Germinal Center Function in the Spleen during Simian HIV Infection in Rhesus Monkeys

Abstract

Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with C→T and G→A replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.

Dynamics of Memory B-Cell Populations in Blood, Lymph Nodes, and Bone Marrow during Antiretroviral Therapy and Envelope Boosting in Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaques

“…As we saw no lasting increase in PC/PB counts in the bone marrow and no overall changes in lymph node memory B cells, we interpret this result as reflecting germinal center loss or dysfunction in the chronically infected macaques. It is known that germinal centers are damaged and reduced in size and number during HIV and SIV infections (33,35). Thus, although B cells were able to respond to the protein boost, long-term B-cell memory did not develop.…”

B cell repertoire expansion occurs in meningeal ectopic lymphoid tissue

“…This strongly suggested that antigen-driven SHM and selection contributed to B cell repertoires in mELT, similar to the spleen as a highly active site of B cell antigen training. Although IgG replacement mutations were clearly overrepresented in the CDRs in the spleen and in mELT, we found additional positions with high mutational activity within FRs ( Figure 5, A-D) (29). Replacement mutations in IgM-expressing B cells, which may occur before or after GC reaction, were more randomly distributed along the entire IgM-VH in all tissues examined ( Figure 5F), possibly representing trial-and-error BCR mutations, with less selection in pre-GC or non-class-switched B cells.…”

Nonhuman Primate Models of Human Immunology

“…A high degree of conservation of BCR heavy and light chain variable regions between humans and macaques has facilitated molecular analysis of the B cell maturation in rhesus macaques (10,36,55). These tools facilitated studies of B cell repertoire maturation during simian immunodeficiency virus infection, and demonstrated that, as described for humans, germinal centers support antigen-driven somatic hypermutation of antibodies that follows a pattern and a rate of nucleotide substitution and selection that closely mimics that described for humans (57).…”