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Comparative Study
. 2011 Aug 1;187(3):1243-53.
doi: 10.4049/jimmunol.1100016. Epub 2011 Jun 29.

CD28 expressed on malignant plasma cells induces a prosurvival and immunosuppressive microenvironment

Jayakumar R Nair  1 , Louise M Carlson, Chandana Koorella, Cheryl H Rozanski, Gerald E Byrne, P Leif Bergsagel, John P Shaughnessy Jr, Lawrence H Boise, Asher Chanan-Khan, Kelvin P Lee
Affiliations
Comparative Study

CD28 expressed on malignant plasma cells induces a prosurvival and immunosuppressive microenvironment

Jayakumar R Nair et al. J Immunol. .

Abstract

Interactions between the malignant plasma cells of multiple myeloma and stromal cells within the bone marrow microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal bone marrow-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce prosurvival signals to the myeloma cells and also induce niche production of supportive soluble factors. However, despite their central importance, the specific molecular and cellular components involved remain poorly characterized. We now report that the prototypic T cell costimulatory receptor CD28 is overexpressed on myeloma cells during disease progression and in the poor-prognosis subgroups and plays a previously unrecognized role as a two-way molecular bridge to support myeloid stromal cells in the microenvironment. Engagement by CD28 to its ligand CD80/CD86 on stromal dendritic cell directly transduces a prosurvival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal dendritic cell to produce the prosurvival cytokine IL-6 (involving novel cross-talk with the Notch pathway) and the immunosuppressive enzyme IDO. These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, point to similar interaction for normal plasma cells, and suggest novel therapeutic strategies to target malignant and pathogenic (e.g., in allergy and autoimmunity) plasma cells.

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Conflict of interest statement

