D. van Baarle

Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasia

Oncogenic human papillomavirus (HPV)‐infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (Tregs) might be involved in the failure of the immune system to control the development of HPV‐induced cancer. We investigated frequencies, phenotype and activity of Tregs in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4+/CD25high T cell frequencies in peripheral blood and CD4+ T cell fraction. These CD4+/CD25high T cells represent Tregs as demonstrated by their low proliferation rate, low interferon (IFN)‐γ/interleukin (IL)‐10 ratio, high expression of CD45RO, GITR, CTLA‐4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16+ cervical cancer patients, in‐vitro depletion of CD25+ T cells resulted in increased IFN‐γ T cell responses against HPV16 E6‐ and E7 peptides. Thus, increased frequencies of Tregs in cervical cancer patients may indeed suppress HPV‐specific immunity. Longitudinal analysis of CD4+/CD25high T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of Tregs in cervical cancer. These results imply that Tregs may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by Tregs will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia.

Role of incidental and/or cured intestinal parasitic infections on profile of CD4+ and CD8+ T cell subsets and activation status in HIV-1 infected and uninfected adult Ethiopians

Intestinal parasitic infections have been suggested to cause persistent immune activation leading to an unbalanced immune state. Such a state has been proposed to be a major factor in the pathogenesis of AIDS in an African context. The present study investigated the effect of incidental parasitic infection and treatment on the profile of T cell differentiation and activation markers on CD4+ and CD8+ T cells from HIV‐1 infected and uninfected adult Ethiopians. Cryopreserved PBMCs from 64 subjects (41 HIV‐negative and 23 HIV‐positive) with follow‐up visits at 6‐monthly intervals were used to compare the effect of incidental intestinal parasites and their treatment upon T cell subset profiles and activation status. The samples were stained with antibodies to various T cell differentiation and activation markers allowing naive, memory, effector, memory/effector, activated and resting CD4+ and CD8+ T cell subsets to be quantified by triple‐colour FACScan. Incidental intestinal parasitic infections resulted in a significant increase in memory CD4+ T cell numbers both in HIV‐negative and HIV‐positive subjects (P < 0·05). There was also a significant increase in the percentage of CD8+ HLA‐DR+ T cells (P < 0·05) in HIV‐positive subjects co‐infected with parasites. In HIV‐negative subjects, a significant decline in activated cells and a significant increase in resting CD8+ T cells (P < 0·05) was observed after treatment for parasites. These data suggest that intestinal parasitic infections could result in the alteration of T cell subset counts and also in the up‐regulation of T cell activation markers in

Detailed analysis of Epstein–Barr virus‐specific CD4+ and CD8+ T cell responses during infectious mononucleosis

We studied simultaneously Epstein–Barr virus (EBV)‐specific CD4+ and CD8+ T cell responses during and after infectious mononucleosis (IM), using a previously described 12‐day stimulation protocol with EBNA1 or BZLF1 peptide pools. Effector function of EBV‐specific T cells was determined after restimulation by measuring intracellular interferon‐γ production. During IM, BZLF1‐specifc CD4+ T cell responses were dominant compared with CD8+ T cell responses. EBNA1‐specific CD4+ and CD8+ T cell responses were low and remained similar for 6 months. However, 6 months after IM, BZLF1‐specific CD4+ T cell responses had declined, but CD8+ T cell responses had increased. At diagnosis, EBV‐specific CD8+ T cells as studied by human leucocyte antigen class I tetramer staining comprised a tetramerbrightCD8bright population consisting mainly of CD27+ memory T cells and a tetramerdimCD8dim population consisting primarily of CD27‐ effector T cells. The remaining EBV‐specific CD8+ T cell population 6 months after the diagnosis of IM consisted mainly of tetramerbrightCD8bright CD27+ T cells, suggesting preferential preservation of memory T cells after contraction of the EBV‐specific T cell pool.

HCV-specific T-cell responses in injecting drug users : evidence for previous exposure to HCV and a role for CD4+ T cells focussing on nonstructural proteins in viral clearance

Summary.  In order to understand the parameters associated with resolved hepatitis C virus (HCV)‐infection, we analysed the HCV‐specific T‐cell responses longitudinally in 13 injecting drug‐users (IDUs) with a prospectively identified acute HCV infection. Seven IDUs cleared HCV and six IDUs remained chronically infected. T‐cell responses were followed in the period needed to resolve and a comparable time span in chronic carriers. Ex vivo T‐cell responses were measured using interferon‐γ Elispot assays after stimulation with overlapping peptide pools spanning the complete HCV genome. CD4+ memory‐T‐cell responses were determined after 12‐day stimulation with HCV proteins. The maximum response was compared between individuals. The T‐cell responses measured directly ex vivo were weak but significantly higher in resolvers compared to chronic carriers, whereas the CD4+ memory‐T‐cell response was not different between resolvers and chronic carriers. However, HCV Core protein was targeted more often in chronic carriers compared to individuals resolving HCV infection. CD4+ T‐cell responses predominantly targeting nonstructural proteins were associated with resolved HCV infection. Interestingly, observation of memory‐T‐cell responses present before the documented HCV‐seroconversion suggests that reinfections in IDUs occur often. The presence of these responses however, were not predictive for the outcome of infection. However, a transition of the HCV‐specific CD4+ memory‐T‐cell response from targeting Core to targeting nonstructural proteins during onset of infection was associated with a favourable outcome. Therefore, the specificity of the CD4+ memory‐T‐cell responses measured after 12‐day expansion seems most predictive of resolved infection.

