Pokrath Hansasuta

Recognition of HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific.

The recognition of MHC class I molecules by killer cell immunoglobulin‐like receptors (KIR) is central to the control of NK cell function and can also modulate the CTL activation threshold. Among KIR receptors, KIR3DL2 is thought to interact with HLA‐A3 and ‐A11, although direct evidence has been lacking. In this study, we show that HLA‐A3 and ‐A11 tetramers specifically bind to KIR3DL2*001 transfectants and that this recognition is peptide‐specific. Single amino acid substitutions in the nonamer peptide underline a critical role for residue 8 in recognition of KIR3DL2. However, the role of this interaction in vivo still remains to be established.

Dynamics of T Cell Responses in HIV Infection

Cytotoxic CD8+ T cells play a major role in the immune response against viruses. However, the dynamics of CD8+ T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8+ T cells, in relation to Ag-specific CD4+ T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8+ T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.

Broadly cross-reactive HIV-specific cytotoxic T-lymphocytes in highly-exposed persistently seronegative donors.

HIV-specific cytotoxic T-lymphocytes (CTL) are believed to play a key part in the control of virus levels throughout HIV infection. An important goal of a potential prophylactic vaccine against HIV is therefore to elicit a strong CTL response which is broadly cross-reactive against a diverse range of HIV strains. We have detected HIV-specific CTL in two groups of highly-exposed but persistently seronegative female sex workers in Africa which show extensive cross-reactivity between different viral sequences. In a small group of women exposed to both HIV-1 and HIV-2 in Gambia, studied over 4 years, we have repeatedly detected HLA-B35-restricted CTL which exhibit cross-reactivity between the HIV-1 and HIV-2 sequences of the CTL epitopes. In women with particularly intense exposure to what are likely to be multiple clades of HIV-1 in Nairobi Kenya, we have detected CTL directed towards epitopes conserved between HIV-1 clades. In neither group is there any evidence that variation in CCR5 sequence or expression is responsible for their apparent resistance to HIV infection. However, in seropositive donors from Oxford infected with African strains of HIV-1, we have defined CTL responses which are specific for particular clades and have mapped some unique A clade CTL epitopes, together with others to highly-conserved regions of the virus. Further information about the extent of cross-reactive CTL immunity will be important for future vaccine design and evaluation.

How important is the 'quality' of the cytotoxic T lymphocyte (CTL) response in protection against HIV infection?

Cytotoxic T lymphocyte (CTL) responses have been associated with protection from HIV-1 infection in people with a high degree of exposure to HIV and who show no serological evidence of HIV infection (HEPS, highly exposed persistently seronegative). However, it remains unclear how protective CTL responses could apparently develop in a minority of people, whilst the great majority of HIV-infected people make strong CTL responses yet progress to AIDS and death. In this paper we review the data which supports the hypothesis that the quality of the T-cell response, rather than its magnitude, may be an important factor that merits further investigation.

Persistent HIV-1-specific cellular responses despite prolonged therapeutic viral suppression

Design Antiretroviral therapy (ART) currently represents the best way to avert the lethal consequences of chronic persistent HIV-1 infection. It leads to significant reductions of plasma viremia, often to undetectable levels, but it can also be linked with the reduction and disappearance of detectable HIV-specific CD8 T-cell responses. Results Here we describe a group of patients in whom ongoing replication of HIV, particularly transcription of Nef mRNA species, was detected despite prolonged and clinically successful antiretroviral treatment. Modest, but significant, numbers of HIV-specific CD8 T cells and CD4 T-cell responses were found in these subjects, with the strongest responses directed towards Nef epitopes. Detailed phenotypic analysis of the HIV-specific CD8 cells demonstrated low perforin levels and persistent expression of CD27, a phenotype associated with incomplete differentiation of cytotoxic T lymphocytes (CTL). Conclusion This immature CTL phenotype has been described previously in association with chronic HIV disease, but its continued persistence is surprising in the setting of prolonged viral suppression on therapy and the presence of HIV-specific CD4 cell activity.

