[B lymphocytes and plasma cells as drivers of rheumatic diseases]. / B-Lymphozyten und Plasmazellen als Treiber rheumatischer Erkrankungen.

Various research groups at the German Rheumatism Research Center in Berlin, in close cooperation with the Department of Rheumatology and Clinical Immunology of the Medical Clinic at the Charité, have made important contributions to the significance of B cells and plasma cells in rheumatic diseases, which are relevant not only for rheumatology but for all clinical specialties in which antibody-mediated diseases play a role. In particular, the research addresses impaired B cell homeostasis, the importance of the IgM Fc receptor in the regulation of autoimmunity, the role of long-lived memory plasma cells in maintaining autoimmunity and ensuring its survival in specific niches organized by stromal cells in bone marrow and inflamed tissues. The research results have contributed to a better understanding of the immunological and molecular mechanisms in rheumatic diseases and their treatment. The identification of the long-lived memory plasma cell has led to promising treatment approaches with curative potential in autoimmune diseases.

Homeostasis and Durability of T-Cell Memory-The Resting and the Restless T-Cell Memory.

The molecular basis of the persistence of experienced T lymphocytes, also known as "memory T lymphocytes," is still enigmatic. We are beginning to understand their considerable heterogeneity and topographic compartmentalization into memory T cells circulating through the body and those residing in a particular tissue. In some tissues, like murine spleen, subpopulations of memory T cells proliferating in the absence of antigen (homeostatic proliferation) have been described. Other populations are maintained resting in terms of transcription, mobility, and proliferation in dedicated survival niches organized by stromal cells. The survival of these memory T cells is conditional on being in such a niche, where they can persist for a lifetime. Circulating memory T lymphocytes of distinct immune responses slowly decline in numbers over time. The rules governing their entry into and exit from blood, as well as their lifestyle outside of the blood and their relation to resident memory T cells are poorly understood. Homeostasis of circulating, proliferating, and resting memory T cells is obviously controlled by different rheostats: tissue-exit and tissue-entry signals for circulating and proliferation-inducing signals for proliferating memory T cells. For tissue-resident, resting memory T cells, it is the availability of their survival niche. Apparently, this mechanism (i.e., the link between memory T cell and stromal cell) is so robust that it provides efficient T-cell memory over a lifetime in tissues such as the bone marrow.

Multiple developmental pathways lead to the generation of CD4 T-cell memory.

Long-term immunological memory mediated by CD4 T cells provides a rapid protection against previously encountered pathogens or antigens. However, it is still controversial how memory CD4 T cells are generated and maintained. Unclear definitions of T-cell memory may be partially responsible for this controversy. It is becoming clear that diverse pathways are responsible for the differentiation and long-term persistence of memory T cells. We herein discuss the diversity of memory cell generation, describing a novel population of resting memory CD4 T cells and their precursors.

Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells.

The persistence of long-lived memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, which are provided in niches organized by stromal cells. Using an ex vivo system in which we supply the known survival signals, direct cell contact to stromal cells, and the soluble cytokine a proliferation-inducing ligand (APRIL), we have elucidated the critical signaling pathways required for the survival of long-lived plasma cells. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to critical inactivation of Forkhead-Box-Protein O1/3 (FoxO1/3) and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Accordingly, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, by the nuclear factor κB (NF-κB) pathway, blocks activation of the endoplasmic-reticulum-stress-associated initiator caspase 12. Thus, stromal-cell-contact-induced PI3K and APRIL-induced NF-κB signaling provide the necessary and complementary signals to maintain bone marrow memory plasma cells.

Ezh2 controls development of natural killer T cells, which cause spontaneous asthma-like pathology.

