Circulating immune and plasma biomarkers of time to HIV rebound in HIV controllers treated with vesatolimod.

Background: Antiretroviral therapy (ART) for HIV-1 treatment has improved lifespan but requires lifelong adherence for people living with HIV (PLWH), highlighting the need for a cure. Evaluation of potential cure strategies requires analytic treatment interruption (ATI) with close monitoring of viral rebound. Predictive biomarkers for HIV-1 rebound and/or duration of control during ATI will facilitate these HIV cure trials while minimizing risks. Available evidence suggests that host immune, glycomic, lipid, and metabolic markers of inflammation may be associated with HIV-1 persistence in PLWH who are treated during chronic HIV-1 infection. Methods: We conducted post-hoc analysis of HIV controllers who could maintain low levels of plasma HIV-1 without ART in a phase 1b vesatolimod trial. Baseline and pre-ATI levels of immune, glycomic, lipidomic, and metabolomic markers were tested for association with ATI outcomes (time of HIV-1 rebound to 200 copies/mL and 1,000 copies/mL, duration of HIV-1 RNA ≤400 copies/mL and change in intact proviral HIV-1 DNA during ATI) using Spearman's correlation and Cox proportional hazards model. Results: Higher levels of CD69+CD8+ T-cells were consistently associated with shorter time to HIV-1 rebound at baseline and pre-ATI. With few exceptions, baseline fucosylated, non-galactosylated, non-sialylated, bisecting IgG N-glycans were associated with shorter time to HIV rebound and duration of control as with previous studies. Baseline plasma MPA and HPA binding glycans and non-galactosylated/non-sialylated glycans were associated with longer time to HIV rebound, while baseline multiply-galactosylated glycans and sialylated glycans, GNA-binding glycans, NPA-binding glycans, WGA-binding glycans, and bisecting GlcNAc glycans were associated with shorter time to HIV rebound and duration of control. Fourteen bioactive lipids had significant baseline associations with longer time to rebound and duration of control, and larger intact proviral HIV-1 DNA changes; additionally, three baseline bioactive lipids were associated with shorter time to first rebound and duration of control. Conclusion: Consistent with studies in HIV non-controllers, proinflammatory glycans, lipids, and metabolites were generally associated with shorter duration of HIV-1 control. Notable differences were observed between HIV controllers vs. non-controllers in some specific markers. For the first time, exploratory biomarkers of ATI viral outcomes in HIV-controllers were investigated but require further validation.

HIV rebound in HIV controllers is associated with a specific fecal microbiome profile.

HIV infection is associated with gut dysbiosis, and microbiome variability may affect HIV control when antiretroviral therapy (ART) is stopped. The TLR7 agonist, vesatolimod, was previously associated with a modest delay in viral rebound following analytical treatment interruption in HIV controllers (HCs). Using a retrospective analysis of fecal samples from HCs treated with vesatolimod or placebo (NCT03060447), people with chronic HIV (CH; NCT02858401) or without HIV (PWOH), we examined fecal microbiome profile in HCs before/after treatment, and in CH and PWOH. Microbiome diversity and abundance were compared between groups to investigate the association between specific phyla/species, immune biomarkers, and viral outcomes during treatment interruption. Although there were no significant differences in gut microbiome diversity between people with and without HIV, HCs, and CH shared common features that distinguished them from PWOH. there was a trend toward greater microbiome diversity among HCs. Treatment with vesatolimod reduced dysbiosis in HCs. Firmicutes positively correlated with T-cell activation, while Bacteroidetes and Euryarchaeota inversely correlated with TLR7-mediated immune activation. Specific types of fecal microbiome abundance (e.g. Alistipes putredinis) positively correlated with HIV rebound. In conclusion, variability in the composition of the fecal microbiome is associated with markers of immune activation following vesatolimod treatment and ART interruption.

CyTOF analysis identifies unusual immune cells in urine of BCG-treated bladder cancer patients.

