CD8⁺ T cells Are Preferentially Activated during Primary Low Dose <i>Leishmania major</i> Infection but Are Completely Dispensable during Secondary Anti-<i>Leishmania</i> Immunity

<div><p>We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose <i>Leishmania major</i> infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose <i>L. major</i> infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-<i>Leishmania</i> immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-<i>Leishmania</i> immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during <i>in vitro</i> and <i>in vivo</i> recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary <i>L. major</i> challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-<i>Leishmania</i> immunity following low dose infection, they are completely dispensable during secondary immunity.</p></div

High and low dose infections preferentially expand CD4⁺ and CD8⁺ T cells, respectively.

<p>C57BL/6 mice were infected with low (1×10³) or high (2×10⁶) dose <i>L. major</i> promastigotes in their right hind footpad, sacrificed after 7 days and the draining lymph-node cells were labelled with CFSE dye and co-cultured with <i>L. major</i>-infected bone marrow-derived dendritic cells (BMDC) at a DC: lymph node cell ratio of 1∶100. After 4 days, the percentages of proliferating (CFSE^lo) CD4⁺ and CD8⁺ T cells (A and B) were assessed by flow cytometry. (A) is a representative histogram plots of individual animals while B represents the mean +/− SE of proliferating cells of all the animals (6 mice) within the group. Proliferating (CFSE^lo) IFN-γ (upper panels) and TNF (lower panels) -producing CD4⁺ (C and D) and CD8⁺ (E and F) T cells were also determined by flow cytometry. C and E are representative dot plots of individual animals within the group while D and F represent the mean +/− SE of proliferating cytokine-producing cells of all the animals (6 mice) within the group. The lymph nodes draining the infected feet were collected and digested with collagenase and the cells were then stained with different fluorochrome-conjugated antibodies against different DC subsets and the absolute numbers (upper panel) and percentage (lower panel) of CD11c⁺MHCII⁺ (G) and CD11c⁺CD103⁺CD8α⁺ (H) dendritic cells were determined by flow cytometry. Live cells were first gated on CD11c⁺ cells and further analyzed for MHC class II, CD103 and CD8α expression. Results are representative of 2 independent experiments (n = 6 mice/group) with similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Differential expansion of CD4⁺ and CD8⁺ T cells following high and low dose infections with <i>L. major</i> occur <i>in vivo</i>.

<p>Thy1.2 C57BL/6 mice that healed their primary low dose (1×10³) or high dose (2×10⁶) <i>L. major</i> infections were sacrificed and their spleen cells were labeled with CFSE dye and adoptively transferred (3×10⁷) to Thy1.1 congenic recipient mice. After 24 hr, the recipient mice were challenged with 5×10⁶ parasites, sacrificed after 1 wk and the percentages of proliferating donor (CFSE^lo) Thy1.2⁺ (donor) cells (A) were determined by flow cytometry. In addition, the percentages of proliferating (CFSE^lo) IFN-γ (B) and TNF (C) -producing donor (Thy1.2⁺) cells were also determined by flow cytometry. Results presented are representative of 2 independent experiments (n = 3–4 mice/group) with similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Kinetics of lesion development, cell recruitment in the draining lymph nodes and parasite burden following high and low dose <i>L. major</i> infections.

<p>C57BL/6 mice were infected with low (1×10³) or high (2×10⁶) dose <i>L. major</i> promastigotes in their right hind footpad and lesion size was measured weekly with Vernier callipers (A). At indicated times, some mice were sacrificed and the number of cells in the draining lymph nodes (dLNs) was determined by cell counting (B). Parasite burden in the infected footpads was determined by limiting dilution assay (C). Results presented are representative of 3 independent experiments (n = 4 mice/group) with similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p

The differential expansion of CD4⁺ and CD8⁺ T cells by high and low dose infections, respectively is sustained during recall response.

<p>C57BL/6 mice infected with low dose (1×10³) and high dose (2×10⁶) <i>L. major</i> and allowed to completely resolve (heal) their lesion (>12 wks.). Healed mice were sacrificed and dLN cells were labelled with CFSE dye and co-cultured with <i>L. major</i>-infected BMDCs at a DC:dLN cell ratio of 1∶100. After 4 days, the percentages of proliferating (CFSE^lo) CD4⁺ and CD8⁺ T cells were determined by flow cytometry (A and B). The percentages of IFN-γ (upper panel) and TNF (lower panel) -producing CD4⁺ (C and D) and CD8⁺ (E and F)) T cells within the proliferating (CFSE^lo) population were also assessed. Shown are the representative histogram (A) and dot (C and E) plots of individual animals within the group and bar graphs (B, D and F) showing the means +/− SE of all the animals (3–4 mice) within the group. The absolute numbers of CD11c⁺MHCII⁺ dendritic cells in the draining lymph nodes of high and low dose infected mice were also determined by flow cytometry following Collagenase/Dispase digestion (G). Results presented are representative of 3 independent experiments (n = 3–4 mice/group) with similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p

CD8⁺ T cells are dispensable for secondary anti-<i>Leishmania</i> immunity.

<p>C57BL/6 mice infected with low (1×10³) or high (2×10⁶) dose <i>L. major</i> were allowed to completely resolve their lesions (>12 wks.). Healed mice were treated with anti-CD4 (clone GK1.5) or anti-CD8 (clone TIB 210) mAbs to deplete CD4⁺ or CD8⁺ cells, respectively (A). Control mice received rat IgG1 (isotype control) antibody. After 24 hr., treated mice were challenged with 5×10⁶<i>L. major</i> and DTH response was measured at 72 hr. post-challenge (B). Three weeks after challenge, mice were sacrificed and parasite burden in the challenged footpads was determined by limiting dilution (C). Age-matched naïve mice served as controls. Results presented are representative of 2 independent experiments (n = 3–4 mice/group) with similar results. *, p<0.05; ***, p<0.001.</p

Low and high dose <i>L. major</i> infection leads to comparable protection against secondary virulent challenge.

<p>C57BL/6 mice were infected with low (1×10³) or high dose (2×10⁶) <i>L. major</i> and allowed to completely resolve their lesions (>12 wks.). Healed mice and some naïve age-matched controls were challenged with 5×10⁶ (high dose challenge, A and C) or 1×10³ (low dose challenge, B and D) <i>L. major</i> in the contra-lateral footpad and delayed-type hypersensitivity (DTH) response was determined at 72 hr post-challenge (A and B). Three weeks after challenge, mice were sacrificed and parasite burden in the challenged footpads was determined by limiting dilution assay (C and D). Results presented are representative of 3 independent experiments (n = 3–4 mice/group) with similar results. **, p<0.01. ND, not detected.</p