Human herpes virus 6 reactivation: important predictor for poor outcome after myeloablative, but not non-myeloablative allo-SCT

Hematopoietic SCT (HSCT) is often complicated by viral reactivations. In this retrospective cohort study (January 2004–August 2008), predictors for human herpes virus 6 (HHV6)-reactivation and associations between HHV6-reactivation and clinical outcomes after allogeneic HSCT were studied. HHV6 DNA load in plasma was monitored weekly by quantitative real-time PCR. Associations between the main end point HHV6-reactivation and other end points, that is, acute GVHD (aGVHD) and NRM were analyzed using Cox proportional hazard models. In total, 108 patients receiving either a myeloablative (MA; n=60) or non-myeloablative (NMA; n=48) conditioning regimen were included. Median age was 40 years (range 17–65); median follow-up was 20 months (range 3–36). In 16/60 (27%) patients with MA conditioning regimen, a HHV6 reactivation was observed (mean viral load 50 323 cp/mL) compared with 2/48 (4%) patients with a NMA conditioning regimen with low viral load (mean 1100 cp/mL). In multivariate analysis, MA conditioning was the only predictor for HHV6 reactivation (P=0.02). In addition, HHV6 reactivation was associated with grades 2–4 aGVHD (P<0.001) and NRM (P=0.03). Regular monitoring of HHV6 reactivation after HSCT might be important in MA HSCT patients to enable early initiation of antiviral treatment or to anticipate aGVHD, all of which may improve clinical outcome.

First analysis of human herpesvirus 6T-cell responses: Specific boosting after HHV6 reactivation in stem cell transplantation recipients

Early human herpesvirus 6 (HHV6) reactivation after hematopoietic stem cell transplantation (HSCT) is associated with poor survival. We characterized HHV6 immuneresponses in HSCT patients during lymphopenia. Prospectively, HHV6 DNA-load was measured weekly by realtime-PCR. Numbers of IFNγ-producing HHV6-T-cells were retrospectively determined by enzyme-linked immunospot assay 2 months after HSCT. HHV6-specific T-cell proliferative capacity was analyzed with a newly developed assay using antigen-presenting autologous HHV6-infected PBMC. Fifty-six patients were included (median age 4.6 years; range 0.2-21.2 years). HHV6-reactivation occurred in 29/56 (52%) patients with a median time of 14 (range 1-41) days after HSCT. The median number of IFN-γ producing HHV6-specific T-cells at 2 months and the HHV6-specific CD8+ T-cell proliferative capacity at 6 months after HSCT was increased after HHV6-reactivation compared to non-reactivating patients (P=0.006 and p=0.019). In conclusion, HHV6-specific immuneresponses can be initiated during lymphopenia early after HSCT, which implicates a potential window for development of HHV6-specific (immuno)therapy.

Comprehensive longitudinal analysis of hepatitis C virus (HCV)-specific T cell responses during acute HCV infection in the presence of existing HIV-1 infection

Summary.  The aim of this study was to study the development of HCV‐specific T cell immunity during acute HCV infection in the presence of an existing HIV‐1 infection in four HIV‐1 infected men having sex with men. A comprehensive analysis of HCV‐specific T cell responses was performed at two time points during acute HCV infection using a T cell expansion assay with overlapping peptide pools spanning the entire HCV genome Three patients with (near) normal CD4+ T cell counts (range 400–970 × 106/L) either resolved (n = 1) or temporary suppressed HCV RNA. In contrast, one patient with low CD4+ T cell counts (330 × 106/L), had sustained high HCV RNA levels. All four patients had low HCV‐specific CD8+ T cell responses, and similar magnitudes of CD4+ T cell responses. Interestingly, individuals with resolved infection or temporary suppression of HCV‐RNA had HCV‐specific CD4+ T cell responses predominantly against nonstructural (NS) proteins. While the individual with high HCV RNA plasma concentrations had CD4+ T cell responses predominantly directed against Core. Our data show that an acute HCV infection in an HIV‐1 infected person can be suppressed in the presence of HCV‐specific CD4+ T cell response targeting non‐structural proteins. However further research is needed in a larger group of patients to evaluate the role of HIV‐1 on HCV‐specific T cell responses in relation to outcome of acute HCV infection.

T-cell response to viral antigens in adults and children with common variable immunodeficiency and specific antibody deficiency.