High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical parameter predictive of treatment response, and when treatment fails, the prognosis is very poor. Further, a high percentage of NMIBC patients treated with BCG suffer unwanted effects that force them to stop treatment. Thus, early identification of patients in which BCG treatment will fail is really important. Here, to identify early stage non-invasive biomarkers of non-responder patients and patients at risk of abandoning the treatment, we longitudinally analysed the phenotype of cells released into the urine of bladder cancer patients 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed a large proportion of granulocytes and monocytes, mostly expressing activation markers. A novel population of CD15+CD66b+CD14+CD16+ cells was highly abundant in several samples; expression of these markers was confirmed using flow cytometry and qPCR. A stronger inflammatory response was associated with increased cell numbers in the urine; this was not due to hematuria because the cell proportions were distinct from those in the blood. This pilot study represents the first CyTOF analysis of cells recruited to urine during BCG treatment, allowing identification of informative markers associated with treatment response for sub-selection of markers to confirm using conventional techniques. Further studies should jointly evaluate cells and soluble factors in urine in larger cohorts of patients to characterise the arms of the immune response activated in responders and to identify patients at risk of complications from BCG treatment.

Natural Killer Cell Receptors and Ligands Are Associated With Markers of HIV-1 Persistence in Chronically Infected ART Suppressed Patients.

The latent HIV-1 reservoir represents a major barrier to achieving a long-term antiretroviral therapy (ART)-free remission or cure for HIV-1. Natural Killer (NK) cells are innate immune cells that play a critical role in controlling viral infections and have been shown to be involved in preventing HIV-1 infection and, in those who are infected, delaying time to progression to AIDS. However, their role in limiting HIV-1 persistence on long term ART is still uncharacterized. To identify associations between markers of HIV-1 persistence and the NK cell receptor-ligand repertoire, we used twin mass cytometry panels to characterize the peripheral blood NK receptor-ligand repertoire in individuals with long-term antiretroviral suppression enrolled in the AIDS Clinical Trial Group A5321 study. At the time of testing, participants had been on ART for a median of 7 years, with virological suppression <50 copies/mL since at most 48 weeks on ART. We found that the NK cell receptor and ligand repertoires did not change across three longitudinal samples over one year-a median of 25 weeks and 50 weeks after the initial sampling. To determine the features of the receptor-ligand repertoire that associate with markers of HIV-1 persistence, we performed a LASSO normalized regression. This analysis revealed that the NK cell ligands CD58, HLA-B, and CRACC, as well as the killer cell immunoglobulin-like receptors (KIRs) KIR2DL1, KIR2DL3, and KIR2DS4 were robustly predictive of markers of HIV-1 persistence, as measured by total HIV-1 cell-associated DNA, HIV-1 cell-associated RNA, and single copy HIV-RNA assays. To characterize the roles of cell populations defined by multiple markers, we augmented the LASSO analysis with FlowSOM clustering. This analysis found that a less mature NK cell phenotype (CD16+CD56dimCD57-LILRB1-NKG2C-) was associated with lower HIV-1 cell associated DNA. Finally, we found that surface expression of HLA-Bw6 measured by CyTOF was associated with lower HIV-1 persistence. Genetic analysis revealed that this was driven by lower HIV-1 persistence in HLA-Bw4/6 heterozygotes. These findings suggest that there may be a role for NK cells in controlling HIV-1 persistence in individuals on long-term ART, which must be corroborated by future studies.

The TLR7 agonist vesatolimod induced a modest delay in viral rebound in HIV controllers after cessation of antiretroviral therapy.

Toll-like receptor 7 (TLR7) agonists, in combination with other therapies, can induce sustained control of simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) in nonhuman primates. Here, we report the results of a randomized, double-blind, placebo-controlled phase 1b clinical trial of an oral TLR7 agonist, vesatolimod, in HIV-1-infected controllers on antiretroviral therapy (ART). We randomized participants 2:1 to receive vesatolimod (n = 17) or placebo (n = 8) once every other week for a total of 10 doses while continuing on ART. ART was then interrupted, and the time to viral rebound was analyzed using the Kaplan-Meier method. Vesatolimod was associated with induction of immune cell activation, decreases in intact proviral DNA during ART, and a modest increase in time to rebound after ART was interrupted. The delayed viral rebound was predicted by the lower intact proviral DNA at the end of vesatolimod treatment (13 days after the final dose). Inferred pathway analysis suggested increased dendritic cell and natural killer cell cross-talk and an increase in cytotoxicity potential after vesatolimod dosing. Larger clinical studies will be necessary to assess the efficacy of vesatolimod-based combination therapies aimed at long-term control of HIV infection.

