CD8⁺ T cells lacking CD96 produce perforin and IFNγ following stimulation with PMA/ionomycin.

<p>FACS sorted CD96⁺ and CD96^neg were stimulated with PMA/ionomycin and assessed for A) IFNγ and B) perforin production in an ELISPOT assay. Bars represent the mean value ± SD of three independent experiments and show spot forming units (SFU) per 5000 cells. Statistical analysis was performed using Student’s T test *p < 0.05.</p

The absolute number and CD96 MFI of CD96⁺CD8⁺ T cells correlates with CD4⁺ T cell counts.

<p>Association of A) CD96 MFI on CD8⁺ T cells (n = 37) and B) the number of CD96⁺CD8⁺ T cells with CD4⁺ T cell counts (n = 36). Correlations were determined by two-tailed non-parametric Spearman correlations.</p

CD96 T cell expression in HIV-1-infected subjects is down-regulated compared to healthy controls (HC).

<p>PBMCs from elite controllers (EC, n = 20), viremic non-controllers (NC, n = 20) and healthy controls (HC, n = 40) were surface stained for CD96 expression. A) Representative histograms (dark grey = fluorescence minus one (FMO) control, solid black line = NC, light grey = EC, dotted black line = HC) and dot plots. B) Percentage of CD8⁺ T cells expressing CD96. C) Mean fluorescence intensity (MFI) of CD96 on CD8⁺ T cells. D) Percentage of naïve and different CD8⁺ T cell memory populations (TCM = central memory T cell, TEM = effector memory T cell, TEMRA = terminally differentiated effector memory T cell) expressing CD96. E) CD96 MFI on CD8⁺ T cells within each memory subset. The bars represent the mean and error bars represent the range from minimum to maximum value. Statistical analysis was performed using Kruskal Wallis tests with Dunn’s post-test *p < 0.05, **p < 0.01, ***p < 0.001.</p

CD96 expression is down-modulated by LPS stimulation and up-regulated by TCR engagement.

<p>A) Percentage of CD38⁺ HLA-DR⁺ CD8⁺ T cells as a measure of immune activation. B) Association of CD96 MFI on CD8⁺ T cells and percentage of CD38⁺ HLA-DR⁺ CD8⁺ T cells (n = 40). C) Percentage of CD96 expression and D) CD96 MFI on CD8⁺ T cell following stimulation with either LPS, PHA, IL-12/18 and anti-CD3/CD28 for 24 hrs compared to unstimulated cells. Statistical analysis was performed using Student’s T test *p < 0.05, **p < 0.01, ***p < 0.001. Correlations were determined by two-tailed non-parametric Spearman correlations.</p

Efficient merozoite phagocytosis at low antibody concentrations.

<p>A) Increasing numbers of EtBr labeled merozoites were incubated with a pool of immune plasma from PNG children and then added to THP-1 cells. The % phagocytosis was determined by flow cytometry. The ratio of 4∶1 was chosen for subsequent assays, and is indicated by the arrow above. B) Titration of a pool of immune plasma from PNG children for opsonising activity using the 4∶1 merozoite:THP-1 cell ratio. % phagocytosis was determined by flow cytometry. The chosen dilution, 1/30,000, is indicated by an arrow above.</p

Merozoite opsonisation proceeds rapidly at low antibody concentrations.

<p>Merozoites were added to wells containing THP-1 cells simultaneously (white bars), or after 40 mins of preincubation (grey bars), with varying dilutions of an immune plasma pool. % phagocytosis was determined by flow cytometry.</p

Phagocytosis by THP-1 cells is antibody and Fc Receptor dependent.

<p>A) EtBr stained merozoites were incubated with i) non-immune plasma or ii) immune PNG plasma, and were added to THP-1 cells. THP-1 cells were gated by forward and side scatter, and EtBr fluorescence was determined by flow cytometry. A non-immune control sample was used to set the EtBr positive gate. B) THP-1 cells were stained with anti-CD14 antibody and treated with trypan blue (TB) buffer which quenched all surface FITC fluorescence (black:THP-1 cells only, white: CD14-FITC stained THP-1, grey: CD14-FITC stained THP-1 with TB buffer). C) FITC stained merozoites were opsonised with i) non-immune or ii) immune PNG plasma and added to THP-1 cells. FITC fluorescence was measured by flow cytometry before and after quenching, and phagocytosed merozoites were resistant to quenching (black:THP-1 cells only, white: fluorescence with FITC stained merozoites, grey: fluorescence with FITC stained merozoites after TB buffer). D) The % phagocytosis measured for EtBr stained merozoites was equivalent to FITC stained merozoites after quenching. Opsonised EtBr stained and FITC stained merozoites were added to THP-1 cells at 3∶1 and 10∶1 merozoite:THP-1 ratios. E) Phagocytosis is active and Fc Receptor dependent. THP-1 cells were treated with cytochalasin D (CytoD) or blocked with non-specific IgG prior to addition to immune plasma opsonised merozoites in the phagocytosis assay. Each point represents the mean ± standard error. <b>*</b>, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.005.</p

Isolated merozoites maintain surface coat integrity.

<p>A) E64-treated schizonts were filtered to release free merozoites and haemozoin crystals, and the filtrate was passed over magnetic columns. Merozoite purification was confirmed by Giemsa-stained smears of i) filtrate, ii) retained haemozoin and iii) purified merozoites. B) Merozites were stained with EtBr and enumerated by flow cytometry. C) Washed Merozoites retained surface proteins MSP-3, MSP-6 and AMA-1 by western blot (S: purified Schizonts; M: filtered merozoites; SM: EtBr stained merozoites). D) Merozoite surface proteins are maintained during merozoite isolation and wash steps as shown by surface localisation of MSP-3 and MSP1₁₉ by immunofluorescence microscopy. Antigens were stained with Alexa-594 and the nucleus with DAPI (panels in order Alexa549; DAPI; brightfield; Alexa549/DAPI; merge).</p

Direction of exposure and T cell IFN-γ response correlations.

<p>IFN-γ T cell responses from the viremic partner (VP) group were plotted against the corresponding average number of unprotected receptive (A) or insertive (B) anal exposures to a partner's HIV-1. Significant correlations (denoted by *) were determined by two-tailed nonparametric spearmans correlation, spearman r (r), and linear regression analysis was plotted as straight lines.</p

Total T cell activation was not influenced by exposure.

<p>The expression level of activation markers CD38 and HLA-DR were compared between VP group (squares, n = 6) and NVP group (circles, n = 10). The expression of CD38 on total CD4⁺ (A) and total CD8⁺ (B) T cells showed no significant difference. No significant difference was seen in CD4⁺ T cell co-expression of CD38 and HLA-DR (C). However, CD8⁺ T cells from the VP group co-expressed significantly higher of CD38 and HLA-DR compared to the NVP group (denoted by *) (D).</p