Attenuation of HIV-associated human B cell exhaustion by siRNA downregulation of inhibitory receptors.

Authors:
Kardava L, Moir S, Wang W, Ho J, Buckner CM, Posada JG, O'Shea MA, Roby G, Chen J, Sohn HW, Chun TW, Pierce SK, Fauci AS.
In:
Source: J Clin Invest
Publication Date: (2011)
Issue: 121(7): 2614-24
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Basic Research
Cells used in publication:
B cell, human
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofector® 96-well Systems
Experiment

siRNA transfection. Transient transfection of B cells was performed using the Lonza nucleofection 96-well plate system according to the manufacturer’s specifications. Briefly, 1 × 106 cells were resuspended in 20 µl of nucleofector solution, mixed with 500 nM of control nontargeting or gene-specific ON-TARGETplus SMARTpool siRNAs from Dharmacon, with the exception of CD22 siRNA, which was obtained from Ambion (Supplemental Table 1). Nucleofection was performed using program EO-117. Cells were rapidly transferred to preheated complete medium (RPMI 1640/10% FBS) and incubated for 24–96 hours at 37°C. Viability of cells was evaluated by vital dye exclusion (Guava Technologies).

Abstract

Chronic immune activation in HIV-infected individuals leads to accumulation of exhausted tissue-like memory B cells. Exhausted lymphocytes display increased expression of multiple inhibitory receptors, which may contribute to the inefficiency of HIV-specific antibody responses. Here, we show that downregulation of B cell inhibitory receptors in primary human B cells led to increased tissue-like memory B cell proliferation and responsiveness against HIV. In human B cells, siRNA knockdown of 9 known and putative B cell inhibitory receptors led to enhanced B cell receptor-mediated (BCR-mediated) proliferation of tissue-like memory but not other B cell subpopulations. The strongest effects were observed with the putative inhibitory receptors Fc receptor-like-4 (FCRL4) and sialic acid-binding Ig-like lectin 6 (Siglec-6). Inhibitory receptor downregulation also led to increased levels of HIV-specific antibody-secreting cells and B cell-associated chemokines and cytokines. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive or tonic signaling. Furthermore, the extent of FCLR4 knockdown effects on BCR-mediated proliferation varied depending on the costimulatory ligand, suggesting that inhibitory receptors may engage specific pathways in inhibiting B cell proliferation. These findings on HIV-associated B cell exhaustion define potential targets for reversing the deleterious effect of inhibitory receptors on immune responses against persistent viral infections.