CD27− B-Cells Produce Class Switched and Somatically Hyper-Mutated Antibodies during Chronic HIV-1 Infection

Class switch recombination and somatic hypermutation occur in mature B-cells in response to antigen stimulation. These processes are crucial for the generation of functional antibodies. During HIV-1 infection, loss of memory B-cells, together with an altered differentiation of naïve B-cells result in production of low quality antibodies, which may be due to impaired immunoglobulin affinity maturation. In the current study, we evaluated the effect of HIV-1 infection on class switch recombination and somatic hypermutation by studying the expression of activation-induced cytidine deaminase (AID) in peripheral B-cells from a cohort of chronically HIV-1 infected patients as compared to a group of healthy controls. In parallel, we also characterized the phenotype of B-cells and their ability to produce immunoglobulins in vitro. Cells from HIV-1 infected patients showed higher baseline levels of AID expression and increased IgA production measured ex-vivo and upon CD40 and TLR9 stimulation in vitro. Moreover, the percentage of CD27−IgA+ and CD27−IgG+ B-cells in blood was significantly increased in HIV-1 infected patients as compared to controls. Interestingly, our results showed a significantly increased number of somatic hypermutations in the VH genes in CD27− cells from patients. Taken together, these results show that during HIV-1 infection, CD27− B-cells can also produce class switched and somatically hypermutated antibodies. Our data add important information for the understanding of the mechanisms underlying the loss of specific antibody production observed during HIV-1 infection

HIV-1 infected patients present with expanded populations of blood CD27⁻ IgA⁺ and CD27⁻ IgG⁺ B-cells and show inverse patterns of SHM.

<p>The percentage of CD27⁻IgA⁺ (A) and CD27⁻IgG⁺ (B) among total IgA/G expressing B-cells is significantly expanded in HIV-1 infected patients (right panel) as compared with healthy controls (left panel). (C) The number of somatic hypermutations in the VH region of mRNA transcripts of CD27⁻ B cells (left panel) is increased in HIV-1 infected patients (black bars), as compared with healthy controls (white bars), while an opposite trend is shown for CD27⁺ B cells (right panel), where the number of somatic hypermutations in the VH region of mRNA transcripts is decreased in HIV-1 infected patients (black bars) as compared with healthy controls (white bars). The number below the bars indicates the number of clones analysed.</p

Mutations in VH3-Cγ transcripts from sorted CD27−IgD+ and CD27+IgD− B cells from HIV-1 infected patients and healthy controls.

<p>The SHM pattern analysis was performed in the distinct clones.</p>*<p>Replacements/silent mutations.</p>**<p>Complementarity-determining region.</p>***<p>Framework region.</p

IgA and IgG production <i>in vitro</i> in HIV-1 infected patients and healthy controls.

<p>(A) The levels of IgA before (left panel) and after <i>in vitro</i> stimulation (anti-CD40 mAb, IL4 and IL10 or CpG, middle and right panels) are higher in patients (black bars) as compared with controls (white bars). (B) The levels of IgG before (left panel) and after <i>in vitro</i> stimulation (middle and right panels) are similar in patients (black bars) as compared with controls (white bars).</p

AID expression in HIV-1 infected patients and healthy controls.

<p>(A) The <i>ex-vivo</i> levels of AID mRNA expression (left panel) are higher in patients (black bars) as compared with controls (white bars) but they reach similar levels upon <i>in vitro</i> stimulation (middle and right panels). The baseline levels of AID mRNA expression correlate with the percentage of CD27⁺ B cells in healthy controls (B) while there is no correlation for HIV-1 infected patients (C). The anti-CD40 stimulation was performed with an anti-CD40 mAb, IL4 and IL10 while TLR9 stimulation with CpG.</p