CCL5-independent helper T lymphocyte responses to immuno-dominant pneumococcal surface protein A epitopes

Abstract

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several antigens, in addition to the polysaccharide capsule, have been implicated in both the virulence and protective immunity against Streptococcus pneumoniae; one of the best-studied S. pneumoniae antigens is pneumococcal surface protein A (PspA). Recently, it was shown that genetic polymorphisms could diminish CCL5 expression, which results in increased susceptibility to and progression of infectious diseases. We previously showed CCL5 blockade reduced PspA-specific humoral and cellular pneumococcal immunity, during S. pneumoniae strain EF3030-induced carriage, by diminishing IFN-γ and enhancing IL-10 secretion by effector T cells. We also identified immuno-dominant helper T lymphocyte (HTL) epitopes in PspA peptide 19-23 (PspA(199-246)), which caused comparatively more cytokine secretion and proliferation responses by splenic and cervical lymph node (CLN) CD4(+) T cells from mice previously challenged with S. pneumoniae strain EF3030. In this study, we sought to determine if PspA(199-246)-specific CD4(+) T cells responses were resistant to the effect of CCL5 deficiency. In short, T cell responses against these HTL epitopes were resistant to CCL5 inhibition, than compared to cells from control or naïve mice, and unaffected by reduced co-stimulatory molecule expression caused by CCL5 blockade. CCL5 deficiency also corresponded with a higher number of IL-10(+) CD11b(+) CD11c(Lo) and CD11b(+) CD11c(Hi) cells and lower IFN-γ expression by similar cells, than compared to controls. These data confirm CCL5 is an essential factor for optimal pneumococcal adaptive immunity and show CD4(+) T cell responses to PspA(199-246) are largely resistant to CCL5 deficiency.

Figure 1. Proliferation and cytokine response of PspA peptide-specific systemic and local CD4⁺ T cells that express CCL5 during pneumococcal carriage

Cervical lymph node (CLN) and spleen lymphocytes were isolated from female F1 (C57BL/6 × BALB/c) naïve mice (open box) and 28 days after intranasal challenge with Streptococcus pneumoniae strain EF3030 and treated with control (solid box) or anti-CCL5 (hashed box) antibody (Ab) solutions. CD4⁺ T cells were incubated with 1μM of PspA peptides plus mitomycin C-treated naïve syngeneic feeder cells, for 3 days, at a ratio of 5:1 × 10⁶ cells. Proliferation was measured by BrdU incorporation, which was measured by ELISA. The data presented are the mean OD₄₅₀. Stars (★) indicate statistically significant (p < 0.01) increases between naïve versus control and anti-CCL5 antibody-treated groups. Whereas asterisks (*) indicate statistically significant (p < 0.01) increases between control antibody- and anti-CCL5 antibody-treated groups. Experimental groups consisted of 10 mice and experiments were repeated three times. The data presented are the mean ± SEM optical densities of quadruplicate cultures from each group. IL-10 and IFN-γ production of cultured supernatants was determined by luminex capable of detecting > 2 pg/ml.

Figure 2. Flow cytometry analysis of CD28 and CD40L expression by CD4⁺ T cells following pneumococcal challenge

Representative plots from three separate experiments are shown where spleen- and cervical lymph node (CLN)-derived CD4⁺ T cells from female F1 (C57BL/6 × BALB/c) mice, treated with control antibody (Ab, solid line) or anti-CCL5 Ab (dotted line) solutions, were isolated 7 and 14 days after intranasal challenge with Streptococcus pneumoniae strain EF3030. Mean fluorescence intensity (MFI) and fluorescence intensity histograms of CD28 and CD40L expression by CD4⁺ cells are illustrated and were analyzed using Flow Jo version 8.3 software. Underlined MFI values represent bacterial-challenged, anti-CCL5 antibody-treated groups.

Figure 3. Flow cytometry analysis of CD80 and CD86 expression by CD11b⁺ and B220⁺ cells following pneumococcal challenge

Representative plots from three separate experiments are shown where spleen- and cervical lymph node-derived CD4⁺ T cells from Female F1 (C57BL/6 × BALB/c) mice, treated with control or anti-CCL5 antibody (Ab) solutions, were isolated 7 and 14 days after intranasal challenge with Streptococcus pneumoniae strain EF3030. Mean fluorescence intensity (MFI) and fluoroscence intensity histograms of CD80 or CD86 expression by CD11b⁺ and B220⁺ cells are illustrated and were analyzed using Flow Jo version 8.3 software. Underlined values represent MFI recorded bacterial-challenged, anti-CCL5 antibody-treated groups.

Figure 4. Change in IL-10- and IFN-γ-expressing splenic and cervical lymph node CD4⁺ T cells following pneumococcal challenge

Female F1 (C57BL/6 × BALB/c) mice were intranasally challenged with 10⁷ CFUs of Streptococcus pneumoniae strain EF3030 in a 15μl volume of Ringer’s solution. Anti-CCL5 antibody (open histogram) or control antibody (solid histogram) antibody were administered by intraperitoneal route every 3 days, starting 2 days before challenge. Anti-CCL5 antibody- and control antibody-treated groups consisted of 10 mice each and studies were repeated 3 times. Panel A shows the mean fluorescence intensity (MFI) and fluorescence intensity histograms of IL-10 expression by cervical lymph node (CLN)- and spleen-derived CD4⁺ T cells from anti-CCL5 and control antibody-treated groups as well as isotype control antibody staining (dotted histogram) of pooled lymphocytes from these groups, which were analyzed using Flow Jo version 8.3 software. Underlined values represent MFI recorded bacterial-challenged, anti-CCL5 antibody-treated groups. Panel B shows the mean fluorescence intensity (MFI) and fluorescence intensity histograms of IFN-γ expression by CLN- and spleen-derived CD4⁺ T cells from anti-CCL5 and control antibody-treated groups as well as isotype control antibody staining (dotted histogram) of pooled lymphocytes from these groups, which were analyzed using Flow Jo version 8.3 software. Underlined values represent MFI recorded bacterial-challenged, anti-CCL5 antibody-treated groups

Figure 5. Change in IL-10⁺ and IFN-γ⁺ splenic and cervical lymph node CD11c⁺ leukocytes following pneumococcal challenge

Female F1 (C57BL/6 × BALB/c) mice were intra-nasally challenged with 10⁷ CFUs of S. pneumoniae strain EF3030 in a 15μl volume of Ringer’s solution. Anti-CCL5 antibody or control antibodies were administered by intra peritoneal route every 3 days, starting 2 days before challenge with Pneumococci. Splenic and cervical lymph node (CLN) lymphocytes from anti-CCL5 and control antibody-treated groups as well as isotype control antibody groups were stained and analyzed by flow cytometry. Experimental and control groups consisted of 10 mice each and studies were repeated 3 times. Representative density plots along with percentages of IL-10⁺, IFN-γ⁺ and CD11b⁺ CD11c^Hi or CD11b⁺ CD11c^Lo populations are shown of CD11b-gated cells isolated 7 or 14 days after bacterial challenge.