Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM) cells in cattle naturally infected with bovine tuberculosis

Abstract

Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+)IL-2(+)TNF-α(+) and IFN-γ(+) TNF-α(+) response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hi)CD45RO(+)CD62L(lo) T-effector memory (T(EM)) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM) cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

Figure 1. Identification of recombinant antibodies with specificity for bIL-2.

PBMC from a cow naturally infected with M. bovis were cultured in the presence of PPD-B to allow screening of candidate bIL-2 by ICS flow cytometry. Panel A shows the histogram gating strategy used to interrogate responses in singlet, live CD4⁺ lymphocytes. Panel B shows the measurement of detectable IL-2 and/or IFN-γ within the CD4⁺ population for each of 6 candidate IL-2 antibody clones. The clone number is shown in the top left corner of each histogram and the percentage of CD4⁺ cell in which co-expression of IFN-γ and IL-2 could be detected is shown in the top right of each histogram. Data are representative of 1 of 2 independent experiments.

Figure 2. Identification of multifunctional IFN-γ, IL-2 and TNF-α CD4⁺ cells in natural M. bovis infection.

PBMC from naturally M. bovis infected cattle were cultured in the presence of either PPD-B or medium and the co-expression of IFN-γ, IL-2 and TNF-α determined by ICS flow cytometry. Histograms were gated on singlet, live lymphocytes and then all CD4⁺ cells analysed for all combinations of cytokine productivity. The upper histograms show the total proportion of CD4⁺ cells staining for expression of IFN-γ, IL-2 or TNF-α following stimulation with PPD-B (left panel) or medium control (right panel). Subgating of the IFN-γ⁺ and IFN-γ⁻ CD4⁺ cells provides histograms that represent all possible functionalities for the expressing of the 3 cytokines, as shown in the lower histograms. The numbers indicate percentage of CD4⁺ cells and data are representative of 1 of 10 naturally infected cattle.

Figure 3. Characterisation of antigen-specific multifunctional CD4⁺ responses in cattle naturally infected with M. bovis.

PBMC from naturally M. bovis infected cattle were cultured in the presence of either PPD-B or medium and the co-expression of IFN-γ, IL-2 and TNF-α determined by ICS flow cytometry. The percentage of PPD-B stimulated CD4⁺ cells expressing all possible combinations of each the 3 cytokines is represented as a stacked bar graph for each of 10 naturally infected cattle (medium control response subtracted).

Figure 4. Multifunctional CD4⁺ cells demonstrate a better improved quality of effector cytokine response.

PBMC from naturally M. bovis infected cattle were cultured in the presence of PPD-B and the expression of IFN-γ, IL-2 and TNF-α determined by ICS flow cytometry. The Median Fluorescence Intensity (MFI) for all single, double and triple cytokine producing CD4⁺ cells were determined. Symbols represent the mean MFI (± SEM) of each of the indicated CD4⁺ functional phenotypes for 10 naturally infected cattle. Significant differences between single and multifunctional phenotypes was determined by Repeat Measures ANOVA with Tukey multicomparison post-analysis test analysis (* ρ<0.05, ** ρ<0.01, *** ρ<0.001).

Figure 5. Characterisation of memory markers reveals a characteristic T_EM phenotype for multifunctional CD4⁺ cells in naturally infected cattle.

PBMC from naturally M. bovis infected cattle were stimulated with PPD-B and stained for production of cytokine by ICS flow cytometry. Histograms were first gated on singlet, live lymphocytes and gating strategies applied to allow phenotyping of cells for expression of CD44, CD45RO and CD62L. The quadrant gate defining expression of CD44 and CD62L (top panel), CD45RO and CD62L (middle panel) or CD44 and CD45RO (bottom panel) was determined using the total live-lymphocyte population, as shown for each surface marker combination in the histograms on the far left of the panel. This quadrant gating strategy was used to determine the surface phenotype of total CD4⁺IFN-γ⁺, CD4⁺IL-2⁺, CD4⁺TNF-α⁺ and triple functional CD4⁺ cells, as shown in the other histograms. The percentage of cytokine producing CD44^hiCD62L^lo, CD45RO⁺CD62L^lo and CD44^hiCD45RO⁺ is shown for each of these CD4⁺ populations. The data is from one representative animal of 5 analysed.