Non-survivors produce less EBV-specific IFN-γ, and from difference T cell subsets.

<p>Among IFN-γ producers, there was no difference in the magnitude of CD4⁺ T cell IFN-γ production in response to EBNA-1 or PfSEA-1 stimulation between eBL survivor and non survivors (A), non-survivors produced less CD8⁺ T cell IFN-γ from EBNA-1 stimulated cells (B). Among EBNA-1-specific CD8⁺ T cell IFN-γ producers, survivors produced more CD8⁺ T cell IFN-γ from the CD45RA^-CCR7^- effector memory (T_EM) cell subset than non-survivors (C). Among PfSEA-1-specific CD8⁺ T cell IFN-γ producers, there was no difference between survivors and non-survivors in the cell type that produced IFN-γ (D). Among IFN-γ T cell responders in both survivors and non-survivors, CD25⁺Foxp3⁺ Treg frequency negatively correlated with CD8⁺ T cell EBNA-1 IFN-γ production (E).</p

Regulatory T Cells in Endemic Burkitt Lymphoma Patients Are Associated with Poor Outcomes: A Prospective, Longitudinal Study

<div><p>Deficiencies in Epstein-Barr virus (EBV)-specific T cell immunosurveillance appear to precede the development of endemic Burkitt lymphoma (eBL), a malaria-associated pediatric cancer common in sub-Saharan Africa. However, T cell contributions to eBL disease progression and survival have not been characterized. Our objective was to investigate regulatory and inflammatory T cell responses in eBL patients associated with clinical outcomes. By multi-parameter flow cytometry, we examined peripheral blood mononuclear cells from 38 eBL patients enrolled in a prospective cohort study in Kisumu, Kenya from 2008–2010, and 14 healthy age-matched Kenyan controls. Children diagnosed with eBL were prospectively followed and outcomes categorized as 2-year event-free survivors, cases of relapses, or those who died. At the time of diagnosis, eBL children with higher CD25⁺Foxp3⁺ regulatory T (Treg) cell frequencies were less likely to survive than patients with lower Treg frequencies (p = 0·0194). Non-survivors also had higher absolute counts of CD45RA⁺Foxp3^lo naïve and CD45RA^-Foxp3^hi effector Treg subsets compared to survivors and healthy controls. Once patients went into clinical remission, Treg frequencies remained low in event-free survivors. Patients who relapsed, however, showed elevated Treg frequencies months prior to their adverse event. Neither concurrent peripheral blood EBV load nor malaria infection could explain higher Treg cell frequencies. CD8⁺ T cell PD-1 expression was elevated in all eBL patients at time of diagnosis, but relapse patients tended to have persistently high PD-1 expression compared to long-term survivors. Non-survivors produced more CD4⁺ T-cell IL-10 in response to both Epstein-Barr Nuclear Antigen-1 (EBNA-1) (p = 0·026) and the malaria antigen <i>Plasmodium falciparum</i> Schizont Egress Antigen-1 (p = 0·0158) compared to survivors, and were concurrently deficient in (EBNA-1)-specific CD8⁺ T-cell derived IFN-γ production (p = 0·002). In addition, we identified the presence of Foxp3^-IL10⁺ regulatory Type 1 cells responding to EBNA-1 in contrast to the malaria antigen tested. These novel findings suggest that poor outcomes in eBL patients are associated with a predominantly immuno-regulatory environment. Therefore, Treg frequencies could be a predictive biomarker of disease progression and manipulation of Treg activity has potential as a therapeutic target to improve eBL survival.</p></div

T cells in eBL patients have higher PD-1 expression than in healthy controls.