BACKGROUND: Natural killer T (NKT) cells express a T-cell receptor that recognizes endogenous and environmental glycolipid antigens. Several subsets of NKT cells have been identified, including IFN-γ-producing NKT1 cells, IL-4-producing NKT2 cells, and IL-17-producing NKT17 cells. However, little is known about the factors that regulate their differentiation and respective functions within the immune system. OBJECTIVE: We sought to determine whether the polycomb repressive complex 2 protein enhancer of zeste homolog 2 (Ezh2) restrains pathogenicity of NKT cells in the context of asthma-like lung disease. METHODS: Numbers of invariant natural killer T (iNKT) 1, iNKT2, and iNKT17 cells and tissue distribution, cytokine production, lymphoid tissue localization, and transcriptional profiles of iNKT cells from wild-type and Ezh2 knockout (KO) iNKT mice were determined. The contribution of NKT cells to development of spontaneous and house dust mite-induced airways pathology, including airways hyperreactivity (AHR) to methacholine, was also assessed in wild-type, Ezh2 KO, and Ezh2 KO mice lacking NKT cells. RESULTS: Ezh2 restrains development of pathogenic NKT cells, which induce spontaneous asthma-like disease in mice. Deletion of Ezh2 increased production of IL-4 and IL-13 and induced spontaneous AHR, lung inflammation, mucus production, and IgE. Increased IL-4 and IL-13 levels, AHR, lung inflammation, and IgE levels were all dependent on iNKT cells. In house dust mite-exposed animals Ezh2 KO resulted in enhanced AHR that was also dependent on iNKT cells. CONCLUSION: Ezh2 is a central regulator of iNKT pathogenicity and suppresses the ability of iNKT cells to induce asthma-like pathology.

Salmonella SiiE prevents an efficient humoral immune memory by interfering with IgG+ plasma cell persistence in the bone marrow.

Serum IgG, which is mainly generated from IgG-secreting plasma cells in the bone marrow (BM), protects our body against various pathogens. We show here that the protein SiiE of Salmonella is both required and sufficient to prevent an efficient humoral immune memory against the pathogen by selectively reducing the number of IgG-secreting plasma cells in the BM. Attenuated SiiE-deficient Salmonella induces high and lasting titers of specific and protective Salmonella-specific IgG and qualifies as an efficient vaccine against Salmonella A SiiE-derived peptide with homology to laminin ß1 is sufficient to ablate IgG-secreting plasma cells from the BM, identifying laminin ß1 as a component of niches for IgG-secreting plasma cells in the BM, and furthermore, qualifies it as a unique therapeutic option to selectively ablate IgG-secreting plasma cells in autoimmune diseases and multiple myeloma.

Humoral Immunity vs. Salmonella.

In primary infection with Salmonella, it has been reported-without consideration of Salmonella's functions-that humoral immunity plays no role in the clearance of bacteria. In fact, Salmonella targets and suppresses several aspects of humoral immunity, including B cell lymphopoiesis, B cell activation, and IgG production. In particular, the suppression of IgG-secreting plasma cell maintenance allows the persistence of Salmonella in tissues. Therefore, the critical role(s) of humoral immunity in the response to Salmonella infection, especially at the late phase, should be re-investigated. The suppression of IgG plasma cell memory strongly hinders vaccine development against non-typhoidal Salmonella (NTS) because Salmonella can also reduce humoral immune memory against other bacteria and viruses, obtained from previous vaccination or infection. We propose a new vaccine against Salmonella that would not impair humoral immunity, and which could also be used as a treatment for antibody-dependent autoimmune diseases to deplete pathogenic long-lived plasma cells, by utilizing the Salmonella's own suppression mechanism of humoral immunity.

Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memory CD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM) and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that, following a critical number of cell divisions, memory precursors downregulate CCR7 and upregulate IL-2Rß, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM.

Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections.

The bone marrow (BM) consists of multiple, structured micro-environmental entities-the so called niches, which contain hematopoietic cells as well as stromal cells. These niches fulfill a variety of functions, such as control of the hematopoietic stem cell pool, differentiation of hematopoietic cells, and maintenance of immunological memory. However, due to the molecular and cellular complexity and a lack of suitable histological multiplexing methods, the composition of the various BM niches is still elusive. In this study, we apply multiepitope-ligand-cartography (MELC) on bone sections from mice. We combine multiplexed immunofluorescence histology data with various object-based segmentation approaches in order to define irregularly shaped, net-like structures of stromal cells. We confirm MELC as a robust histological method and validate our automated segmentation algorithms using flow cytometry and manual evaluation. By means of MELC multiplexing, we reveal heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network. Moreover, we demonstrate by quantification a preferential contact of B cell subsets as well as of plasma cells to processes of CXCL12-expressing stromal cells, compared with stromal somata. In summary, our approach is suitable for spatial analysis of complex tissue structures.

