In this study, we have examined the longitudinal levels of several serum molecules associated with B cell and/or immune system activation, over a period of several years preceding AIDS-NHL diagnosis. These included two B cell-stimulatory cytokines, IL6 and IL10, and sCD23, a soluble Fc receptor for IgE that has cytokine-like B cell-stimulatory properties (41
). Additionally, we assessed serum levels of IgE, which is induced by Th2 cytokines, as well as serum levels of sCD27 and sCD30, two TNF-receptor superfamily molecules that reflect immune system activation (43
), and CRP, a marker for inflammation, the production of which is driven by pro-inflammatory cytokines.
Elevated serum levels of IL6, CRP, sCD27, and sCD30, were consistently seen at all time points tested preceding lymphoma diagnosis, when comparing all AIDS-NHL cases (n=179) to HIV+ controls (). Even after adjustment for CD4 T cell count (), CD4 slope, or HIV VL, to control for differences possibly related to HIV disease progression rather than AIDS-NHL, an increased association clearly persisted between the development of AIDS-NHL and elevated serum levels at all time points for IL6, sCD27, and sCD30. Increased frequency of detectable IL10 was also seen preceding AIDS-NHL cases both with and without CD4 adjustment, but only at the time closest to lymphoma diagnosis. With CD4 adjustment, CRP was no longer significant at 0–1 year prior to NHL, which may reflect an increasing contribution of more general HIV disease-related inflammation obscuring the earlier relationship with development of AIDS-NHL and/or may be due to the smaller number of samples available closest to lymphoma diagnosis. Similarly, elevated serum levels of sCD23 only at >3 years pre-lymphoma may suggest rising levels in HIV+ controls with time obscure earlier differences. IgE showed no significant association with AIDS-NHL in any analysis, in contrast to an early report (36
), but consistent with later studies (38
), which is likely due to differences in study design and differences in the immunoassays utilized. Hence, even when taking into account lower CD4 counts (or more rapid CD4 decline or higher baseline VL) over the same length of time of HIV infection, our data indicated the presence of a sustained environment of B cell and immune system hyper-activation for more than three years in those subjects who went on to develop AIDS-NHL.
A little more than half of the AIDS-NHL cases in the overall analyses (100/179) were diagnosed with lymphoma after some other AIDS-defining condition. We compared this subset of cases to an independent set of controls matched on time of AIDS diagnosis, and saw elevated serum levels of IL6, sCD27, and sCD30, but only at 0–1 and/or 1–3 years prior to lymphoma diagnosis. This suggests that although a prior AIDS-defining condition may disrupt an earlier pre-lymphoma B cell stimulatory environment and/or confound our ability to detect it, evidence of increased immune activation does emerge, especially in the year immediately preceding AIDS-NHL diagnosis. In light of differences seen between systemic and CNS AIDS-NHL cases (as discussed below), less immune activation might also be attributable at least in part to the fact that PCNSL occurred in a higher proportion of post-AIDS diagnosis cases (44%) than in the overall AIDS-NHL cases (32%).
IL6 is a pluripotent cytokine that can stimulate B cell proliferation and differentiation, and promote cell survival and tumor growth (52
). IL6 production is driven by multiple stimuli, including exposure to lipopolysaccharide (LPS) or to HIV virions (56
). IL6 also is associated with proinflammatory responses, as well as with Th17 responses, which are associated with autoimmunity (59
). Interestingly, those forms of autoimmunity that involve B cell activation and are mediated by autoantibody production have been seen to be associated with an enhanced risk for the development of B cell NHL (61
). Previous cross-sectional studies have documented elevated IL6 preceding AIDS-NHL (28
). The observation that elevated serum levels of IL6 are repeatedly associated with AIDS-NHL for more than three years prior to diagnosis is consistent for a role for IL6-driven B cell stimulation in the development and/or growth of these lymphomas.
CD30 and CD27 are the receptors for CD30 ligand (CD153) and CD70, respectively, which are TNF-like immune stimulatory molecules (62
). CD30 is characteristically expressed on Hodgkin lymphoma Reed-Sternberg cells, but is rarely expressed by NHL cells. sCD30 is produced by activated T cells, and may be a marker for Th2 immune responses that support B cell activation and differentiation into antibody-secreting plasma cells (66
). CD27 is expressed on the surface of B cells following activation, and is a marker for memory B cells (67
). In prior cross-sectional studies, we noted strong correlations between these two related molecules, and have reported elevated serum levels of both sCD30 and sCD27 preceding the diagnosis of AIDS-NHL (32
). Like IL6, the consistent elevation of these molecules for more than three years pre-lymphoma provide strong evidence for the role of sustained B cell activation in the development of AIDS-NHL. Elevated serum sCD30 also has been reported preceding the diagnosis of non-AIDS NHL (71
), suggesting that an environment rich in B cell activation may be a feature that precedes many B cell lymphomas, regardless of HIV status.
IL10 is a B cell stimulatory cytokine that also can suppress Th1 responses, including anti-viral cell-mediated responses (72
). Our observations of IL10 in this study are consistent with our previous work, in which we found elevated serum levels of IL10 immediately preceding AIDS-NHL, as well as an association between IL10 genotype and the development of AIDS-NHL (29
). An elevated frequency of detectable IL10 only at the time point closest lymphoma diagnosis (<1 year pre-NHL) suggests that this cytokine is either tumor-produced, consistent with a report that IL10 is an autologous growth factor for AIDS-NHL (75
), and/or is produced by, or in response to, the inflammatory microenvironment of the tumor.
When the results from those who developed PCNSL were compared to those who had systemic non-CNS NHL, it was seen that significantly elevated serum levels of B cell activation-associated molecules were seen only with the development of systemic lymphomas. This suggests that elevated serum levels of molecules associated with B cell activation are not associated with an increased risk for the development of PCNSL, which is characterized by EBV infection of tumor cells. Rather, elevated levels of B cell activation-associated molecules were seen preceding the development of those forms of AIDS-NHL (systemic AIDS-NHL, including BL and DLBCL) that contain molecular lesions believed to result from errors in B cell activation-associated DNA-modifying events. These findings are consistent with the view that EBV+ PCNSL result primarily from loss of effective immunoregulation of EBV infection, while systemic lymphomas, which are often EBV-negative tumors, are associated with chronic B cell activation over a prolonged period of time, resulting in the accumulation of genetic lesions due to IgH
CSR or SHM (5
). The recent observation that elevated levels of AICDA
expression occur pre-AIDS-NHL, but only in those who developed non-CNS lymphomas, is consistent with this conclusion (39
). AICDA is a B cell activation associated DNA mutating molecule that plays a central role in both IgH
CSR and SHM in germinal center B cells (77
). Interestingly, AICDA
expression can be induced in B cells by exposure to HIV (78
), as well as other lymphomagenic viruses, including EBV and HCV (79
), suggesting a direct role for HIV in the induction of B cell activation. Studies to quantify AICDA
expression and its association with HIV levels in the same cases and controls included in this study are planned.
The assessment of additional cytokines and immune stimulatory molecules other than those that were quantified in this study would have been informative. However, we focused on those molecules for which we had compelling preliminary evidence in prior preliminary cross-sectional studies, and for which robust immunoassays were available, given the limited volume of pre-NHL diagnosis serum that is available. As multiplexed immunoassays are becoming more sensitive and reliable, we hope to assess additional biomarkers in the future.
In summary, levels of B cell stimulatory cytokines and of molecules associated with immune activation are elevated several years preceding the diagnosis of AIDS-NHL. These results are consistent with the hypothesis that chronic B cell hyper-activation contributes to the development of these lymphomas.