TCRβ Combinatorial Immunoreceptor Expression by Neutrophils Correlates with Parasite Burden and Enhanced Phagocytosis during a Plasmodium berghei ANKA Malaria Infection

Abstract

Recent studies have demonstrated that a subpopulation of neutrophils express the TCRαβ combinatorial immunoreceptor in humans and mice. Here, we report that a Plasmodium berghei ANKA murine malaria infection induces expansion of TCRβ expressing CD11b⁺ Ly6G⁺ neutrophils in the spleen during the early phase of infection. Measurement of TCRβ transcript and protein levels of neutrophils in wild-type versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of nonspecific antibody staining or passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells. Remarkably, on day 3 postinfection, we observed a highly significant correlation between the proportion of neutrophils that express TCRβ and peripheral blood parasite burden. In addition, TCRβ⁺ neutrophils phagocytose parasitized erythrocytes with 4-fold greater efficiency than TCRβ^- neutrophils. Together these results signify that TCR expression by the neutrophil plays an important role in the regulation of parasite burden by enhancing the phagocytic capacity of the neutrophil.

Keywords: Plasmodium berghei ANKA; TCRβ; neutrophil.

FIG 1

Measurement of neutrophils during a Plasmodium berghei ANKA infection in ECM-susceptible C57BL/6 and -resistant BALB/c mice. Mice were infected with 10⁶ Plasmodium berghei ANKA (Pb-A) parasites. (A to D) Parasitemia was enumerated in the two strains of mice (A), and neutrophils were quantitated by measuring the expression of CD11b and Ly6G on live lymphocyte singlets in spleen tissue (B) and plotted as proportion (C) and absolute number (D) over the course of a strain ANKA infection. (E to G) Neutrophils were also quantitated in perfused brain tissue (E), and the percentage (F) and absolute number (G) of brain-sequestered leukocytes that were CD11b⁺ Ly6G⁺ neutrophils was also determined in perfused brain tissue of susceptible C57BL/6 mice on days 0, 3, and 6 postinfection and resistant BALB/c mice on day 6 postinfection. n = 5 (spleen) and n = 4 (brain) for each time point for each strain of mouse, and results presented are representative of three independent experiments.

FIG 2

Plasmodium berghei ANKA induces expression of TCRβ on neutrophils in C57BL/6 and BALB/c mice. Mice were infected with 10⁶ Plasmodium berghei ANKA (Pb-A) parasites. The proportion of neutrophils that were TCRβ⁺ CD3ε⁻ (A) was then determined and graphed as proportion (B) and absolute number (C) during a strain ANKA infection in C57BL/6 and BALB/c mice. (D) TCRβ and CD3ε expression was also measured on brain-sequestered neutrophils of susceptible C57BL/6 mice on days 0, 3, and 6 postinfection and resistant BALB/c mice on day 6 postinfection. Findings are presented as proportion (E) and absolute number (F). A fluorescence-minus-one control for TCRβ was used for gating purposes. n = 5 (spleen) and n = 4 (brain) for each time point for each strain of mouse, and results presented are representative of three independent experiments.

FIG 3

Comparison of TCRβ expression on neutrophils in C57BL/6 wild-type versus nude and rag1 knockout mice. Mice were infected with 10⁶ Plasmodium berghei ANKA (Pb-A) parasites, and TCRβ expression was compared in gated CD11b⁺ Ly6G⁺ neutrophils isolated from wild-type (A and D) versus nude (B and E) and rag1 knockout (C and F) spleen tissue. The proportion of neutrophils that express TCRβ and the absolute number of TCRβ-expressing neutrophils was compared in naive mice (G and H) and in strain ANKA-infected mice on day 6 postinfection (I and J). Absence of peripheral T cells in nude and Rag1 knockout mice did not reduce TCRβ expression by neutrophils. A fluorescence-minus-one control was used to select the gate for TCRβ. n = 5 for each time point for each group of mice, and results presented are representative of two independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01 (P values calculated using the Mann-Whitney U test).

