Berit Neumann

Characterization of B and plasma cells in blood, bone marrow, and secondary lymphoid organs of rhesus macaques by multicolor flow cytometry

B cells, as an important part of the humoral immune response, are generated in the BM, migrate to secondary lymphoid organs, and upon activation, differentiate into antibody‐producing memory B cells or plasma cells. Despite the pivotal roles that they play in different diseases, a comprehensive characterization in healthy rhesus macaques, which serve as valuable models for a variety of human diseases, is still missing. With the use of multiparameter flow cytometry, we analyzed B cells in BM collected from two locations, i.e., the iliac crest (BMca) and the femur (BMfem), PB, as well as secondary lymphoid organs of healthy rhesus macaques. We assessed the frequencies of immature and mature B cells, as well as CD19+ CD20– CD38+/++ CD138+/++ plasmablasts/plasma cells. Furthermore, we found site‐specific differences in the expression of markers for B cell activation and proliferation, chemokine receptors and Igs, as well as the distribution of memory B cell subpopulations. As secondary lymphoid organs harbor the highest frequencies of naive B cells, expression of CD80, CD95, and Ki67 was lower compared with B cells in the periphery and BM, whereas expression of IgD, CXCR4 (CD184), and CCR7 (CD197) was higher. Interestingly, BMca differed from BMfem regarding frequencies of B cells, their expression of CD80 and CXCR4, T cells, and plasma cells. In summary, these data identify baseline values for the above‐mentioned parameters and provide the foundation for future studies on B and plasma cells in different diseases.

Noncytolytic CD8+ Cell Mediated Antiviral Response Represents a Strong Element in the Immune Response of Simian Immunodeficiency Virus-Infected Long-Term Non-Progressing Rhesus Macaques

The ability of long term non progressors to maintain very low levels of HIV/SIV and a healthy state, involves various host genetic and immunological factors. CD8+ non-cytolytic antiviral response (CNAR) most likely plays an important role in this regard. In order to gain a deeper insight into this unique phenomenon, the ability of CD8+ T cells to suppress viral replication in vitro was investigated in 16 uninfected, longitudinally in 23 SIV-infected long-term non-progressing (LTNPs), and 10 SIV-infected rhesus macaques with progressing disease. An acute infection assay utilizing CD4+ cells from MHC-mismatched monkeys to avoid cytolytic responses was employed. The study has identified CNAR as a long-term stable activity that inversely correlated with plasma viral load. The activity was also detected in CD8+ cells of uninfected macaques, which indicates that CNAR is not necessarily a virus specific response but increases after SIV-infection. Physical contact between CD4+ and CD8+ cells was mainly involved in mediating viral inhibition. Loss of this activity appeared to be due to a loss of CNAR-expressing CD8+ cells as well as a reduction of CNAR-responsive CD4+ cells. In contrast, in vitro viral replication did not differ in CD4+ cells from un-infected macaques, CNAR(+) and CNAR(-) LTNPs. A role for transitional memory cells in supporting CNAR in the macaque model of AIDS was questionable. CNAR appears to represent an important part of the immune response displayed by CD8+ T cells which might be underestimated up to now.

Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques

Cryopreservation of peripheral blood mononuclear cells (PBMCs) is common for large clinical trials for which phenotypical characterization of lymphocytes is retrospectively performed in specialized core laboratories. It is therefore essential to assess the comparability between fresh and frozen samples. No side-by-side comparison of B and plasma cells of rhesus macaques (RM), which serve as useful models for several human diseases has been conducted until now. Hence, we performed an extensive comparative analysis between fresh and thawed mononuclear cells (MNCs) from blood and various tissues of healthy RM to analyze for the possible effects of cryopreservation on phenotype and functionality. Our data demonstrate that -80°C cryopreservation induces profound changes compared to fresh ex vivo-derived material. Percentages of B cells were stable in PBMCs, but were increased in all organs analyzed. The expression of CD27, a marker for differentiation between naïve and memory B cells, was massively reduced in PBMCs and MNC from organs with the most severe changes observed in cells from bone marrow (BM). Additionally, similar low percentages of CD27(+) memory B cells were detected in PBMCs and BM samples stored in liquid nitrogen. Therefore, cryopreservation is not suitable for the phenotypical and functional characterization of B cells. Further optimization of cryoconservation protocols monitoring the surface expression of CD27, which was identified as a marker for the quality of cryopreserved material of RM, will be essential.

Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques

ABSTRACT Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope (Env) proteins are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin reactivity, and a balance might be required for efficient mucosal transmission.

