Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

German Rheumatism Research Center, a Leibniz Institute

Human bocavirus (HBoV) was recently described as a new member of the Parvoviridae family, and its possible association with respiratory illness in infants has been discussed. To date, HBoV genomes have been detected worldwide in respiratory tract samples obtained from children with pulmonary diseases, whereas only limited data on virus-specific immunity are available, mainly because of the lack of recombinant viral antigens.

CD4+ T helper cell responses against human bocavirus viral protein 2 viruslike particles in healthy adults.

Human bocavirus (HBoV) was recently detected in samples from children and infants with infections of the respiratory tract. Here we analyze the prevalence of IgG and IgM antibodies against HBoV virus-like VP2 particles in healthy adult blood donors and children using a newly established standardized enzyme-linked immunosorbent assay. Virus-specific IgG antibodies were frequently detected in infants with active viremia and respiratory illness (10/24, 42%) and in young children without detectable HBoV genomes in their blood (27/52, 52%). In sera obtained from healthy adults, ubiquitous VP2-specific antibodies were found in 280/299 (94%) cases. HBoV-specific IgM antibodies were detected in 10/24 (42%) of sera samples obtained from HBoV DNA-positive children, and in 6/24 (25%) the sera displayed equivocal responses. In contrast, VP2-specific IgM was not detectable in samples obtained from 52 children without detectable amounts of HBoV genomes in their blood. Only 2/299 sera samples from healthy adult blood donors were found to be IgM-positive (1%), and equivocal IgM responses were observed in 9/299 (3%) individuals. In conclusion, a high IgG seroprevalence of HBoV in the adult population was observed, whereas the presence of virus-specific IgM was associated with viremia. These data show that ELISA test systems for the detection of HBoV-specific antibodies are a valuable tool for serological diagnosis of this new emerging pathogen.

Humoral immune response against human bocavirus VP2 virus-like particles.

CD4(+) T lymphocytes are key to immunological memory. Here we show that in the memory phase of specific immune responses, most of the memory CD4(+) T lymphocytes had relocated into the bone marrow (BM) within 3-8 weeks after their generation-a process involving integrin alpha2. Antigen-specific memory CD4(+) T lymphocytes highly expressed Ly-6C, unlike most splenic CD44(hi)CD62L(-) CD4(+) T lymphocytes. In adult mice, more than 80% of Ly-6C(hi)CD44(hi)CD62L(-) memory CD4(+) T lymphocytes were in the BM. In the BM, they associated to IL-7-expressing VCAM-1(+) stroma cells. Gene expression and proliferation were downregulated, indicating a resting state. Upon challenge with antigen, they rapidly expressed cytokines and CD154 and efficiently induced the production of high-affinity antibodies by B lymphocytes. Thus, in the memory phase of immunity, memory helper T cells are maintained in BM as resting but highly reactive cells in survival niches defined by IL-7-expressing stroma cells.

Professional memory CD4+ T lymphocytes preferentially reside and rest in the bone marrow.

Immunological memory is still one of the enigmas of modern immunology. We poorly understand the generation of memory cells from their precursors, the lifestyle of memory cells or their maintenance, reactivation and termination. Here, we discuss the recent evidence suggesting that memory plasma cells, as defined in this review, and memory Th cells are maintained in the bone marrow, resting (in terms of proliferation) in survival niches organized by dedicated stroma cells, which control the homeostasis of immunological memory.

Organization and maintenance of immunological memory by stroma niches.

Memory T-helper (Th) lymphocytes are crucial for the maintenance of acquired immunity to eliminate infectious pathogens. We have previously demonstrated that most memory Th lymphocytes reside and rest on stromal niches of the bone marrow (BM). Little is known, however, regarding the molecular basis for the generation and maintenance of BM memory Th lymphocytes. Here we show that CD69-deficient effector CD4 T lymphocytes fail to relocate into and persist in the BM and therefore to differentiate into memory cells. Consequently, CD69-deficient CD4 T cells fail to facilitate the production of high-affinity antibodies and the generation of BM long-lived plasma cells in the late phase of immune responses. Thus, CD69 is critical for the generation and maintenance of professional memory Th lymphocytes, which can efficiently help humoral immunity in the late phase. The deficit of immunological memory in CD69-deficient mice also highlights the essential role of BM for the establishment of Th memory.

Type II membrane protein CD69 regulates the formation of resting T-helper memory.