A Novel Immunodominant CD8+ T Cell Response Restricted by a Common HLA-C Allele Targets a Conserved Region of Gag HIV-1 Clade CRF01_AE Infected Thais

Background CD8+ T cell responses play an important role in the control of HIV-1. The extensive sequence diversity of HIV-1 represents a critical hurdle to developing an effective HIV-1 vaccine, and it is likely that regional-specific vaccine strains will be required to overcome the diversity of the different HIV-1 clades distributed world-wide. Unfortunately, little is known about the CD8+ T cell responses against CRF01_AE, which is responsible for the majority of infections in Southeast Asia. Methodology/Principal Findings To identify dominant CD8+ T cell responses recognized in HIV-1 clade CRF01_AE infected subjects we drew upon data from an immunological screen of 100 HIV-1 clade CRF01_AE infected subjects using IFN-gamma ELISpot to characterize a novel immunodominant CD8+ T cell response in HIV-1 Gag restricted by HLA-Cw*0102 (p24, 277YSPVSILDI285, YI9). Over 75% of Cw*0102+ve subjects targeted this epitope, representing the strongest response in more than a third of these individuals. This novel CD8 epitope was located in a highly conserved region of HIV-1 Gag known to contain immunodominant CD8 epitopes, which are restricted by HLA-B*57 and -B*27 in clade B infection. Nonetheless, viral escape in this epitope was frequently observed in Cw*0102+ve subjects, suggestive of strong selection pressure being exerted by this common CD8+ T cell response. Conclusions/Significance As HLA-Cw*0102 is frequently expressed in the Thai population (allelic frequency of 16.8%), this immunodominant Cw*0102-restricted Gag epitope may represent an attractive candidate for vaccines specific to CRF01_AE and may help facilitate further studies of immunopathogenesis in this understudied HIV-1 clade.

Delayed differentiation of potent effector CD8+ T cells reducing viremia and reservoir seeding in acute HIV infection

Potent CD8+ T cells endowed with effector functions able to kill HIV-producing cells and reduce the seeding of the HIV reservoir are only detected at peak viremia in acute HIV infection. Peak HIV viremia pushes CD8+ T cells Aside from CD4+ T cell death, the immune system in chronically infected HIV patients is dysfunctional, including the inability of CD8+ T cells to control the virus. Animal studies with simian immunodeficiency virus have suggested that early CD8+ T cell responses may be capable of reducing viral burden, but getting access to patient samples at the earliest stage of infection is challenging. Takata et al. examined a large cohort of acutely infected patients that were given antiretroviral therapy (ART) upon enrollment in the study to evaluate T cell activation and HIV viral load over time, allowing them to parse out immune function (or dysfunction) based on acute stages of infection. They saw that CD8+ T cell responses were a little slow to ramp up but that activated CD8+ T cells present after initiation of ART could reduce the magnitude of the viral reservoir. These findings confirm that targeting CD8+ T cells at the early stage of infection could lead to viral eradication. CD8+ T cells play a critical role in controlling HIV viremia and could be important in reducing HIV-infected cells in approaches to eradicate HIV. The simian immunodeficiency virus model provided the proof of concept for a CD8+ T cell–mediated reservoir clearance but showed conflicting evidence on the role of these cells to eliminate HIV-infected cells. In humans, HIV-specific CD8+ T cell responses have not been associated with a reduction of the HIV-infected cell pool in vivo. We studied HIV-specific CD8+ T cells in the RV254 cohort of individuals initiating ART in the earliest stages of acute HIV infection (AHI). We showed that the HIV-specific CD8+ T cells generated as early as AHI stages 1 and 2 before peak viremia are delayed in expanding and acquiring effector functions but are endowed with higher memory potential. In contrast, the fully differentiated HIV-specific CD8+ T cells at peak viremia in AHI stage 3 were more prone to apoptosis but were associated with a steeper viral load decrease after ART initiation. Their capacity to persist in vivo after ART initiation correlated with a lower HIV DNA reservoir. These findings demonstrate that HIV-specific CD8+ T cell magnitude and differentiation are delayed in the earliest stages of infection. These results also demonstrate that potent HIV-specific CD8+ T cells contribute to the reduction of the pool of HIV-producing cells and the HIV reservoir seeding in vivo and provide the rationale to design interventions aiming at inducing these potent responses to cure HIV infection.

Prevalence and significance of plasma Epstein-Barr Virus DNA level in nasopharyngeal carcinoma

