Adrian B. McDermott

A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis

The 51Cr release assay has traditionally been used to investigate effector cell cytotoxic function against labeled targets, but this method has inherent problems that include hazards associated with radioactivity, cell labeling and high spontaneous release. Here we describe a novel flow cytometric assay which addresses and improves upon the problems currently encountered with the 51Cr release assay. The fluorometric assessment of T lymphocyte antigen specific lysis (FATAL) assay employs dual staining (PKH-26 and CFSE) to identify and evaluate the target population. We found that the PKH-26/CFSE combination efficiently labeled target cells. Evaluation of the spontaneous leakage from dye labeled target cells was forty fold lower than the spontaneous leakage seen with the 51Cr release assay. The FATAL assay permitted a more accurate assessment of the effector: target ratio, and detected low levels of cytotoxic T lymphocyte (CTL) mediated lysis. There was a strong correlation between the 51Cr release and FATAL assays, when performed in parallel with identical effector and target cells (r(2)=0.998, P=<0.0001). This novel method of detecting cytolysis represents a qualitative and quantitative improvement over standard 51Cr release analysis. The FATAL assay will be of value to further investigate mechanisms of cytolysis by effector cell populations.

CD8(+) T cells in preventing HIV infection and disease.

The complex interplay between the host immune response and HIV has been the subject of intense research over the last 25 years. HIV and simian immunodeficiency virus (SIV) CD8+ T cells have been of particular interest since they were demonstrated to be temporally associated with reduction in virus load shortly following transmission. Here, we briefly review the phenotypic and functional properties of HIV-specific and SIV-specific CD8+ T-cell subsets during HIV infection and consider the influence of viral variation with specific responses that are associated with disease progression or control. The development of an effective HIV/AIDS vaccine combined with existing successful prevention and treatment strategies is essential for preventing new infections. In the context of previous clinical HIV/AIDS vaccine trials, we consider the challenges faced by therapeutic and vaccine strategies designed to elicit effective HIV-specific CD8+ T cells.

Accumulation of follicular CD8+ T cells in pathogenic SIV infection

LN follicles constitute major reservoir sites for HIV/SIV persistence. Cure strategies could benefit from the characterization of CD8+ T cells able to access and eliminate HIV-infected cells from these areas. In this study, we provide a comprehensive analysis of the phenotype, frequency, localization, and functionality of follicular CD8+ T cells (fCD8+) in SIV-infected nonhuman primates. Although disorganization of follicles was a major factor, significant accumulation of fCD8+ cells during chronic SIV infection was also observed in intact follicles, but only in pathogenic SIV infection. In line with this, tissue inflammatory mediators were strongly associated with the accumulation of fCD8+ cells, pointing to tissue inflammation as a major factor in this process. These fCD8+ cells have cytolytic potential and can be redirected to target and kill HIV-infected cells using bispecific antibodies. Altogether, our data support the use of SIV infection to better understand the dynamics of fCD8+ cells and to develop bispecific antibodies as a strategy for virus eradication.

Longitudinal dynamics of the HIV-specific B cell response during intermittent treatment of primary HIV infection

Background Neutralizing antibodies develop in natural HIV-1 infection. Their development often takes several years and may rely on chronic virus exposure. At the same time recent studies show that treatment early in infection may provide opportunities for immune preservation. However, it is unknown how intermittent treatment in early infection affects development of the humoral immune response over time. We investigate the effect of cART in early HIV infection on the properties of the memory B cell compartment following 6 months of cART or in the absence of treatment. The patients included participated in the Primo-SHM trial where patients with an early HIV-1 infection were randomized to no treatment or treatment for 24 or 60 weeks. Methods Primo-SHM trial patients selected for the present study were untreated (n = 23) or treated for 24 weeks (n = 24). Here we investigate memory B cell properties at viral set-point and at a late time point (respectively median 54 and 73 weeks) before (re)-initiation of treatment. Results At viral set-point, the memory B cell compartment in treated patients demonstrated significantly lower fractions of antigen-primed, activated, memory B cells (p = 0.006). In contrast to untreated patients, in treated patients the humoral HIV-specific response reached a set point over time. At a transcriptional level, sets of genes that showed enhanced expression in memory B cells at viral setpoint in untreated patients, conversely showed rapid increase of expression of the same genes in treated patients at the late time point. Conclusion These data suggest that, although the memory B cell compartment is phenotypically preserved until viral setpoint after treatment interruption, the development of the HIV-specific antibody response may benefit from exposure to HIV. The effect of viral exposure on B cell properties is also reflected by longitudinal changes in transcriptional profile in memory B cells over time in early treated patients.