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

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.
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
96-well Shuttle™ System

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).


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.