Several T cell abnormalities have been described in common variable immunodeficiency (CVID), a B cell disorder of mainly unknown origin. A subset of CVID patients suffers from frequent reactivations of herpes viruses. We studied T cell function in CVID [and in a subset of paediatric patients with specific antibody deficiency (SAD)] by measuring T cell proliferation and cytokine production in response to herpes virus‐antigens in paediatric CVID patients (n = 9) and paediatric SAD patients (n = 5), in adult CVID patients (n = 14) and in healthy controls. Paediatric CVID patients, but not SAD patients, displayed moderately increased CD8+ T cell proliferation in response to cytomegalovirus, human herpes virus type 6B (HHV6‐B) and herpes simplex virus compared to controls. CD8+ T cell responses in adult CVID patients tended to be increased in response to cytomegalovirus and herpes simplex virus. In response to stimulation with herpes virus antigens, the proinflammatory cytokines interleukin (IL)‐1β, IL‐6, tumour necrosis factor (TNF)‐α and interferon inducible protein (IP)‐10 were produced. Overall, no major differences were detected in cytokine production upon stimulation between patients and controls, although higher IL‐10 and IL‐12 production was detected in paediatric patients. In conclusion, cellular immunity against herpes virus antigens appears undisturbed in CVID patients, although defects in subpopulations of CVID patients cannot be excluded.

Plasma HCV-RNA decline in the first 48 h identifies hepatitis C virus mono-infected but not HCV/HIV-coinfected patients with an undetectable HCV viral load at week 4 of peginterferon-alfa-2a/ribavirin therapy

Summary.  During peginterferon‐alfa‐2a/ribavirin therapy, plasma hepatitis C virus (HCV)‐RNA decreases with a rapid first phase and a slower second phase. We compared the viral load decrease and slope in the first 48 h in patients with a rapid viral response (RVR, i.e. HCV‐RNA < 50 IU/mL at week 4) with patients not achieving an RVR. From 23 HCV‐infected (14 mono‐infected and nine HCV/HIV‐coinfected) genotype 1 or 4 positive peginterferon‐alfa‐2a/ribavirin‐treated patients, plasma HCV‐RNA was determined at baseline, 48 h, weeks 1, 2, 4, 8, 12, 48 and 72. The HCV viral load decrease (Δ0–48), the slope (λ1) and the efficiency factor (ε) were determined in the first 48 h after the start of therapy. Five (36%) HCV mono‐infected patients and three (33%) HIV/HCV‐coinfected patients achieved an RVR whereas six (43%) HCV mono‐infected patients and five (56%) HIV/HCV‐coinfected patients reached a sustained viral response (SVR). In contrast to HIV/HCV‐coinfected patients, five HCV mono‐infected patients with an RVR showed both a larger Δ0–48 and steeper λ1 (−1.77log10 IU/mL ± 0.66 and −2.04/day ± 0.76) compared to nine non‐RVR patients (−0.66log10 IU/mL ± 0.39; P = 0.019 and −0.76/day ± 0.41; P = 0.019). When divided by SVR, a greater Δ0–48 and steeper λ1 were also seen in both HCV mono‐infected and HIV/HCV‐coinfected patients. Thus, in the first 48 h after the start of therapy, HCV mono‐infected patients with an RVR have a larger viral load decrease, steeper viral slope and a higher efficiency factor as compared with non‐RVR patients.

High level of perforin expression in T cells: an early prognostic marker of the severity of herpesvirus reactivation after allogeneic stem cell transplantation in adults.

BACKGROUND Epstein-Barr virus (EBV) and cytomegalovirus reactivations are frequent complications of hematopoeitic allogeneic stem cell transplantation (SCT) because of a lack of T cell control after immunosuppression. Early diagnosis of reactivation and subsequent preemptive therapy relies on frequent viral load measurement. Additional virus-specific T cell reconstitution data could improve the predictive value of viral load detection for viral complications after transplantation. Here, we studied perforin expression in CD8(+) T cells as a measure of cytotoxic T cell capacity in relation to the occurrence of viral reactivation. METHODS In a prospective study, we monitored 40 patients during the first 3 months after transplantation and measured viral loads in combination with intracellular perforin expression in CD8(+) T cells. RESULTS Median perforin expression in CD8(+) T cells throughout follow-up was higher in patients with viral reactivations than in patients without viral reactivations (4.9% vs 2.3%; P = .001). The median percentage of perforin-expressing CD8(+) T cells in patients with high viral reactivations exceeding 1000 copies/mL (10.7%) was statistically significantly higher than that in patients with minor reactivations of 50-1000 copies (4.0%), that in patients with detectable EBV loads that did not exceed the detection limit of 50 copies/mL (2.9%), and that in patients without reactivations (0.8%). Patients with high viral reactivations reached a high percentage of perforin-expressing CD8(+) T cells (>10.2%) more often and faster than did patients with low viral loads (1000 copies/mL) or without viral reactivations. High perforin expression preceded high viral loads. CONCLUSION Perforin-expressing CD8(+) T cells may be useful as an easy-to-measure prognostic marker for identifying patients at risk for severe viral reactivation very soon after SCT.