LAG-3 silencing and its effect on CD4⁺ T cells within the <i>Mtb</i>-infected co-culture.

<p>This data illustrates the mean frequency of CD4⁺ T cells positive for LAG-3 (<b>A</b>, <b>B</b>), IFN-γ (<b>C</b>, <b>D</b>), IL-10 (<b>E</b>, <b>F</b>), and Treg frequency (<b>G</b>,<b>H</b>) over the course of 96h in the <i>Mtb</i>-infected macrophage co-culture. In all images, gray squares indicate the <i>Mtb</i>-infected co-culture, where CD4⁺ T cell were untreated, and the black circles represent the <i>Mtb</i>-infected co-culture, where CD4⁺ T cell were silenced for LAG-3 before being added to the culture. In <b>A</b>, <b>C</b>, <b>E</b>, and <b>G</b> the CD4⁺ T cells used for co-culture were derived from blood of <i>Mtb</i>-infected rhesus macaques, whereas in <b>B</b>, <b>D</b>, <b>F</b>, and <b>H</b> the CD4⁺ T cells were isolated from lung of <i>Mtb</i>-infected rhesus macaques. Multiple t-tests corrected for multiple comparisons using the Holm-Sidak method were utilized to determine significance between time points. Horizontal bars represent the SEM. *<i>P</i> < 0.05.</p

Interaction between macaque macrophages and CD4⁺ T-cells during co-culture is shown using multilabel confocal microscopy.

<p>Immunostaining of cells positive for <i>Mtb</i> (red), macrophages (green), nuclei (blue) and a merge images (far right) (A). A representative image of macrophage (green):CD4⁺ T-cell (white) co-culture (lower left panel) and an infected macrophage with <i>Mtb</i> and associated with T-cell (lower right panel) (B); scale bars- 100 μm (A), 20 μm and 40 μm (B).</p

The effect of LAG-3 silencing on cytokine production in co-cultures supplemented with CD4⁺ T-cells isolated the lung of <i>Mtb</i>-infected rhesus macaques.

<p>(<b>A</b>) Concentrations of IFN-γ, (<b>B</b>) IL-6 and (<b>C</b>) CXCL11 in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture at 48h post-infection. (<b>D</b>) The presence of MIF in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture at 24h post-infection. All samples were measured in pg/ml. Statistical significance was determined using a one-way ANOVA in Prism 6, using multiple comparisons to compare each mean, with the Tukey multiple comparison test; the mean and SEM are represented by horizontal bars. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

The effect of LAG-3 silencing on cytokine production in co-cultures supplemented with CD4⁺ T-cells from the blood of <i>Mtb</i>-infected rhesus macaques.

<p>(<b>A</b>) Production of IFN-γ in <i>Mtb</i>-infected macrophage culture, untreated co-cultures, and LAG-3 silenced co-cultures at 0, 24, and 48h post-infection. (<b>B</b>) The presence of IL-6 and (<b>C</b>) CXCL11 in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture at 48h post-infection. (<b>D</b>) Levels of MIF in <i>Mtb</i>-infected macrophage culture, untreated co-culture, and LAG-3 silenced co-culture supernatant at 24h post-infection. All samples were measured in pg/ml. Statistical significance was determined using a one-way ANOVA in Prism 6, using multiple comparisons to compare each mean, with the Tukey multiple comparison test; the mean and SEM are represented by horizontal bars. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

Log₂ average fold change values of gene expression for select immune function genes amongst biological replicate samples from <i>Mtb</i>-infected macrophages (24h) as well as the two co-culture sets (<i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells; <i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells where LAG-3 expression was silenced by RNAi).

<p>Log₂ average fold change values of gene expression for select immune function genes amongst biological replicate samples from <i>Mtb</i>-infected macrophages (24h) as well as the two co-culture sets (<i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells; <i>Mtb</i>-infected macrophages (24h) + infected lung CD4⁺ T-cells where LAG-3 expression was silenced by RNAi).</p

Image_4_Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.jpg

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells – which may be activated, increased in number, or resident in specific tissues – are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.</p

The effect of LAG-3 silencing on CD4⁺ T-cell killing of <i>Mtb</i> in a differentiated macrophage culture supplemented with CD4⁺ T-cells.

<p>Log₁₀-bacterial burden at 0, 24, 48 72, and 96h post-infection in differentiated <i>Mtb</i>-infected macrophage cultures as well as co-cultures supplemented with CD4⁺ T-cells from blood (A) or lung (B) of <i>Mtb</i>-infected animals. The CD4⁺ T-cells were either untreated, or were silenced for LAG-3 with siRNA. The macrophage only cultures are indicated by solid circles, macrophage cultures supplemented with CD4⁺ T-cells are represented by solid squares, and macrophage cultures supplemented with LAG-3 silenced CD4⁺ T-cells are represented by solid triangles. Statistical significance was determined using a two-way ANOVA in Prism 6, with matched values stacked into sub columns, and using multiple comparisons to compare effects within each row (time) by comparing each experimental condition (macrophage, co-culture or LAG-3 RNAi co-culture) with every other condition at that time-point, with the Tukey multiple comparison test; the mean and SEM are represented by horizontal bars. *p < 0.05, **p < 0.01.</p

Image_6_Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.jpg

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells – which may be activated, increased in number, or resident in specific tissues – are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.</p

Global transcriptomic impact of silencing LAG-3 expression in macrophage-T-cell co-cultures during <i>Mtb</i> infection.

<p>Hierarchical clustering of specific gene ontology (GO) categories keywords or pathways revealed that immune activation/inflammation genes were represented to significantly higher levels in macrophages infected with <i>Mtb</i> and to lower levels in co-cultures (<b>A</b>). Mitochondrial electron transport related categories were significantly overrepresented in co-cultures of macrophages and lung derived CD4⁺ T-cells (<b>B</b> and <b>C</b>), but the over representation of these functions was ameliorated in co-cultures where LAG-3 expression was silenced by RNAi (<b>B</b> and <b>C</b>). The red color represents higher significance while the blue color represents lower significance. The values represented are negative log₁₀ of significance p-values of accumulation using DAVID.</p

Image_3_Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.jpg

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells – which may be activated, increased in number, or resident in specific tissues – are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.</p