FIG 4

Plasmodium berghei ANKA infection induces expression of TCRβ mRNA by neutrophils. Mice were infected with 10⁶ strain ANKA parasites, and TCRβ transcription was assessed by the PrimeFlow RNA assay. (A and E) Abundant levels of TCRβ mRNA were observed in positive-control CD3ε⁺ T cells isolated from spleen tissue of naive and strain ANKA wild-type mice. TCRβ mRNA was also quantitated in gated CD3ε⁻ CD11b⁺ Ly6G⁺ neutrophils isolated from naive (day 0) and strain ANKA-infected (day 6) C57BL/6 wild-type (B and F), nude (C and G), and rag1 knockout (D and H) spleen tissue. Results are presented as proportion of neutrophils that express TCRβ transcript (I) and absolute number of TCRβ transcript-expressing neutrophils (J). A fluorescence-minus-one (FMO) control was used to select the gate for TCRβ transcript, and values plotted were calculated by subtracting the background in each FMO control from its corresponding sample. Results shown are representative of two independent experiments. *, P < 0.05; **, P < 0.01 (P values calculated using the Mann-Whitney U test).

FIG 5

Analysis of the TCR Vβ repertoire of splenic neutrophils during Plasmodium berghei ANKA (Pb-A) infection of C57BL/6 and BALB/c mice. Mice were infected with strain ANKA, and expression of 15 Vβ TCR subtypes was then assessed by flow cytometry. (A and B) Vβ subtype expression was measured on gated CD11b⁺ Ly6G⁺ splenic neutrophils in naive (C and D) and strain ANKA-infected (E and F) C57BL/6 and BALB/c mice. An FITC channel fluorescence-minus-one control was used to select the most appropriate gate that was applied to all Vβ subtypes. n = 5 for each time point for each strain of mice, and results presented are representative of two independent experiments.

FIG 6

Repertoire of productive TCRβ gene rearrangements. Mice were infected with strain ANKA, and unbiased molecular analysis of T cell receptor expression was performed on TCRβ⁺ CD3ε⁻ CD11b⁺ Ly6G⁺ neutrophils sorted from spleen tissue of four moribund mice. Although neutrophil TCRβ undergoes productive gene rearrangements, neutrophil TCRβ has a restricted repertoire and displays preferred usage.

FIG 7

TCRβ expression by the neutrophil correlates with parasite burden on day 3 postinfection with Plasmodium berghei ANKA. On day 3 postinfection, the percentage of live singlet CD11b⁺ Ly6G⁺ neutrophils in the spleen that are TCRβ⁺ CD3ε⁻ was correlated with peripheral parasitemia (parasitized erythrocytes/total erythrocytes × 100) in five individual mice. Results presented are representative of four independent experiments. P ≤ 0.01; Pearson r correlation R² value of 0.92.

FIG 8

TCRβ⁺ neutrophils display enhanced phagocytosis of pRBC compared to TCRβ⁻ neutrophils. (A to D) The ability of CD11b⁺ Ly6G⁺ neutrophils (A) that are TCRβ⁺ versus TCRβ⁻ (B) to phagocytose pRBC (C and D) after 90 min was analyzed in an in vitro phagocytosis assay consisting of a 1:1 ratio of pRBC (labeled with CellTrace Far Red), and splenocytes (stained with neutrophil and T cell markers) isolated from C57BL/6 mice on day 3 postinfection with strain ANKA. (E) The percentage of neutrophils that phagocytosed pRBC was compared in TCRβ⁺ versus TCRβ⁻ neutrophils. (F) In addition, the pRBC MFI was compared in TCRβ⁺ versus TCRβ⁻ neutrophils that had undergone phagocytosis to determine if TCRβ expression influenced the quantity of pRBC phagocytosed by a single neutrophil. A fluorescence-minus-one control was used to select the gate for TCRβ. n = 3 for each phagocytosis assay, and results presented are a replicate from three independent experiments.