Targeting and hematopoietic suppression of human CD34+ cells by measles virus

Abstract

The major cause of mortality in measles is generalized suppression of cell-mediated immunity that persists following virus clearance and results in secondary infections. The mechanisms contributing to this long-term immunosuppression are not clear. Herein we present evidence that measles virus (MV) disrupts hematopoiesis by infecting human CD34+ cells and human bone marrow stroma. MV infection does not affect the hematopoietic capability of hematopoietic stem cells (HSCs) directly; rather, the infection impairs the ability of stroma to support development of HSCs. These results suggest that MV-mediated defects in hematopoiesis contribute to the long-term immunosuppression seen in measles.

FIG. 1.

Infection of CD34⁺ cells by wild-type MV. (a) Flow cytometric analysis of CD34⁺ cells from human UCB with CD34⁺ and CD38⁺ fluorochrome-conjugated antibodies. (b) Expression of CD46⁺ on CD34⁺ cells from human UCB (unfilled histogram) compared to that from an isotype control (shaded histogram). (c) Expression of SLAM on CD34⁺ cells from human UCB (unfilled histogram) compared to that from an isotype control (shaded histogram). (d) Infection of CD34⁺ cord blood cells by MV-JW. Cell surface expression of the MV HA glycoprotein versus that of CD46⁺ is shown. Left panel: mock-infected control (CTRL); right panel: MV-JW infected. The percentage of double-positive (CD46⁺/MV-HA⁺) cells in each sample is noted in the upper-right quadrant of each panel. (e) Production of MV from infected CD34⁺ cells over a 24-day time course measured by a TCID₅₀ assay. (f) Inhibition of MV infection of CD34⁺ cells by antibodies against CD46 or SLAM. Inhibition of viral entry by using an isotype control antibody (isotype ctrl Ab), anti-CD46 MAb MCI20.6 (P = 0.4 compared to control), anti-SLAM antibody IPO-3, or both antibodies together (P < 0.2 compared to control) is shown. Virus production in the cell supernatant at 4 days postinfection was measured by TCID₅₀ assay and is expressed as the log TCID₅₀ per milliliter of supernatant. Assays were performed in triplicate and are expressed as means ± standard errors.

FIG. 2.

Suppression of hematopoiesis in bulk culture LTC-IC assay. (a) Titers of MV from supernatants of MV-JW as measured by TCID₅₀ assay. Results are representative of 2 independent experiments. (b) Cell numbers in bulk culture LTC-ICs during 5-week cocultivation with human BM stromal cells. Results of two separate experiments are shown. Filled squares, MV-JW cultures; unfilled circles, control cultures. (c) Quantification of total CFCs (including CFU-GM and BFU-E) harvested from week 5 of bulk culture LTC-IC. CFCs from LTC-IC control and MV-JW are shown. Bars indicate means of triplicate culture results ± 1 standard deviation, P < 0.002. (d to i) Light micrographs of colonies formed from CFCs harvested from bulk LTC-IC cultures. Panel d, control and CFU-GM cells; panel e, control and BFU-E; panel f, MV-JW and CFU-GM; panel g, MV-JW and BFU-E; panel h, UV-MV and CFU-GM; panel I, UV-MV and BFU-E. (j) RT-PCR of colonies from control or MV-JW- or MV-UV-infected LTC-IC culture, detecting either the MV-N message (top) or, as a positive control, human β-actin message (bottom). dH₂O, no template for PCR; MV-ctrl, RNA template prepared from MV-JW infected human PBMC; peN1, control plasmid DNA template carrying MV-N gene.

FIG. 3.

Suppression of hematopoiesis requires MV infection of BM stromal cells. (a to c) Light micrographs depicting CPE observed in bulk LTC-IC cultures at week 8. Panel a, control; panel b, MV-JW infected; panel c, MV-UV infected. (d) RT-PCR detecting MV-N message in bulk culture LTC-IC cocultivated with either human or mouse stroma at week 2 or week 5 postinfection. Top: RT-PCR of MV-N message from mock-infected control (C), MV-UV-infected (UV), and MV-JW-infected (JW) cells. Bottom: positive control PCR for human β-actin. H₂O, no PCR template; peN1, plasmid DNA template carrying MV-N gene. (e) CFC assay demonstrating loss of CFC progenitor cells derived from 5-week bulk culture LTC-IC cocultivated with human stroma (hLTC-IC; P < 0.002) compared to control or mouse stroma (mLTC-IC). Bars indicate means of triplicate cultures ± 1 standard deviation.