CytoGLMM: conditional differential analysis for flow and mass cytometry experiments.

BACKGROUND: Flow and mass cytometry are important modern immunology tools for measuring expression levels of multiple proteins on single cells. The goal is to better understand the mechanisms of responses on a single cell basis by studying differential expression of proteins. Most current data analysis tools compare expressions across many computationally discovered cell types. Our goal is to focus on just one cell type. Our narrower field of application allows us to define a more specific statistical model with easier to control statistical guarantees. RESULTS: Differential analysis of marker expressions can be difficult due to marker correlations and inter-subject heterogeneity, particularly for studies of human immunology. We address these challenges with two multiple regression strategies: a bootstrapped generalized linear model and a generalized linear mixed model. On simulated datasets, we compare the robustness towards marker correlations and heterogeneity of both strategies. For paired experiments, we find that both strategies maintain the target false discovery rate under medium correlations and that mixed models are statistically more powerful under the correct model specification. For unpaired experiments, our results indicate that much larger patient sample sizes are required to detect differences. We illustrate the CytoGLMM R package and workflow for both strategies on a pregnancy dataset. CONCLUSION: Our approach to finding differential proteins in flow and mass cytometry data reduces biases arising from marker correlations and safeguards against false discoveries induced by patient heterogeneity.

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Natural killer cell phenotype is altered in HIV-exposed seronegative women.

Highly exposed seronegative (HESN) individuals present a unique setting to study mechanisms of protection against HIV acquisition. As natural killer (NK) cell activation and function have been implicated as a correlate of protection in HESN individuals, we sought to better understand the features of NK cells that may confer protection. We used mass cytometry to phenotypically profile NK cells from a cohort of Beninese sex workers and healthy controls. We found that NK cells from HESN women had increased expression of NKG2A, NKp30 and LILRB1, as well as the Fc receptor CD16, and decreased expression of DNAM-1, CD94, Siglec-7, and NKp44. Using functional assessments of NK cells from healthy donors against autologous HIV-infected CD4+ T cells, we observed that NKp30+ and Siglec-7+ cells had improved functional activity. Further, we found that NK cells from HESN women trended towards increased antibody-dependent cellular cytotoxicity (ADCC) activity; this activity correlated with increased CD16 expression. Overall, we identify features of NK cells in HESN women that may contribute to protection from HIV infection. Follow up studies with larger cohorts are warranted to confirm these findings.

Characterization of the Impact of Daclizumab Beta on Circulating Natural Killer Cells by Mass Cytometry.

Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56bright subpopulation of NK cells that is correlated with the drug's therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions.

TIGIT is upregulated by HIV-1 infection and marks a highly functional adaptive and mature subset of natural killer cells.

OBJECTIVE: Our objective was to investigate the mechanisms that govern natural killer (NK)-cell responses to HIV, with a focus on specific receptor--ligand interactions involved in HIV recognition by NK cells. DESIGN AND METHODS: We first performed a mass cytometry-based screen of NK-cell receptor expression patterns in healthy controls and HIV individuals. We then focused mechanistic studies on the expression and function of T cell immunoreceptor with Ig and ITIM domains (TIGIT). RESULTS: The mass cytometry screen revealed that TIGIT is upregulated on NK cells of untreated HIV women, but not in antiretroviral-treated women. TIGIT is an inhibitory receptor that is thought to mark exhausted NK cells; however, blocking TIGIT did not improve anti-HIV NK-cell responses. In fact, the TIGIT ligands CD112 and CD155 were not upregulated on CD4 T cells in vitro or in vivo, providing an explanation for the lack of benefit from TIGIT blockade. TIGIT expression marked a unique subset of NK cells that express significantly higher levels of NK-cell-activating receptors (DNAM-1, NTB-A, 2B4, CD2) and exhibit a mature/adaptive phenotype (CD57, NKG2C, LILRB1, FcRγ, Syk). Furthermore, TIGIT NK cells had increased responses to mock-infected and HIV-infected autologous CD4 T cells, and to PMA/ionomycin, cytokine stimulation and the K562 cancer cell line. CONCLUSION: TIGIT expression is increased on NK cells from untreated HIV individuals. Although TIGIT does not participate directly to the response to HIV-infected cells, it marks a population of mature/adaptive NK cells with increased functional responses.

