Fig 2: CD79a activation on myeloid cells alters cytokine expression and activates signaling pathways.The secretion of cytokines and chemokines by Ly6C+ immature BM myeloid cells isolated from SCID mice and cultured for 48 h under the indicated conditions was assessed using a membrane-based cytokine array. (A) Arrays of conditioned media from culture of BM cells treated with anti- CD79a(v-20), isotype control, or 4T1 tumor cell CM, together with a control array for 4T1 CM alone. (B) Relative levels of proteins with the most significant change (indicated by boxes in A) were quantitated by densitometry. (C, D) Validation of changes in IL-6 and CCL22 by Quantikine ELISA. Both the arrays and the ELISA are representative of 2 independent experiments. (E) Induction of downstream signaling events triggered by cross-linking CD79a with anti CD79a(v-20) on BM cells from SCID mice was assessed by western blot.

Fig 3: CD79a activation on myeloid cells affects migration, differentiation and T-cell suppression.(A) The effect of stimulation with anti CD79a(v-20) on the migration of FACS-sorted Ly6C+ immature myeloid cells from naive bone marrow was tested in a Transwell® system for 48 h. Results are mean +/- SEM; n = 3 determinations. (B) Bone marrow cells from naïve SCID mice were stimulated with anti- CD79a(v-20) (5 µg/ml) for 96 h with or without added GM-CSF in low (2%) normal mouse serum. The effect of anti CD79a(v-20) on myeloid cell differentiation status was assessed using immature myeloid (CD11b+Gr1+), granulocytic (Gr1+) and macrophage (F4/80+) markers. Data shown are representative of two independent experiments. (C) The effect of immature myeloid cells stimulated with anti CD79a on proliferation of CD4 T cells. Immunopurified naïve CD4+ T cells (“T”) labeled with CSFE were stimulated with anti CD3/CD28 with the addition of the indicated ratios of FACS-sorted immature BM myeloid cells (“M”) with or without the addition of anti CD79a(v-20) antibody. Cell divisions were measured by flow cytometry analysis for CFSE dilution. Dose response for effect on T-cell division of myeloid cells added at the indicated ratio, with the addition of anti CD79a(v-20) or isotype-matched control antibody is shown. (D) The effect of LLC tumor cell conditioned medium (CM) (ratio 1/5 v/v), myeloid cells and the combination of both on T cells proliferation. Data shown are representative of two independent experiments.

Fig 4: Expression of CD79a mRNA and protein in bone marrow cells from B-cell deficient mice.(A) RNA was extracted from BM cells of naïve SCID mice and from spleen of naïve Balb/c mice and CD79a and CD79b mRNA expression were measured by qRT-PCR. RNA levels were normalized to PPIAand presented relative to the level of CD79b mRNA in SCID BM. Results are mean +/- SEM; n = 3. (B) BM-derived leukocytes from naïve SCID mice were analyzed by flow cytometry for intracellular epitopes of CD79a using the anti-mouse CD79a clone F11-172. (C) To assess the extracellular expression of CD79a, BM cells from mice bearing LLC tumors were co-stained with anti CD79-11 together with the polyclonal anti-CD79a(v-20), which showed a weaker pattern of staining than CD79-11. Boxes indicate the myeloid cells. (D) CD79a/b protein in splenocytes from naïve and 4T1 tumor-bearing mice was assessed by western blot under reducing conditions. Data shown are representative of three independent experiments.

Fig 5: Tumor cell-secreted factors upregulate CD79a on myeloid cells and induce their expansion and migration.(A) The ability of tumor cells of varying metastatic potential to induce expression of CD79a on immature myeloid cells from bone marrow of naïve mice was assessed using a Transwell® system (0.4 µm pore size) that prevented contact between tumor and bone marrow-derived cells. BM cells were co-incubated with tumor cells of medium alone for 48 h and then BM cells were analyzed by flow cytometry. (B) Representative FACS plots and histograms showing the effect of 4T1 secreted factors on the expansion of BM myeloid cells expressing CD79a, tested using a Transwell® system as in A. (C) Migration of BM cells in response to 4T1 secreted factors was tested in a Transwell® system as above, but using a 3.0 µm pore-size membrane to allow migration of BM cells towards the 4T1 cells in the well below. Cells that migrated through the membrane into the well below were collected, counted and analyzed by FACS for CD79a expression. Results are mean +/- SEM; n = 4 determinations.