Plasma cells play a crucial role for the humoral immune response as they represent the body's factories for antibody production. The differentiation from a B cell into a plasma cell is controlled by a complex transcriptional network and happens within secondary lymphoid organs. Based on their lifetime, two types of antibody secreting cells can be distinguished: Short-lived plasma cells are located in extrafollicular sites of secondary lymphoid organs such as lymph node medullary cords and the splenic red pulp. A fraction of plasmablasts migrate from secondary lymphoid organs to the bone marrow where they can become long-lived plasma cells. Bone marrow plasma cells reside in special microanatomical environments termed survival niches, which provide factors promoting their longevity. Reticular stromal cells producing the chemokine CXCL12, which is known to attract plasmablasts to the bone marrow but also to promote plasma cell survival, play a crucial role in the maintenance of these niches. In addition, hematopoietic cells are contributing to the niches by providing other soluble survival factors. Here, we review the current knowledge on the factors involved in plasma cell differentiation, their localization and migration. We also give an overview on what is known regarding the maintenance of long lived plasma cells in survival niches of the bone marrow. © 2013 International Society for Advancement of Cytometry.

Tracking plasma cell differentiation and survival.

In the bone marrow (BM), memory plasma cells (PCs) survive for long time periods in dedicated microenvironmental survival niches, resting in terms of proliferation. Several cell types, such as eosinophils and reticular stromal cells, have been reported to contribute to the survival niche of memory PCs. However, until now it has not been demonstrated whether the niche is formed by a fixed cellular microenvironment. By intravital microscopy, we provide for the first time evidence that the direct contacts formed between PCs and reticular stromal cells are stable in vivo, and thus the PCs are sessile in their niches. The majority (∼ 80%) of PCs directly contact reticular stromal cells in a non-random fashion. The mesenchymal reticular stromal cells in contact with memory PCs are not proliferating. On the other hand, we show here that eosinophils in the vicinity of long-lived PCs are vigorously proliferating cells and represent a dynamic component of the survival niche. In contrast, if eosinophils are depleted by irradiation, newly generated eosinophils localize in the vicinity of radiation-resistant PCs and the stromal cells. These results suggest that memory PC niches may provide attraction for eosinophils to maintain stability with fluctuating yet essential accessory cells.

Static and dynamic components synergize to form a stable survival niche for bone marrow plasma cells.

It is believed that memory CD8(+) T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL-7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8(+) T cells individually colocalize with IL-7(+) reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4-1BB (CD137), IL-2, or IFN-γ, despite the expression of CD69 on about 30% of the cells. Ninety-five percent of the memory CD8(+) T cells in BM are in G0 phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G2 of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8(+) memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8(+) memory T cells. Taken together, the present results suggest that CD8(+) memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL-7 receptor signaling.

Memory CD8(+) T cells colocalize with IL-7(+) stromal cells in bone marrow and rest in terms of proliferation and transcription.

Long-term antibody production is a key property of humoral immunity and is accomplished by long-lived plasma cells. They mainly reside in the bone marrow, whose importance as an organ hosting immunological memory is becoming increasingly evident. Signals provided by stromal cells and eosinophils may play an important role for plasma cell maintenance, constituting a survival microenvironment. In this joint study of experiment and theory, we investigated the spatial colocalization of plasma cells, eosinophils and B cells by applying an image-based systems biology approach. To this end, we generated confocal fluorescence microscopy images of histological sections from murine bone marrow that were subsequently analyzed in an automated fashion. This quantitative analysis was combined with computer simulations of the experimental system for hypothesis testing. In particular, we tested the observed spatial colocalization of cells in the bone marrow against the hypothesis that cells are found within available areas at positions that were drawn from a uniform random number distribution. We find that B cells and plasma cells highly colocalize with stromal cells, to an extent larger than in the simulated random situation. While B cells are preferentially in contact with each other, i.e., form clusters among themselves, plasma cells seem to be solitary or organized in aggregates, i.e., loosely defined groups of cells that are not necessarily in direct contact. Our data suggest that the plasma cell bone marrow survival niche facilitates colocalization of plasma cells with stromal cells and eosinophils, respectively, promoting plasma cell longevity. © 2015 International Society for Advancement of Cytometry.

Sandra Zehentmeier

Immunodynamics

German Rheumatism Research Center, a Leibniz Institute

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

Sandra Zehentmeier

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Immunodynamics

German Rheumatism Research Center, a Leibniz Institute

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

Immunodynamics