failed to organise across the gap in the peripheral callus displaying a highly disordered pattern of deposition. Conclusions Overall our data show that lack of an adaptive immune system does not lead to better, but rather deregulated and suboptimal bone healing. These features were recapitulated in mice that specifically lacked T and not B cells.

Lymphocytes control bone fracture healing by programming the mineralisation capacity of migratory osteogenic precursors

Backgroundand objectives Inflammation of the bone microenvironment in arthritic conditions leads to extensive infiltration of immune cells causing destruction of articular cartilage and bone. Similarities characterise bone fracture. Here, sustained inflammation leads to synthesis of bone and cartilage, massive infiltration of hematopoietic cells and controlled tissue resorption terminating into net bone formation. Thus, to better understand how bone inflammation leads to successful repair instead of autoimmunity, the authors have drawn a comprehensive cellular study of post-fracture bone regeneration by confocal microscopy and other histological techniques. Materials and methods Experimental closed femoral fracture was used to induce bone regeneration in C57BL/six mice and congenic RAG −/− KO (lymphopenic), TCR βδ chain −/− KO (T cell deficient) and JHT −/− KO (B cell deficient) mice. Confocal microscopy was used to detect the presence of T, B, macrophages, eosinophils, activated osteoclasts, osteogenic precursors and osteblasts in the fracture callus. Histomorphometry was used to quantify bone regeneration. Cell culture was used to measure mineralisation capacity at steady state of bone marrow derived osteogenic precursors in WT and lymphopenic mice. Results Our data show that during fracture repair the majority of lymphocytes, macrophages and eosinophils appear late upon ensuing endochondral bone formation. Interestingly, lymphopenic mice had the capacity to mineralise cartilage and undergo subsequent resorption earlier than WT controls in vivo. Accordingly, by histomorphometric comparison of fracture repair in different immunodeficient animal models, the authors found that the speed of endochondral bone formation is T cell dependent. Confocal microscopy also showed that accelerated mineralisation in RAG mice occurred well before lymphocytes could be detected within the callus of WT animals. However, the authors clearly visualised in the callus of RAG and normal mice the concomitant immigration of osteogenic precursors from surrounding tissues. Remarkably, bone marrow osteogenic cells from RAG mice, taken in normal conditions, displayed abnormal mineralisation capacity than normal controls upon cell culture. This suggests that lymphocytes control bone regeneration by programming the basal activity and capacity to respond to damage of osteoblast precursors. Conclusions Bone repair involves the migration of bone marrow osteogenic precursors into the bloodstream to reach fracture sites. Immune cells infiltrate the fracture callus during the mid-late stages of bone regeneration and they consist mainly of B lymphocytes. T cells control the velocity of endochondral bone formation by programming the mineralisation capacity of osteogenic cells.

A4.7 T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion

Background and objectives Fracture healing is a regenerative process and is one of the two processes in the human body in which an organ is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process and appear to recruit accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. Materials and methods In order to answer this question, we analysed femoral fracture healing in mice by confocal microscopy, flow cytometry and molecular biology. Results Upon fracture, we could detect generalised increased frequencies of T cells in the bone marrow of injured mice which persisted throughout healing. By closely analysing the fractured bone, we found that surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralisation. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby newly forming bone. During healing, B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation associated with high OPG production capacity. During bone regeneration, both osteoblasts and osteoclasts were found to undergo direct cell-cell contact with lymphocytes. The latter were also found to secrete OPG in the callus and their appearance also correlated with a progressive increase in the ratio of OPG vs. RANKL gene expression during fracture repair. Conclusions Our data strongly suggests a regulatory role of T and B cells not only in the initial phase of healing but specifically also in the later stages of fracture remodelling.

A8.10 Human memory t cells from the bone marrow are resting and maintain long-lasting memory

Objectives Memory T cells are essential components of immunological memory, but little is known about their maintenance. Recirculating memory T cells of blood and secondary lymphoid organs slowly decay in numbers. In mice, we have recently identified professional resting memory CD4+ T cells located in the BM and stably maintained in dedicated survival niches. The cells are resting, despite expression of CD69. Here we analyse and compare paired human peripheral blood (PB) and BM samples for phenotype, specificity and cytokine profiles of memory CD4+ and CD8+ T cells. Material and Methods Mononuclear cells were isolated from paired PB and BM samples from individuals undergoing hip replacement surgery. Phenotypic analysis and cytokine profile of memory T-cells in response to CMV pp65, tetanus toxoid and measles were measured by flow cytometry. Proliferation and cell cycle status were analysed using Ki-67 and propidium iodide staining, respectively. Global transcription of cells was assessed by microarray analysis. Results Significantly higher numbers of memory T cells were found in BM than in PB. BM memory T cells displayed polyfunctionality, secreted a multitude of cytokines, and specificities for all recall antigens tested. They maintained long-lasting memory, even when it was not detectable in blood. CD69-expressing memory T cells were detected in BM but not in PB. Ki-67- and propidium iodide-positive cells were significantly lower in CD69+ memory T cells from BM compared to PB. BM CD69+ memory T cells showed a resting global gene expression profile. Conclusions BM is home to resting, professional memory CD4+ and CD8+T cells, providing systemic, stable longterm memory.