HIV infection is associated with a greatly elevated risk for the development of non-Hodgkin lymphoma (NHL), which while diminished, remains elevated in the highly active antiretroviral therapy (HAART) era. Chronic B cell activation, driven by contact with HIV virions, B cell-stimulatory cytokines, viruses (EBV, HPV, HCV), and by high levels of antigenic stimulation occurs in HIV infected persons, and it is seen at even higher levels in those who go on to develop AIDS-NHL. Evidence from multiple studies indicates that elevated serum levels of several B cell-stimulatory cytokines and biomarkers are seen preceding AIDS-NHL, as well as in immunocompetent persons that develop NHL. Phenotypic changes in circulating B cells also are seen preceding AIDS-NHL, including the expression of AICDA, and of cell-surface molecules and miRNA that are associated with activated B cells. HAART only partially normalizes the immune system of treated HIV⁺ persons as they still show clear evidence for ongoing inflammation and immune activation in, even those who show complete suppression of HIV viremia. Together, this provides ample evidence to support the notion that chronic activation of B cells contributes to the genesis of B cell lymphomas.

Background

AIDS-related non-Hodgkin lymphoma (AIDS-NHL) is a common AIDS-defining cancer. Prior studies suggest that chronic B-cell activation precedes AIDS-NHL diagnosis. Activation of B cells by multiple factors, including Toll-like receptor (TLR) signaling, leads to the expression of activation-induced cytidine deaminase (AID), a DNA mutating molecule that can contribute to oncogene translocations/mutations, leading to NHL. The goal of this study was to determine whether surface markers expressed on activated and/or germinal center B cells, and AID expression, were elevated on circulating B cells preceding AIDS-NHL and to determine if TLR signaling contributes to this activated B-cell phenotype.

Methods

Stored viable peripheral blood mononuclear cell specimens, obtained before AIDS-NHL diagnosis, were assessed by multicolor flow cytometry. Additionally, B cells isolated from peripheral blood mononuclear cell were exposed to TLR ligands in vitro, after which B-cell phenotype was assessed by flow cytometry.

Results

An elevated fraction of B cells expressing CD10, CD71, or CD86 was seen in those who went on to develop AIDS-NHL. AID expression was detected in some who developed AIDS-NHL, but not in HIV+ or HIV- controls. TLR2-stimulated purified B cells exhibited the activated B-cell phenotype observed in HIV+ subjects before AIDS-NHL diagnosis.

Conclusions

These results indicate that an elevated fraction of B cells display an activated/germinal center phenotype in those HIV+ subjects who go on to develop AIDS-NHL and suggest that TLR2-mediated activation may play a role in HIV infection-associated B-cell activation, potentially contributing to the genesis of AIDS-NHL.

To assess the intra-individual and inter-individuals biological variation and the effect of aging on lymphocyte T-cells subsets.We assessed lymphocyte phenotypes (CD3, CD4, and CD8 T-cells) in 89 HIV-1-infected and 88 uninfected white non-Hispanic men every 6 months, to examine the biological variation for those measurements, and the average change in lymphocyte phenotype over 34 years.The markers showed significant intra-individuality in HIV-infected and uninfected individuals with index of individuality of <1.4. No mean changes were seen over the 34 years, with the exception of percentage CD4T-cells in HIV-uninfected individuals.In the pre-HAART era, HIV-infected individuals experienced an increase in mean absolute CD3 T-cell numbers (11.21 cells/μL, P = 0.02) and absolute CD8 T-cell numbers (34.57 cell/μl, P < .001), and in the percentage of CD8 T-cells (1.45%, P < .001) per year and a significant decrease in mean absolute CD4 T-cell numbers (23.68 cells/μl, P < .001) and in the percentage of CD4 T-cells (1.49%, P < .001) per year.In the post-HAART era, no changes in mean levels were observed in absolute CD3 T-cell count (P = .15) or percentage (P = .99). Significant decreases were seen in mean count (8.56 cells/μl, P < .001) and percentage (0.59%, P < .001) of CD8 T-cells, and increases in mean absolute count (10.72 cells/μl, P < .001) and percentage (0.47%, P < .001) of CD4 T-cells.With the exception of CD4 (%), no average changes per year were seen in lymphocyte phenotype of HIV-uninfected men. The results of coefficients of variation of intra and inter-individuals of this study can be useful for HIV-1 infection monitoring and in addition the observation could be a useful guide for intra- and inter-individual coefficient variations, and establishing quality goal studies of different blood biomarkers in healthy and other diseases.

The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcγRI, FcγRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and CH2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcγRI and FcγRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.