Human natural killer cells mediate adaptive immunity to viral antigens.

Adaptive immune responses are defined as antigen sensitization-dependent and antigen-specific responses leading to establishment of long-lived immunological memory. Although natural killer (NK) cells have traditionally been considered cells of the innate immune system, mounting evidence in mice and nonhuman primates warrants reconsideration of the existing paradigm that B and T cells are the sole mediators of adaptive immunity. However, it is currently unknown whether human NK cells can exhibit adaptive immune responses. We therefore tested whether human NK cells mediate adaptive immunity to virally encoded antigens using humanized mice and human volunteers. We found that human NK cells displayed vaccination-dependent, antigen-specific recall responses in vitro, when isolated from livers of humanized mice previously vaccinated with HIV-encoded envelope protein. Furthermore, we discovered that large numbers of cytotoxic NK cells with a tissue-resident phenotype were recruited to sites of varicella-zoster virus (VZV) skin test antigen challenge in VZV-experienced human volunteers. These NK-mediated recall responses in humans occurred decades after initial VZV exposure, demonstrating that NK memory in humans is long-lived. Our data demonstrate that human NK cells exhibit adaptive immune responses upon vaccination or infection. The existence of human memory NK cells may allow for the development of vaccination-based approaches capable of establishing potent NK-mediated memory functions contributing to host protection.

A novel human IL2RB mutation results in T and NK cell-driven immune dysregulation.

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rß, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rß, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rß surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rß-/- animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rß expression and signaling in T and NK cells.

Mass Cytometry Analytical Approaches Reveal Cytokine-Induced Changes in Natural Killer Cells.

BACKGROUND: Natural killer (NK) cells have antiviral and antitumor activity that could be harnessed for the treatment of infections and malignancies. To maintain cell viability and enhance antiviral and antitumor effects, NK cells are frequently treated with cytokines. Here they performed an extensive assessment of the effects of cytokines on the phenotype and function of human NK cells. METHODS: They used cytometry by time-of-flight (CyTOF) to evaluate NK cell repertoire changes after stimulation with interleukin (IL)-2, IL-15 or a combination of IL-12/IL-15/IL-18. To analyze the high dimensional CyTOF data, they used several statistical and visualization tools, including viSNE (Visualization of t-Distributed Stochastic Neighbor Embedding), Citrus (Cluster identification, characterization, and regression), correspondence analysis, and the Friedman-Rafsky test. RESULTS: All three treatments (IL-2, IL-15, and IL-12/IL-15/IL-18) increase expression of CD56 and CD69. The effects of treatment with IL-2 and IL-15 are nearly indistinguishable and characterized principally by increased expression of surface markers including CD56, NKp30, NKp44, and increased expression of functional markers, such as perforin, granzyme B, and MIP-1ß. The combination of IL-12/IL-15/IL-18 induces a profound shift in the repertoire structure, decreasing expression of CD16, CD57, CD8, NKp30, NKp46, and NKG2D, and dramatically increasing expression of IFN-γ. CONCLUSIONS: CyTOF provides insights into the effects of cytokines on the phenotype and function of NK cells, which could inform future research efforts and approaches to NK cell immunotherapy. There are several analytical approaches to CyTOF data, and the appropriate method should be carefully selected based on which aspect of the dataset is being explored. © 2016 International Clinical Cytometry Society.

Polymorphism in apoptotic BAX (-248G>A) gene but not in anti-apoptotic BCL2 (-938C>A) gene and its protein and mRNA expression are associated with cervical intraepithelial neoplasia.