Frequencies of lymphoid T-follicular helper cells obtained longitudinally by lymph node fine-needle aspiration correlate significantly with viral load in SIV-infected rhesus monkeys.

T‐follicular helper (TFH) cells are an important population in lymph nodes (LNs) contributing to the generation of highly specific B cells. For SIV studies in rhesus macaques (RM), analysis of LN is necessary, but restricted due to invasive sampling. We applied the minimally invasive LN fine‐needle aspiration (LN‐FNA) and examined dynamics of TFH cells during SIV infection.

OMIP-026: Phenotypic analysis of B and plasma cells in rhesus macaques

OUR purpose was a broad phenotypic analysis of B and plasma cells regarding differentiation status, activation, proliferation, and chemokine receptor expression in rhesus macaques. We developed two staining panels, which were tested on fresh samples of whole blood or peripheral blood mononuclear cells (PBMCs), bone marrow collected from the iliac crest and femur, lymph nodes, spleen, and tonsils (Figure 1, Table 1). A 10-color-panel was developed to mainly focus on B cells, whereas a 11-color panel concentrated on plasmablasts/plasma cells. Both panels are also applicable for whole blood and bone marrow samples from African green monkeys.

Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity

HIV-1 causes chronic inflammation and AIDS in humans, whereas related simian immunodeficiency viruses (SIVs) replicate efficiently in their natural hosts without causing disease. It is currently unknown to what extent virus-specific properties are responsible for these different clinical outcomes. Here, we incorporate two putative HIV-1 virulence determinants, i.e., a Vpu protein that antagonizes tetherin and blocks NF-κB activation and a Nef protein that fails to suppress T cell activation via downmodulation of CD3, into a non-pathogenic SIVagm strain and test their impact on viral replication and pathogenicity in African green monkeys. Despite sustained high-level viremia over more than 4 years, moderately increased immune activation and transcriptional signatures of inflammation, the HIV-1-like SIVagm does not cause immunodeficiency or any other disease. These data indicate that species-specific host factors rather than intrinsic viral virulence factors determine the pathogenicity of primate lentiviruses.In contrast to HIV, simian immunodeficiency viruses (SIV) do not cause disease in their hosts, and the reasons for this are unclear. Here, Joas et al. incorporate two putative HIV virulence factors into SIV and study effects in infected monkeys, suggesting that species-specific host factors are responsible for HIV pathogenesis.

Elevated granzyme B(+) B-cell level in SIV-infection correlate with viral load and low CD4 T-cell count.

Granzyme B‐expressing (GrB+) B cells are thought to contribute to immune dysfunctions in HIV patients, but so far their exact role is unknown. This report demonstrates for the first time the existence of GrB+ B cells in SIV‐infected rhesus macaques, which represent the most commonly used nonhuman primate model for HIV research. Similar to HIV patients, we found significantly higher frequencies of these cells in the blood of chronically SIV‐infected rhesus monkeys compared with uninfected healthy ones. These frequencies correlated with plasma viral load and inversely with absolute CD4 T‐cell counts. When investigating GrB+ B cells in different compartments, levels were highest in blood, spleen and bone marrow, but considerably lower in lymph nodes and tonsils. Analysis of expression of various surface markers on this particular B‐cell subset in SIV‐infected macaques revealed differences between the phenotype in macaques and in humans. GrB+ B cells in SIV‐infected rhesus macaques exhibit an elevated expression of CD5, CD10, CD25 and CD27, while expression of CD19, CD185 and HLA‐DR is reduced. In contrast to human GrB+ B cells, we did not observe a significantly increased expression of CD43 and CD86. B‐cell receptor stimulation in combination with IL‐21 of purified B cells from healthy animals led to the induction of GrB expression. Furthermore, initial functional analyses indicated a regulatory role on T‐cell proliferation. Overall, our data pave the way for longitudinal analyses including studies on the functionality of GrB+ B cells in the nonhuman primate model for AIDS.

Long-term efficient control of SIV infection in macaques is associated with an intact intestinal barrier

Hallmarks of SIV infection are early depletion of gut CD4 T cells and diminished intestinal integrity. Comprehensive studies on colon biopsies of SIV‐infected macaques efficiently controlling infection revealed that in contrast to viremic and failing controllers, elite controllers show preserved CD4 T cells, and low viral load, apoptosis, and inflammation.

Comprehensive panel of cross-reacting monoclonal antibodies for analysis of different immune cells and their distribution in the common marmoset (Callithrix jacchus).

Common marmosets are extensively used in immunological and pharmacological research, and the usage of methods such as flow cytometry gain increasing importance.