Disclosures: JPS is the founder and owns stock in Signal Genetics, LLC. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1. CD28 expression in multiple myeloma
(A) Flow cytometric analysis of CD138 and CD28 expression within the mononuclear population (MNC, gated on FSC/SSC channels) of total bone marrow cells from normal bone marrow donors (n=7) or newly diagnosed MM patients (n=7). A representative flow analysis for each is shown. (B) Gene expression analysis for CD28 expression in plasma cells from normal, MGUS, SM and MM patients and (C) between the 8 different disease subtype within the MM group: activation of cyclin D family members (CD-1, CD-2), MMSET (MS), c-MAF and MAF-B (MF), hyperdiploidy (HY), proliferation (PR), low bone disease (LB), and myeloid contamination (MY). Statistics for B and C figure are given in Table II.
Figure 2
Figure 2. Pro-survival effect of DC co-culture on myeloma cells
A. Flow cytometric analysis using pan-myeloid marker CD11b for analyzing myeloid population within the live mononuclear cell gate of bone marrow aspirates from normal and myeloma patients. B. Fluorescent microscopy of CFSE stained U266 cells in co-culture with DC (arrow), photographed without (DIC) or with FITC filters at 20X (top panel) and 40X (bottom panel). C. Viability of MM1.S cells (5 ×1 04/well) co-cultured with DC (5 × 104, 2.5 × 104 and 1.25 × 104/well) in the presence of 5 µM ATO or 10 µM melphalan in RPMI 1640 media containing 10% FBS. Each column represents the mean of 3 replicates and a representative experiment is shown. D. Viability of MM1.S or primary purified myeloma cells from 3 patients (Pt 1, Pt 2 and Pt 3) in cultured alone or with DC in serum free media (SFM) for 48 hrs. E. Viability of MM1.S or primary purified myeloma cells (Pt 4) cultured in contact with DC or separated by a 1 µm membrane using Transwells, under serum free conditions for 72 hrs. This was experimented was repeated with other patient myeloma cells and a representative experiment is shown. *p ≤ 0.05, **p<0.01, NS- not significant.
Figure 3
Figure 3. CD28 and CD80/CD86 mediate DC support of myeloma cell survival
Viability of myeloma cells as determined by AnnexinV/7AAD flow cytometry after 48 hr culture either alone or co-cultured with DC in the indicated cell-death inducing conditions with or without blocking reagents αCD28 (Fab)2 fragments to block CD28, or CTLA4-Ig± CD28-Ig to block DC CD80/CD86. A. MM1.S cells ± DC were cultured with or without blocking reagents in serum free media. B. MM1.S cells alone or co-cultured with DC treated with melphalan (20 µM) or dexamethasone (10 µM). *p ≤ 0.05, **p≤ 0.001.
Figure 4
Figure 4. Myeloma cells induce DC production of IL-6 in a CD28 and Notch dependent manner
A. IL-6 levels were determined by ELISA in supernatants of DC alone, DC + CTLA4-Ig (positive control), DC + MM1.S coculture ± αCD28 (Fab)2 fragments. B. Top panel - IL-6 levels in supernatants as determined by ELISA. Middle panel - Semi-quantitative RT-PCR analysis of total RNA from DC alone, MM1.S alone or DC + MM1.S co-cultures for expression of the DC-specific marker Fascin-1 or IL-6. Bottom panel - The IL-6 mRNA levels are shown relative to Fascin-1 levels by densitometric analysis. C. DC or MM1.S cells were left unfixed or fixed in paraformaldehyde, cultured in the indicated conditions and assayed for IL-6 production. D. IL-6 induction in co-cultures of DC with primary CD28+ (Pt 5, Pt 6) and CD28-low (Pt 7) myeloma cells. E. Top panel -IL-6 levels in supernatants as determined by ELISA. Middle panel - total RNA from DC, DC + MM1.S or DC + Pt 8 cocultures was used for semi-quantitative RT-PCR analysis of Fascin-1 and IL-6. Bottom panel - the IL-6 mRNA levels are shown relative to Fascin-1 levels by densitometric analysis. F. Top panel - Expression of Notch-1 and its Jagged ligands (Jagged 1 and Jagged 2) on DC and MM1.S were analyzed by flow cytometric analysis (solid histogram – isotype control, open histogram – test antibody). Bottom panel - the effect of blocking Notch signaling with the gamma secretase inhibitor (GSI) DAPT compared to αCD28 (Fab)2 fragments on DC IL-6 production induced by co-culture with MM1.S. *p<0.01, **p<0.001.
Figure 5
Figure 5. Biological activity of IL-6 in the MM-DC co-culture supernatants
A. Proliferation of RPMI 8226 cells cultured in supernatants conditioned by DC alone, U266 alone or DC +U266 co-cultures (48 hrs) ± dexamethasone (2.5 µM) ± neutralizing rabbit polyclonal antibodies against IL-6 was assessed by [H3]TdR incorporation. B. Viability of RPMI 8226 cells cultured with supernatants from DC + MM1.S co-cultures for 48 hr with or without dexamethasone (1 µM) ± neutralizing anti-IL 6 antibodies. *p<0.01. Media conditioned with MM cells alone for 48 hr or media alone incubated in the same plate for 48 hr was used as controls in both experiments.
Figure 6
Figure 6. Induction of the immunosuppressive enzyme indoleamine 2, 3 dioxygenase in MM-DC cocultures
A. IDO activity in cultures of DC alone, DC + IFNγ, DC + U266 + IFNγ, U266 alone or DC + CTLA4-Ig + IFNγ (as a positive control) in RPMI 1640 media supplemented with 100 µM tryptophan was measured by the level of kynurenine generated. B. Induction of IDO activity in co-cultures of DC with primary myeloma cells (Pt 9, Pt 10) + IFNγ. C. IDO activity in co-cultures of DC + U266 + IFNγ ± αCD28 (Fab)2 fragments. D. Western blot analysis of IDO expression. Tubulin was used as loading control, HEK293-IDO cells as a positive control. E. Flow cytometric analysis of DC + MM co-cultures stained for intracellular IDO to identify the IDO producing cell type. F. DC that were previously cocultured with U266 and IFNγ were isolated and re-cocultured with naïve resting allogeneic T cells for 7 days. DC in media alone were used as controls. T cells from these cocultures were then analyzed for CD4 and CD25 expression (top panel) or gated on CD4 and then analyzed for CD25 and FoxP3 expression (bottom panel) using FSCExpress V3.0. G. IDO activity in co-cultures of DC + U266 cells after 48 hours (left panel). The effect of IDO mediated tryptophan depletion and inhibition of IDO activity (IDOi) in co-cultures on PMA + ionomycin-induced T cell proliferation (right panel). *p≤0.01, **p≤0.005.
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