<p>Gating strategy for the identification of PD-1⁺ cells (A). Frequency of CD4⁺ and CD8⁺PD-1⁺ cells at the time of diagnosis in eBL non-survivors, eBL survivors and healthy controls (B and C, respectively). In general PD1 expression on CD4 and CD8 T cells was higher in eBL non-survivors compared to age-matched malaria exposed yet health controls. eBL survivors had intermediate levels of PD1 expressed on their CD4 T cells yet levels as high as non-survivors for their CD8 T cells. Patients who relapsed tended to have peaks of elevated CD8⁺PD-1⁺ frequencies over time (D). Designated with a red ♦ = BL001 (relapse), designated with a red ● = BL002 (relapse), designated with a red ■ = BL003 (relapse), designated with a blue ▲ = BL004 (event-free survivor), designated with a blue ▼ = BL005 (event-free survivor) designated with a blue ■ = BL006 (event-free survivor). FMO; Fluorescence Minus One.</p

Elevated Treg frequencies in patients prior to relapse.

<p>Frequencies of CD25⁺Foxp3⁺ Treg cells (A) and EBV viral load (B) at monthly follow-up time points in 3 event-free survivors and 3 patients who relapsed. Proposed Treg frequency threshold indicative of future relapse was established by measuring Treg frequencies in 8 other event-free survivors 4–9 months post discharge, and frequencies never rose above 0.5% (dotted line). Designated as a red ♦ = BL001 (relapse), designated as a red ● = BL002 (relapse), designated as a red ■ = BL003 (relapse), designated as a blue ▲ = BL004 (event-free survivor), designated as a blue ▼ = BL005 (event-free survivor) designated as a blue ■ = BL006 (event-free survivor).</p

CD25⁺Foxp3⁺ Treg cells are associated with poor outcome.

<p>Identification of CD25⁺Foxp3⁺ Treg cells by flow cytometry (A). At the time of diagnosis, frequencies and (B) absolute cell counts (C) of CD25⁺Foxp3⁺ cells are higher in eBL patients who died than in survivors and healthy controls.</p

Higher numbers of both CD45RA⁺Foxp3^lo naïve Treg (nTreg cells) and CD45RA^-Foxp3^hi effector Treg (eTreg) cells are associated with poor outcome.

<p>(A) Gating strategy differentiating nTreg and eTreg cells from CD45RA^-Foxp3^lo non-Treg cells. At the time of diagnosis, eBL non-survivors have higher absolute cell counts of both nTreg (B) and eTreg (C) cell subtypes compared to eBL survivors and healthy controls.</p

EBV persistence without its EBNA3A and 3C oncogenes in vivo

<div><p>The oncogenic Epstein Barr virus (EBV) infects the majority of the human population and usually persists within its host for life without symptoms. The EBV oncoproteins nuclear antigen 3A (EBNA3A) and 3C (EBNA3C) are required for B cell transformation in vitro and are expressed in EBV associated immunoblastic lymphomas in vivo. In order to address the necessity of EBNA3A and EBNA3C for persistent EBV infection in vivo, we infected NOD-<i>scid</i> γ_c^null mice with reconstituted human immune system components (huNSG mice) with recombinant EBV mutants devoid of EBNA3A or EBNA3C expression. These EBV mutants established latent infection in secondary lymphoid organs of infected huNSG mice for at least 3 months, but did not cause tumor formation. Low level viral persistence in the absence of EBNA3A or EBNA3C seemed to be supported primarily by proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression without elevated lytic replication. In vitro, EBNA3A and EBNA3C deficient EBV infected B cells could be rescued from apoptosis through CD40 stimulation, mimicking T cell help in secondary lymphoid tissues. Thus, even in the absence of the oncogenes EBNA3A and 3C, EBV can access a latent gene expression pattern that is reminiscent of EBV persistence in healthy virus carriers without prior expression of its whole growth transforming program.</p></div

EBV infected cells proliferate without EBNA3A or EBNA3C in vivo.

<p><b>(A)</b> Representative immunofluorescence staining for EBNA2 (red), Ki67 (green) and DAPI (blue) (original magnification, 200x) in splenic sections of huNSG mice infected with wt, 3AKO or 3CKO 5 or 6 weeks p.i.. <b>(B)</b> Quantification of <b>A</b> with the frequency of Ki67⁺ EBNA2⁺ cells of all EBNA2⁺ cells (n = 4-15/group). Pooled data from 2 experiments with mean ± SEM, Mann-Whitney U test.</p

EBV persists without EBNA3A or EBNA3C in vivo.