Abstract Epstein-Barr virus (EBV) DNA has been recognized as a promising tumor marker for nasopharyngeal carcinoma (NPC). This study aims to demonstrate the prevalence of plasma EBV DNA and its temporal correlation with treatment outcomes in the modern era. A total of 204 patients with Stage I–IVB NPC treated with intensity-modulated radiotherapy (IMRT) were enrolled. Quantitative plasma EBV DNA measurement was performed before treatment (pre-IMRT), on the fifth week of radiation (mid-IMRT), at 3 months after radiation (post-IMRT), then every 6 months until disease relapse. Progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan–Meier method. Plasma EBV DNA was detected in 110 patients (53.9%), with a median pre-IMRT EBV DNA level of 8005 copies/ml. Significant correlation was noted between pre-IMRT EBV DNA level and disease stage, but not between pre-IMRT EBV DNA level and World Health Organization classification. With a median follow-up time of 35.1 months, the 3-year PFS and OS rates were higher in the group with undetectable pre-IMRT EBV DNA level compared with in the group in which it was detectable. When classified according to disease stage and pre-IMRT EBV DNA, patients with early disease and detectable pre-IMRT EBV DNA experienced poorer survival than those with locally advanced disease and undetectable pre-IMRT EBV DNA. According to the dynamic changes in EBV DNA level between pre-IMRT and mid/post IMRT, survival was significantly higher in patients who achieved an undetectable level following treatment. On multivariate analysis, post-IMRT EBV DNA level was the strongest predictor of all treatment outcomes (P < 0.001). Our study demonstrated the clinical significance of the plasma EBV DNA level at specific time points, as well as of the dynamic changes in the EBV DNA level. Disappearance of plasma EBV DNA after treatment was associated with better survival.

Clinical Outcome of HIV Viraemic Controllers and Noncontrollers with Normal CD4 Counts Is Exclusively Determined by Antigen-Specific CD8 + T-Cell-Mediated HIV Suppression

In this cross-sectional study we evaluated T-cell responses using several assays to determine immune correlates of HIV control that distinguish untreated viraemic controllers (VC) from noncontrollers (NC) with similar CD4 counts. Samples were taken from 65 ART-naïve chronically HIV-infected VC and NC from Thailand with matching CD4 counts in the normal range (>450 cells/μl). We determined HIVp24-specific T-cell responses using standard Interferon-gamma (IFNγ) ELISpot assays, and compared the functional quality of HIVp24-specific CD8+ T-cell responses using polychromatic flow cytometry. Finally, in vitro HIV suppression assays were performed to evaluate directly the activity of CD8+ T cells in HIV control. Autologous CD4+ T cells were infected with primary patient-derived HIV isolates and the HIV suppressive activity of CD8+ T cells was determined after co-culture, measuring production of HIVp24 Ag by ELISA. The HIVp24-specific T-cell responses of VC and NC could not completely be differentiated through measurement of IFNγ-producing cells using ELISpot assays, nor by the absolute cell numbers of polyfunctional HIVp24-specific CD8+ T cells. However, in vitro HIV suppression assays showed clear differences between VC and NC. HIV suppressive activity, mediated by either ex vivo unstimulated CD8+ T cells or HIVp24-specific T-cell lines, was significantly greater using cells from VC than NC cells. Additionally, we were able to demonstrate a significant correlation between the level of HIV suppressive activity mediated by ex vivo unstimulated CD8+ T cells and plasma viral load (pVL) (Spearman r = -0.7345, p = 0.0003). This study provides evidence that in vitro HIV suppression assays are the most informative in the functional evaluation of CD8+ T-cell responses and can distinguish between VC and NC.

Poor HIV control in HLA-B*27 and B*57/58 noncontrollers is associated with limited number of polyfunctional Gag p24-specific CD8+ T cells.

Objectives:Analysis of immune response in HIV controllers, a unique group of infected individuals who are able to control HIV naturally, has provided us a chance to investigate the roles of host immune responses in HIV control. Design:In this study, the functional quality of HIV Gag p24-specific CD8+ T-cell responses was assessed in two groups of clinically distinct, HLA-B*27, HLA-B*57/58-matched individuals, viremic controllers [plasma HIV load (pVL) ⩽2000 copies/ml) and noncontrollers (pVL >2000 copies/ml) to determine its impacts on natural HIV clinical outcome. Methods:An ex-vivo interferon (IFN)-&ggr; ELISpot assay was used to screen for each individuals HIV Gag p24-specific T-cell responses. Intracellular cytokine staining assay was used to determine their functional quality (as number of cytokine being produced). Results:We found that, in contrast to previous studies, all Thai volunteers with HLA-B*5801 were uniformly noncontrollers. Viremic controllers were observed with a significantly larger number of high functional quality p24-specific CD8+ T cells than noncontrollers (P < 0.05). This superior quality of responses was observed at both total p24 and epitope-specific level. Moreover, the absolute number of high functional quality Gag p24-specific CD8+ T cells was significantly in a negative correlation with pVL (r = −0.6984, P = 0.0006) and also in a positive correlation with CD4+ T-cell count (r = 0.5648, P = 0.0095). Conclusion:We concluded that an adequate number of high functional quality Gag p24-specific CD8+ T cells is strongly associated with a natural HIV controller status.