Immunological memories of the bone marrow.

Memory for antigens once encountered is a hallmark of the immune system of vertebrates, providing us with an immunity adapted to pathogens of our environment. Despite its fundamental relevance, the cells and genes representing immunological memory are still poorly understood. Here we discuss the concept of a circulating, proliferating, and ubiquitous population of effector lymphocytes vs concepts of resting and dormant populations of dedicated memory lymphocytes, distinct from effector lymphocytes and residing in defined tissues, particularly in barrier tissues and in the bone marrow. The lifestyle of memory plasma cells of the bone marrow may serve as a paradigm, showing that persistence of memory lymphocytes is not defined by intrinsic "half-lives", but rather conditional on distinct survival signals provided by dedicated niches. These niches are organized by individual mesenchymal stromal cells. They define the capacity of immunological memory and regulate its homeostasis.

Nonfollicular reactivation of bone marrow resident memory CD4 T cells in immune clusters of the bone marrow.

The bone marrow maintains memory CD4 T cells, which provide memory to systemic antigens. Here we demonstrate that memory CD4 T cells are reactivated by antigen in the bone marrow. In a secondary immune response, antigen-specific T cells of the bone marrow mobilize and aggregate in immune clusters together with MHC class II-expressing cells, mostly B lymphocytes. They proliferate vigorously and express effector cytokines, but they do not develop into follicular T-helper cells. Neither do the B lymphocytes develop into germinal center B cells in the bone marrow. Within 10 days, the immune clusters disappear again. Within 30 days, the expanded antigen-specific memory CD4 T cells return to memory niches and are maintained again individually as resting cells. Thus, in secondary immune responses in the bone marrow T-cell memory is amplified, while in germinal center reactions of secondary lymphoid organs humoral memory is adapted by affinity maturation.

Maintenance of CD8+ memory T lymphocytes in the spleen but not in the bone marrow is dependent on proliferation.

It is current belief that numbers of CD8+ memory T lymphocytes in the memory phase of an immune response are maintained by homeostatic proliferation. Here, we compare the proliferation of CD8+ memory T lymphocytes, generated by natural infections and by intentional immunization, in spleen and bone marrow (BM). Fifty percent of CD8+ memory T lymphocytes in the spleen are eliminated by cyclophosphamide within 14 days, indicating that numbers of at least 50% of splenic CD8+ memory T lymphocytes are maintained by proliferation. The numbers of CD8+ memory T lymphocytes in the BM, however, were not affected by cyclophosphamide. This stability was independent of circulating CD8+ memory T cells, blocked by FTY720, showing that BM is a privileged site for the maintenance of memory T lymphocytes, as resident cells, resting in terms of proliferation.

Crucial role for CD69 in allergic inflammatory responses: CD69-Myl9 system in the pathogenesis of airway inflammation.

CD69 has been known as an early activation marker of lymphocytes; whereas, recent studies demonstrate that CD69 also has critical functions in immune responses. Early studies using human samples revealed the involvement of CD69 in various inflammatory diseases including asthma. Moreover, murine disease models using Cd69-/- mice and/or anti-CD69 antibody (Ab) treatment have revealed crucial roles for CD69 in inflammatory responses. However, it had not been clear how the CD69 molecule contributes to the pathogenesis of inflammatory diseases. We recently elucidated a novel mechanism, in which the interaction between CD69 and its ligands, myosin light chain 9, 12a and 12b (Myl9/12) play a critical role in the recruitment of activated T cells into the inflammatory lung. In this review, we first summarize CD69 function based on its structure and then introduce the evidence for the involvement of CD69 in human diseases and murine disease models. Then, we will describe how we discovered CD69 ligands, Myl9 and Myl12, and how the CD69-Myl9 system regulates airway inflammation. Finally, we will discuss possible therapeutic usages of the blocking Ab to the CD69-Myl9 system.

