Myocardial B cells are a subset of circulating lymphocytes with delayed transit through the heart

Abstract

Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs, without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (>95%) of myocardial B cells remain intravascular, whereas few (<5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell-deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.

Keywords: Adaptive immunity; B cells; Cardiology; Immunology.

Figure 1. Myocardial B cells have disparate origin and, for the most part, are not resident cells.

(A) Flow cytometric analysis of CD45⁺CD19⁺ myocardial cells in a C57/B6J mouse. Most myocardial B cells are CD11b^–. Among the CD19⁺CD11b⁺ myocardial B cells, the majority are IgM⁺CD5^–, and a small portion are IgM⁺CD5⁺. The flow cytometry plots are representative of 3 animals analyzed in different experiments. Mean percentage of total CD19⁺ cells ± SD is reported next to each gate. (B) Flow cytometric analysis of CD45⁺CD19⁺ myocardial cells in C57/B6J B cell–deficient mice (μMT) after BM transplant with WT BM. BM transplant replenished CD11b^– cells more efficiently than CD11b⁺ cells. In the CD11b⁺ compartment, BM transplant did not produce IgM⁺CD5⁺ cells. The flow cytometry plots are representative of 4 animals analyzed in different experiments. Mean percentage of total CD19⁺ cells ± SD is reported next to each gate. (C) Analysis of myocardial CD45⁺CD19⁺ cells from animals conjoined via parabiosis for 3 weeks. Both CD19⁺CD11b⁺ and CD19⁺CD11b^– cells showed 50% chimerism, a finding consistent with the observation that myocardial B cells moved freely between animals. Percent chimerism for Ly6G⁺ neutrophils, CD64⁺Ly6C^lo macrophages, and CD3⁺ T cells is shown for comparison. (D) Analysis of CD45⁺CD19⁺ myocardial B cells in recipient and donor hearts before transplant and on day 4 after heterotopic heart transplant. Before transplant, the heart of the CD45.1 recipient animal contains only CD45.1⁺ B cells, and the donor heart from a CD45.2 animal contains only CD45.2⁺ B cells. Four days after transplant, the recipient heart is mostly unchanged, though it contains a small population of CD45.2⁺ B cells derived from the transplanted heart (left side). The CD45.2 transplanted heart instead has lost almost all of its CD45.2⁺ B cells and now contains mostly CD45.1⁺ recipient-derived B cells. Representative flow cytometry plots from 3 independently transplanted animals. Percentage of total CD19⁺ cells is reported within each gate.

Figure 2. Myocardial B cells recirculate between the heart, the blood, the spleen, and likely other organs.

(A) Myocardial, splenic, and circulating (blood) B cells were analyzed 4 days after heterotopic heart transplant, and the percentage of CD45⁺CD19⁺ cells that were also CD45.2⁺ (donor-derived B cell) was calculated. Graph bars represents the percentage of donor-derived B cells found in the recipient heart and in the donor heart (left), recipient spleen (middle), and recipient peripheral blood (right). The percentage of CD45.2⁺ cells in the absence of the specific antibody (staining control) is reported for reference. Data are expressed as mean ± SD. (B) A Rag1^–/– B cell–deficient mouse was injected with splenocytes from a WT mouse. Three weeks after adoptive transfer, the Rag1^–/– mouse has a detectable population of myocardial B cells. Adoptive transfer of splenocytes reconstituted all the 3 major subgroups of myocardial B cells, CD19⁺CD11b^–, CD19⁺CD11b⁺IgM⁺CD5^–, and CD19⁺CD11b⁺IgM⁺CD5⁺. The flow cytometry plots are representative of data collected from 5 different animals subjected to adoptive transfer for total myocardial B cells and from 3 different animals for the subgroup analysis of myocardial B cells. Average percentage of total CD45⁺ cells is reported in the CD45/CD19 plot, while average percentage of the parent gate is reported in the other plots. Mean ± SD is reported in each plot next to each average value.

Figure 3. Myocardial B cells are mostly intravascular and in intimate contact with the endothelium.