<p><b>(A)</b> Splenic endpoint viral DNA load and <b>(B)</b> viral DNA load per gram lymph node tissue determined by qPCR of huNSG mice infected with either 10⁵ RIU of wt, 3AKO or 3CKO for 5 weeks (spleen: n = 18-21/group, lymph node: n = 8-11/group) or 10⁶ RIU of 3AKO or 3CKO for 6 weeks (spleen: n = 13-14/group, lymph node: n = 12-13/group). Values for mice in which no viral DNA was detected are plotted on the X-axis. <b>(C)</b> Blood DNA viral load over time determined by qPCR of huNSG mice infected with either 10⁵ RIU of wt, 3AKO or 3CKO 5 weeks p.i. (n = 18-21/group) or 10⁶ RIU of 3AKO or 3CKO 6 weeks p.i. (n = 13-14/group). Horizontal dashed line indicates the viral load of 3 times the lower limit of quantification (LLOQ). Horizontal dotted line indicates the LLOQ. <b>(D)</b> Frequency of huNSG mice infected with 10⁵ RIU of 3AKO, 3CKO or wt 5 weeks p.i. or 10⁶ RIU of 3AKO or 3CKO 6 weeks p.i. with EBV DNA copies above (≥200) or below (<200) 3 times the LLOQ in either the blood, the spleen or lymph nodes (n = 10-13/group) as determined by qPCR. Pooled data from 4 low and 2 high infectious dose experiments. *P < 0.05, **P < 0.01, Fisher’s exact test. <b>(E)</b> EBER-ISH (original magnification, 400x) of splenic sections from huNSG mice infected with 10⁶ RIU of 3AKO or 3CKO 6 weeks p.i.. <b>(G)</b> EBER-ISH (original magnification, 200x) of splenic sections from huNSG mice infected with 10⁵ RIU of 3AKO or 3CKO 12 weeks p.i.. <b>(F, H)</b> Quantification of EBER⁺ cells/mm² of <b>E</b> (n = 10-13/group) and <b>G</b> (n = 3-5/group) respectively and of huNSG mice infected with 10⁵ RIU of wt 5 weeks p.i. (n = 4/group) or mock (n = 3-8/group). <b>(I)</b> Frequency of huNSG mice infected with 10⁵ RIU of 3AKO or 3CKO 12 weeks p.i., with EBV DNA copies above (≥200) or below (<200) 3 times the LLOQ in either the blood, the spleen or lymph nodes (n = 3-5/group) as determined by qPCR. Pooled data from 2 experiments. <b>(A-C)</b> Pooled data from 4 low and 2 high infectious dose experiments are displayed with mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni correction for blood viral load and Mann-Whitney U test for splenic viral load, lymph node viral load and EBER-ISH.</p

EBNA3A or EBNA3C deficient EBV infection causes CD8⁺ T cell expansion.

<p><b>(A)</b> The number of splenic CD8⁺ T cells and <b>(B)</b> splenic CD4⁺ T cells of huNSG mice infected with either 10⁵ RIU of wt, 3AKO or 3CKO 5 weeks p.i. (n = 14-21/group) or 10⁶ RIU of 3AKO or 3CKO 6 weeks p.i. (n = 7-13/group) or non-infected control (mock) huNSG mice was determined by flow cytometry, applying the determined frequency to the spleen cell count. <b>(C)</b> The ratio of spleen to body weight (BW) of individual huNSG mice infected with either 10⁵ RIU of wt, 3AKO or 3CKO 5 weeks p.i. (n = 14-21/group) or 10⁶ RIU of 3AKO or 3CKO 6 weeks p.i. (n = 7-13/group) or non-infected control (mock) huNSG mice is depicted. <b>(A-C)</b> Pooled data from 4 low and 2 high infectious dose experiments with mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U test.</p