B Cells Negatively Regulate the Establishment of CD49b(+)T-bet(+) Resting Memory T Helper Cells in the Bone Marrow.

During an immune reaction, some antigen-experienced CD4 T cells relocate from secondary lymphoid organs (SLOs) to the bone marrow (BM) in a CD49b-dependent manner and reside and rest there as professional memory CD4 T cells. However, it remains unclear how the precursors of BM memory CD4 T cells are generated in the SLOs. While several studies have so far shown that B cell depletion reduces the persistence of memory CD4 T cells in the spleen, we here show that B cell depletion enhances the establishment of memory CD4 T cells in the BM and that B cell transfer conversely suppresses it. Interestingly, the number of antigen-experienced CD4 T cells in the BM synchronizes the number of CD49b(+)T-bet(+) antigen-experienced CD4 T cells in the spleen. CD49b(+)T-bet(+) antigen-experienced CD4 T cells preferentially localize in the red pulp area of the spleen and the BM in a T-bet-independent manner. We suggest that B cells negatively control the generation of CD49b(+)T-bet(+) precursors of resting memory CD4 T cells in the spleen and may play a role in bifurcation of activated effector and resting memory CD4 T cell lineages.

Vaccination to gain humoral immune memory.

The concept of immune memory forms the biological basis for vaccination programs. Despite advancements in the field of immune memory and vaccination, most current vaccines are evaluated by magnitude of antigen-specific antibody titers in serum or mucosa after vaccination. It has been shown, however, that antibody-mediated humoral immune memory is established regardless of the magnitude and duration of immune reactions, suggesting that assessment of vaccine efficacy should be performed for several years after vaccination. This long-term investigation is disadvantageous for prevalent and pandemic infections. Long-lived memory plasma cells and memory helper T cells which contribute to humoral immune memory are generated in the bone marrow after migration of memory cell precursors through bloodstream. Thus, it may be a novel evaluation strategy to assess the precursors of memory cells in the blood in the early phase of the immune reaction(s). We here review recent advances on the generation and maintenance of immune memory cells involved in humoral immunity and introduce a current concept of direct and short-term assessment of humoral immune memory formation upon vaccination as a correlate of protection.

Myosin light chains 9 and 12 are functional ligands for CD69 that regulate airway inflammation.

Recent decades have witnessed a rapid worldwide increase in chronic inflammatory disorders such as asthma. CD4+ T helper 2 cells play critical roles in the pathogenesis of allergic airway inflammation, and CD69 expression on activated CD4 T cells is required to induce allergic inflammation in tissues. However, how CD69 mechanistically controls allergic inflammation remains poorly defined. In lymphoid tissues, CD69 regulates cellular retention through inhibition of S1P1 expression and requires no specific ligands to function. In contrast, we show herein that myosin light chain (Myl) 9 and Myl12 are new functional ligands for CD69. Blockade of CD69-Myl9/12 interaction ameliorates allergic airway inflammation in ovalbumin-induced and house dust mite-induced mouse models of asthma. Within the inflamed mouse airways, we found that the expression of Myl9/12 was increased and that platelet-derived Myl9/12 localized to the luminal surface of blood vessels and formed intravascular net-like structures. Analysis of nasal polyps of eosinophilic chronic rhinosinusitis patients revealed that Myl9/12 expression was increased in inflammatory lesions and was distributed within net-like structures in the intravascular space. In addition, we detected Myl9/12 in perivascular spaces where many CD69+ cells were positioned within Myl9/12 structures. Thus, CD69-Myl9/12 interaction is a key event in the recruitment of activated CD69+ T cells to inflamed tissues and could be a therapeutic target for intractable airway inflammatory diseases.

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

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.

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

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.

Reprint of: Environments of B cell development.

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.