(A) Confocal imaging of a section of myocardial tissue from a CD19-tdTomato reporter animal. The 3-dimensional reconstruction of multiple images acquired along the z axes shows that B cells are distributed throughout the myocardium. LSM 880 Indimo, AxioObserver Zeiss Microscope using a Plan-Apochromat 10×/0.8 M27 objective. Z-stack of 69 slices (64.829 uM). (B–E) Confocal images of a frozen section of murine myocardium. CD19-tdTomato, red; CD31, green; DAPI, blue. Myocardial B cells are mostly intravascular and in intimate contact with the endothelium. Taken with an LSM 880 Indimo, AxioObserver Zeiss Microscope using a Plan-Apochromat 63×/1.3 oil DIC UV-IR M27 objective and 2× digital zoom (B). Some B cells are in pairs. Taken with an LSM 880 Indimo, AxioObserver Zeiss Microscope using a Plan-Apochromat 40×/1.3 oil DIC UV-IR M27 objective and a 2.2 digital zoom (C). Few B cells are found in the intraparenchymal/extravascular space, as singlets or doublets. taken with an Olympus Fluoview FV1000 using a PLAPON 60× NA1.4 objective and 2× digital zoom (D); rare B cells are found in transit through the endothelium, taken with the same microscope and objective as D but it is a Z-stack of 23 slices, 0.46 uM/slice (E). (F) Flow cytometric analysis of myocardial CD19⁺CD45⁺ cells 3 minutes after i.v. injection of a CD45.2 antibody. Most myocardial B cells are stained by the i.v. injected antibody, confirming their intravascular location. Only about 3% of cells are not stained by the antibody and are, therefore, extravascular. The flow cytometry plot is representative of 3 independent experiments. Percentage of total CD19⁺ cells is reported next to each gate. The bar graph reports mean percentage of extravascular CD19⁺ cells ± SD.

Figure 4. Myocardial B cells are a subset of circulating B lymphocytes with distinct transcriptome that adheres to the myocardial endothelium.

(A) tSNE plot of 10× single cell sequencing data from CD45⁺CD19⁺ cells sorted from the heart and the blood of the same pool of WT 10-week-old C57/B6J mice. B cells sorted from the heart have a different gene expression profile than B cells sorted from the blood. The experiment was repeated twice. (B) tSNE plot integrated with unsupervised clustering. Myocardial (heart) and circulating (blood) B cells overall contain cells with similar gene expression profile. However, specific clusters of cells, such as cluster 0, are enriched in the heart, while other clusters of cells, such as cluster 1, are enriched in the blood. (C) The heart from a μMT mouse has almost no myocardial B cells (panel on the left, representative plot). After ex vivo perfusion with WT blood and rinsing, the μMT heart contains a sizeable population of CD19⁺ B cells (panel in the middle, n = 3 reporting average percentage of cells in each gate ± SD), which is very similar to that observed in the heart of a WT animal (panel on the right). Representative FACS plots. (D) Still image from intravital microscopy of B cells flowing through a transplanted heart. B cells are depicted in green, while the vasculature is depicted in red. A number of B lymphocytes is still and well visible within the myocardial microvasculature (see Supplemental Video 4). Representative image from videos collected from multiple hearts.

Figure 5. B cell deficiency alters the myocardial leukocyte pool, reduces myocardial mass, and alters left ventricular contractility.

(A) Flow cytometry analysis of myocardial leucocytes in WT and μMT mice. μMT mice have less Ly6C⁺ cells and more CD4⁺ and CD8⁺ T cells. (B). Flow cytometric analysis of WT animals depleted of B cell through administration of anti-CD20 antibody or isotype control. Antibody-mediated B cell depletion is associated with an increase in Ly6G⁺ cells (P = 0.056), CD4⁺ and CD8⁺ cells. (C) Analysis of hearts from 27-week-old C57/B6J mice and syngeneic age/sex-matched μMT mice showed that μMT mice have myocardial fibers with smaller cross-sectional area as assessed by WGA staining. Representative images of WGA staining for each group are shown and cumulative data are reported. (D) μMT mice have lower myocardial mass as assessed by heart weight/tibia length (mg/mm). (E) Echocardiographic analysis of left ventricular function in WT mice and matched μMT mice. μMT mice have higher left ventricular ejection fraction (LVEF) and faster ventricular relaxation (dV/dT-d, volume change per unit of time during diastolic relaxation; ESV, end-systolic volume). (F) Flow cytometry analysis of a sample of human myocardium collected at the time of implantation of a left ventricular assist device and dissociated enzymatically. A clear population of CD19⁺ cells is shown. Representative plot from 3 heart samples analyzed. Percentage of myocardial CD45⁺ cells is reported next to the gate. (G) Histological analysis of human myocardium collected at the time of LVAD placement shows that, similarly to the murine heart, the human heart harbors B cells in the intravascular space, in intimate contact with the endothelium. Magnification, 40×. Pairwise comparisons were performed with 2-tailed t test. Data are expressed as average ± SD. The WT and μMT groups were compared using unpaired 2-tailed t test adjusted for multiple comparisons with the FDR method in A and B. In C, the 2 groups were compared using nested t test *P < 0.05. **P < 0.01.