To assess the intra-individual and inter-individuals biological variation and the effect of aging on lymphocyte T-cells subsets.We assessed lymphocyte phenotypes (CD3, CD4, and CD8 T-cells) in 89 HIV-1-infected and 88 uninfected white non-Hispanic men every 6 months, to examine the biological variation for those measurements, and the average change in lymphocyte phenotype over 34 years.The markers showed significant intra-individuality in HIV-infected and uninfected individuals with index of individuality of <1.4. No mean changes were seen over the 34 years, with the exception of percentage CD4T-cells in HIV-uninfected individuals.In the pre-HAART era, HIV-infected individuals experienced an increase in mean absolute CD3 T-cell numbers (11.21 cells/μL, P = 0.02) and absolute CD8 T-cell numbers (34.57 cell/μl, P < .001), and in the percentage of CD8 T-cells (1.45%, P < .001) per year and a significant decrease in mean absolute CD4 T-cell numbers (23.68 cells/μl, P < .001) and in the percentage of CD4 T-cells (1.49%, P < .001) per year.In the post-HAART era, no changes in mean levels were observed in absolute CD3 T-cell count (P = .15) or percentage (P = .99). Significant decreases were seen in mean count (8.56 cells/μl, P < .001) and percentage (0.59%, P < .001) of CD8 T-cells, and increases in mean absolute count (10.72 cells/μl, P < .001) and percentage (0.47%, P < .001) of CD4 T-cells.With the exception of CD4 (%), no average changes per year were seen in lymphocyte phenotype of HIV-uninfected men. The results of coefficients of variation of intra and inter-individuals of this study can be useful for HIV-1 infection monitoring and in addition the observation could be a useful guide for intra- and inter-individual coefficient variations, and establishing quality goal studies of different blood biomarkers in healthy and other diseases.

BACKGROUND: AIDS-related non-Hodgkin lymphoma (AIDS-NHL) is a common AIDS-defining cancer. Prior studies suggest that chronic B-cell activation precedes AIDS-NHL diagnosis. Activation of B cells by multiple factors, including Toll-like receptor (TLR) signaling, leads to the expression of activation-induced cytidine deaminase (AID), a DNA mutating molecule that can contribute to oncogene translocations/mutations, leading to NHL. The goal of this study was to determine whether surface markers expressed on activated and/or germinal center B cells, and AID expression, were elevated on circulating B cells preceding AIDS-NHL and to determine if TLR signaling contributes to this activated B-cell phenotype. METHODS: Stored viable peripheral blood mononuclear cell specimens, obtained before AIDS-NHL diagnosis, were assessed by multicolor flow cytometry. Additionally, B cells isolated from peripheral blood mononuclear cell were exposed to TLR ligands in vitro, after which B-cell phenotype was assessed by flow cytometry. RESULTS: An elevated fraction of B cells expressing CD10, CD71, or CD86 was seen in those who went on to develop AIDS-NHL. AID expression was detected in some who developed AIDS-NHL, but not in HIV+ or HIV- controls. TLR2-stimulated purified B cells exhibited the activated B-cell phenotype observed in HIV+ subjects before AIDS-NHL diagnosis. CONCLUSIONS: These results indicate that an elevated fraction of B cells display an activated/germinal center phenotype in those HIV+ subjects who go on to develop AIDS-NHL and suggest that TLR2-mediated activation may play a role in HIV infection-associated B-cell activation, potentially contributing to the genesis of AIDS-NHL.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Objectives

In antiretroviral therapy (ART)-treated patients, to determine if AIDS-related non-Hodgkin lymphoma (AIDS-NHL) is preceded by: elevated frequency of potentially malignant abnormal activated/germinal center-like B cells, elevated serum prevalence of B-cell stimulatory Toll-like receptor (TLR) ligands resulting from HIV infection-associated microbial translocation, dysregulated B-cell TLR expression/signaling, and perturbations in the frequency of immunoregulatory cells.

Design

A case-control study nested with a cohort study of HIV-infected women.

Methods

Prediagnostic AIDS-NHL cases (n = 12, collected 1-12 months before diagnosis) and controls (n = 42) from the Women's Interagency HIV Study cohort, were matched for HIV and ART status, age, race, and CD4 lymphocyte count. Serum levels of TLR ligands, the prevalence of malignancy-associated abnormal activated/germinal center-like (CD19CD10CD71CD86AID) B cells, TLR2 expression on B cells, expression of TLR2-modulating micro-RNA, and the frequency of regulatory T and B cells were assessed.

Results

Diagnosis of AIDS-NHL was preceded by a significantly elevated frequency of activated/germinal center-like CD19CD10CD71CD86AID B cells (P = 0.0072), elevated serum prevalence of the TLR2 ligand, and significantly elevated B-cell TLR2 expression (P = 0.0015), positively correlating with the frequency of activated/germinal center-like B cells (rho = 0.7273, P = 0.0144). In cases, a purified subset of activated/germinal center-like B cells exhibited decreased expression of microRNAs that modulate TLR2 signaling, including miR-21, 146a, 146b, and 155. Finally, cases also exhibited significantly elevated frequencies of antitumor immunity inhibitory regulatory B cells (P = 0.0024), but not regulatory T cells.

Conclusions

Our findings suggest that increased microbial translocation and dysregulated TLR expression/signaling, coupled with an elevated frequency of regulatory B cells, precede the diagnosis of AIDS-NHL in HIV-infected ART-treated patients.