Determining mean fractional anisotropy using DDCOSY: preliminary results in biological tissues

Complex materials are ubiquitous in science, engineering and nature. One important parameter for characterising their morphology is the degree of anisotropy. Magnetic resonance imaging offers non-invasive methods for quantitative measurements of the materials anisotropy, most commonly via diffusion tensor imaging and the subsequent extraction of the spatially resolved fractional anisotropy (FA) value. Here, we propose an alternative way of determining the FA as a sample average for cases where spatially resolved methods are not needed or not applicable. It is based on a particular diffusion–diffusion correlation spectroscopy protocol, allowing for the extraction of the mean (i.e. sample averaged) FA value. We demonstrate that mean FA values obtained from three anisotropic biological tissues are consistent with those extracted using diffusion tensor imaging. Moreover, we show that differences of mean FA values in healthy and tumour-bearing mouse brains allow to distinguish these tissue types. We anticipate that the proposed method will be beneficial in the wider context of medical and material science

Oral vaccination with Liporale™-BCG induces CD4⁺ T cell cytokine production in the lung.

<p>Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were stimulated for 6 hours <i>in vitro</i> in the presence of Brefeldin A and monensin then analyzed by flow cytometry. (A) Representative plots show CD4⁺ T cells from the lungs of naïve or BCG vaccinated mice expressing IFNγ, TNFα or IL-2. (B) Bar graphs show the percentage of CD4⁺ T cells from the lungs of naïve or BCG vaccinated mice expressing cytokines at 4, 8 or 30 weeks post immunization. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p<0.05, **p<0.01, ***p<0.001 (one way ANOVA with Tukey post test). Eight and 30 weeks results are representative of 2 independent experiments.</p

Oral vaccination with Liporale™ BCG induces effector and central memory Ag85B-specific CD4⁺ T cells in the spleen.

<p>Lymphocytes from the spleens of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneously vaccinated (BCG s.c.) mice were stained with an Ag85B/MHCII tetramer and enriched for tetramer positive cells by magnetic bead isolation. (A) Representative flow cytometry density plots showing CD62L and CD44 expression on total CD4⁺ T cells from spleens of naïve mice, or Ag85b-specific CD4⁺ T cells from the spleens of BCG vaccinated mice. (B) Bar graphs showing the proportion of naïve (CD62L^hi, CD44^lo), T_EFF/T_EM (CD62L^lo, CD44^hi) or T_CM (CD62L^hi, CD44^hi) CD4⁺ T lymphocytes of total CD4⁺ T cells from naïve mice or Ag85B-specific CD4⁺ T cells from the spleens of BCG vaccinated mice at 4, 8 and 30 weeks post vaccination. Results are displayed as mean + SEM of n = 5 for each group: *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney test). Eight and 30 weeks results are representative of 2 independent experiments.</p

Oral vaccination with Liporale™-BCG induces long-lived CD4⁺ effector T cells in the lung.

<p>Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were analyzed by flow cytometry. (A) Representative flow cytometry plots show the gating strategy used to identify CD4⁺ T cells. (B) Bar graphs show the proportion of naïve (CD62L^hi, CD44^lo), T_EFF/T_EM (CD62L^lo, CD44^hi) or T_CM (CD62L^hi, CD44^hi) CD4⁺ T lymphocytes of total CD4⁺ T cells from the lungs of naïve or BCG vaccinated mice at 4, 8 and 30 weeks post vaccination. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p<0.05, **p<0.01, ***p<0.001 (one way ANOVA with Tukey post test). Eight and 30 weeks results are representative of 2 independent experiments.</p

Oral vaccination with Liporale™-BCG increases the number of Ag85B-specific CD4⁺ T cells in the spleen.

<p>Lymphocytes from the spleens of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneously vaccinated (BCG s.c.) mice were stained with an Ag85B/MHCII tetramer and enriched for tetramer positive cells by magnetic bead isolation. (A) Representative flow cytometry plots show Ag85B-specific CD4⁺ T cells in the spleens of naïve or BCG vaccinated mice at 4, 8 and 30 weeks post immunization. (B) Bar graphs show the number of Ag85B-specific CD4⁺ T cells in the spleens of naïve or BCG vaccinated mice at 4, 8 and 30 weeks post vaccination. Results are displayed as mean +SEM of n = 5 for each group, significance expressed relative to naïve: *p<0.05, **p<0.01, ***p<0.001 (one way ANOVA with Tukey post test). The 8 and 30 weeks results are representative of 2 independent experiments.</p

Blocking CTLA-4 while priming with a whole cell vaccine reshapes the oligoclonal T cell infiltrate and eradicates tumors in an orthotopic glioma model

Vaccine-mediated cancer treatment is unlikely to induce long-term survival unless suppressive mechanisms are overcome. Given the success of antibody-mediated immune checkpoint blockade in relieving regulation of endogenous anti-tumor T cell responses in tumor-burdened hosts, we investigated whether checkpoint blockade could improve the efficacy of responses induced with a whole tumor-cell vaccine. We show that administration of a single dose of blocking antibody was sufficient to significantly enhance antitumor activity of the vaccine, inducing complete radiological regression of established intracranial tumors. The antibody or vaccine alone were ineffective in this setting. The antibody had to be administered before, or close to, vaccine administration, suggesting CTLA-4 blockade had an impact on early priming events. The combined treatment resulted in enhanced trapping of leukocytes in the lymphoid tissues, including T cells that had undergone significant proliferation. There were no obvious changes in the stimulatory function of antigen-presenting cells or the number and function of regulatory T cells, suggesting T cells were the targets of the checkpoint blockade. While tumors regressing under combined treatment were highly infiltrated with a variety of leukocytes, tumor eradication was dependent on CD4+ T cells. Analysis of the TCR repertoire showed that the addition of anti-CTLA-4 at priming reshaped the repertoire of tumor infiltrating T cells. In particular, the oligoclonal populations became greater in magnitude and more diverse in specificity. Using anti-CTLA-4 in a restricted way to promote the priming phase of an anti-cancer vaccine may offer a useful way of harnessing clinical benefit from this powerful agent