HPV is associated with cervical cancer and plays a crucial role in tumor formation. Apoptosis is regulated by different pathways involving genes that either promote (BCL2 gene) or inhibit (BAX gene) cell death. Our goal was to determine whether the BCL2-938C>A (rs2279115) and BAX-248G>A (rs4645878) single nucleotide polymorphisms (SNPs) are associated with squamous intraepithelial neoplasia (SIL) risk, and whether their phenotypic expression was impaired in these lesions. Two hundred and thirty-one cases showing SIL were classified as low SIL (LSIL, n = 101) or high SIL (HSIL, n = 130), and control subjects (n = 266) with no gynecologically proven SIL were recruited. No statistical difference in the genotype and allelic frequency of the BCL-2-938C>A polymorphism was observed among the groups. BCL2-938C/A and A/A homozygotes carriers had higher distribution of BCL-2-expressing cells in stroma in the SIL group. BCL2 mRNA-expression was not correlated with BCL2-938C>A SNPs in both groups. We did find a strong association of the BAX GG genotype and risk for SIL. No difference was observed between LSIL and HSIL groups. In BAX-248G/A and A/A homozygote carriers, the number of BAX-expressing cells was lower the epithelium area in SIL. However, mRNA expression was higher in SIL patients than in the control group. In conclusion, our data provide evidence that allele G carriers in the BAX-248G>A promoter SNP may influence the development of SIL. However, this genotype does not influence the SIL outcome. Additionally, we suggest a possible role of HPV infection in the inhibition of the expression of BAX protein, decreasing cell death, and favoring cervical carcinogenesis.

Serum levels of cytokines and biomarkers for inflammation and immune activation, and HIV-associated non-Hodgkin B-cell lymphoma risk.

BACKGROUND: HIV infection is associated with a marked increase in risk for non-Hodgkin lymphoma (AIDS-NHL). However, the mechanisms that promote the development of AIDS-NHL are not fully understood. METHODS: In this study, serum levels of several cytokines and other molecules associated with immune activation were measured in specimens collected longitudinally during 1 to 5 years preceding AIDS-NHL diagnosis, in 176 AIDS-NHL cases and 176 HIV(+) controls from the Multicenter AIDS Cohort Study (MACS). RESULTS: Multivariate analyses revealed that serum levels of immunoglobulin free light chains (FLC), interleukin (IL)-6, IL-10, IP-10/CXCL10, neopterin, and TNF-α were elevated in those HIV(+) individuals who went on to develop AIDS-NHL. In addition, the fraction of specimens with detectable IL-2 was increased and the fraction with detectable IL-4 was decreased in these subjects. CONCLUSIONS: These results suggest that long-term, chronic immune activation, possibly driven by macrophage-produced cytokines, precedes development of NHL in HIV(+) individuals. IMPACT: FLC, IL-6, IL-10, IP-10/CXCL10, neopterin, and TNF-α may serve as biomarkers for AIDS-NHL. .

Serum levels of the chemokine CXCL13, genetic variation in CXCL13 and its receptor CXCR5, and HIV-associated non-hodgkin B-cell lymphoma risk.

BACKGROUND: CXCL13 and CXCR5 are a chemokine and receptor pair whose interaction is critical for naïve B-cell trafficking and activation within germinal centers. We sought to determine whether CXCL13 levels are elevated before HIV-associated non-Hodgkin B-cell lymphoma (AIDS-NHL), and whether polymorphisms in CXCL13 or CXCR5 are associated with AIDS-NHL risk and CXCL13 levels in a large cohort of HIV-infected men. METHODS: CXCL13 levels were measured in sera from 179 AIDS-NHL cases and 179 controls at three time-points. TagSNPs in CXCL13 (n = 16) and CXCR5 (n = 11) were genotyped in 183 AIDS-NHL cases and 533 controls. OR and 95% confidence intervals (CI) for the associations between one unit increase in log CXCL13 levels and AIDS-NHL, as well as tagSNP genotypes and AIDS-NHL, were computed using logistic regression. Mixed linear regression was used to estimate mean ratios (MR) for the association between tagSNPs and CXCL13 levels. RESULTS: CXCL13 levels were elevated for more than 3 years (OR = 3.24; 95% CI = 1.90-5.54), 1 to 3 years (OR = 3.39; 95% CI = 1.94-5.94), and 0 to 1 year (OR = 3.94; 95% CI = 1.98-7.81) before an AIDS-NHL diagnosis. The minor allele of CXCL13 rs355689 was associated with reduced AIDS-NHL risk (OR(TCvsTT) = 0.65; 95% CI = 0.45-0.96) and reduced CXCL13 levels (MR(CCvsTT) = 0.82; 95% CI = 0.68-0.99). The minor allele of CXCR5 rs630923 was associated with increased CXCL13 levels (MR(AAvsTT) = 2.40; 95% CI = 1.43-4.50). CONCLUSIONS: CXCL13 levels were elevated preceding an AIDS-NHL diagnosis, genetic variation in CXCL13 may contribute to AIDS-NHL risk, and CXCL13 levels may be associated with genetic variation in CXCL13 and CXCR5. IMPACT: CXCL13 may serve as a biomarker for early AIDS-NHL detection.

Assessment of pre-diagnosis biomarkers of immune activation and inflammation: insights on the etiology of lymphoma.

The DNA-modifying processes that are involved in B lymphocyte activation, somatic hypermutation (SHM), and IgH class switch recombination (CSR) have the potential to lead to genetic errors that lead to the genesis of B cell cancers, such as lymphoma. Given the potential contribution of these immune mechanisms to the development of cancer, assessment of the expression of cytokines, and other immune stimulatory molecules that drive B cell activation, prior to lymphoma diagnosis, may provide insights into the etiology of these cancers. Here, we review studies that have examined prediagnosis protein biomarkers for non-Hodgkin lymphoma (NHL), both AIDS-related NHL, as well as NHL seen in immunocompetent populations. Overall, these studies provide support for the notion that B cell hyper-activation is elevated preceding the appearance of AIDS-NHL, particularly those forms of AIDS-NHL that are not driven by EBV infection and that presumably arise from errors in IgH CSR and SHM. In more limited studies, it appears that dysregulation of cytokine production also precedes the diagnosis of NHL in HIV-negative persons. The availability of prediagnosis serum/plasma from cohort studies provides unique opportunities for proteomic approaches to identify novel prediagnosis etiologic biomarkers for NHL.

HIV-associated immune dysfunction and viral infection: role in the pathogenesis of AIDS-related lymphoma.

HIV infection is associated with a much higher risk for the development of non-Hodgkin lymphoma (AIDS-NHL). The principal causes of lymphomagenesis in HIV-infected individuals are thought to be the loss of immune function seen in HIV infection, which results in the loss of immunoregulation of Epstein-Barr virus-infected B cells, as well as HIV infection-associated immune dysregulation, including chronic B-cell activation. In this review, we discuss recent reports that further support the importance of these factors, and we highlight emerging evidence of different mechanisms that potentially drive lymphomagenesis in HIV-infected individuals.

Lack of expression of inhibitory KIR3DL1 receptor in patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes.

BACKGROUND: Natural killer cell-type lymphoproliferative disease of granular lymphocytes is a disorder characterized by chronic proliferation of CD3(-)CD16(+) granular lymphocytes. By flow cytometry analysis, we previously demonstrated a dysregulation in killer immunoglobulin-like receptor (KIR) expression in natural killer cells from patients with this lymphoproliferative disease, the activating KIR receptors being mostly expressed. We also found that patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes usually had KIR genotypes characterized by multiple activating KIR genes. DESIGN AND METHODS: We investigated the mRNA levels of the KIR3DL1 inhibitory and the related KIR3DS1 activating receptors in 15 patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes and in ten controls. These genes are usually expressed when present in the genome of the Caucasian population. RESULTS: We demonstrated the complete lack of KIR3DL1 expression in most of the patients analyzed, with the receptor being expressed in 13% of patients compared to in 90% of controls (P<0.01). Interestingly, studies of the methylation patterns of KIR3DL1 promoter showed a significantly higher methylation status (0.76 ± 0.12 SD) in patients than in healthy subjects (0.49±0.10 SD, P<0.01). The levels of expression of DNA methyl transferases, which are the enzymes responsible for DNA methylation, did not differ between patients and controls. CONCLUSIONS: In this study we showed, for the first time, a consistent down-regulation of the inhibitory KIR3DL1 signal due to marked methylation of its promoter, thus suggesting that together with the increased expression of activating receptors, the lack of the inhibitory signal could also play a role in the pathogenesis of natural killer cell-type lymphoproliferative disease of granular lymphocytes.