Immediate Neutrophil-Variable-T Cell Receptor Host Response in Bacterial Meningitis

Bacterial meningitis is a life-threatening disease that evokes an intense neutrophil-dominated host response to microbes invading the subarachnoid space. Recent evidence indicates the existence of combinatorial V(D)J immune receptors in neutrophils that are based on the T cell receptor (TCR). Here, we investigated expression of the novel neutrophil TCRαβ-based V(D)J receptors in cerebrospinal fluid (CSF) from human patients with acute-phase bacterial meningitis using immunocytochemical, genetic immunoprofiling, cell biological, and mass spectrometric techniques. We find that the human neutrophil combinatorial V(D)J receptors are rapidly induced in CSF neutrophils during the first hours of bacterial meningitis. Immune receptor repertoire diversity is consistently increased in CSF neutrophils relative to circulating neutrophils and phagocytosis of baits directed to the variable immunoreceptor is enhanced in CSF neutrophils during acute-phase meningitis. Our results reveal that a flexible immune response involving neutrophil V(D)J receptors which enhance phagocytosis is immediately initiated at the site of acute bacterial infection.

Isotype control stainings of CSF and peripheral blood. Isotype control staining of unpurified (CSF, left) and various MACS-purified leukocyte samples from CSF or peripheral blood (PB), respectively. Note that consistently no immunostaining was observed when isotypematched control antibodies were used in place of the anti-TCRab (mouse IgG1) primary antibody. Nuclei (blue) are counterstained with DRAQ5 (except the left panel). The samples were collected from patients 1, 2, 6 and 7, respectively. post-acute phase acute phase Patient 4 (N. meningitidis) +7d * Figure S4 CSF-cytology from patient 4 with N. meningitidis meningitis: acute vs. post-acute phase. CSF cytology and TCRβ expression in the acute vs. post-acute phase of N. meningitidis meningitis. Shown are cytospin preparations and TCRβ immunostainings (red) from CSF collected 8 h after onset of symptoms (acute phase) and after 7 days of standard antibiotic therapy (post-acute phase). Note that the TCRβ positive and negative neutrophils are substituted by TCRβ expressing T cells (arrows). Bacteria are highlighted by a yellow asterisk.  Mass-spectrometric identification of a rearranged Vβ-chain variant in CSF neutrophils from patient 3. Mass-spectrometric identification of a rearranged TCR Vβ-chain in CSF neutrophils from patient 3 (E. coli meningitis). Protein lysates from the patient's CSF CD15 + -neutrophils were immunoprecipitated using an anti-TCRβ antibody and the predicted band (boxed) was analyzed by MALDI-TOF mass spectrometry. Peaks 1-5 represent TCR Vβ -specific peptide fragments. Amino acid sequence identities with known TCR Vβ -chains are bolded. They are consistent with a Vβ8-Dβ1 rearranged clonotype.

Figure S9
PB and CSF IL-8 levels in three randomly selected patients Ctr.1-3, healthy age-matched PB controls for patients 8, 9, and 10. IL-8 concentrations and the corresponding percentages of TCRαβ+ neutrophils in the CSF are shown in the bottom panel.
Following page: Repertoire diversities of the TCR variable β-chains that are expressed by PB-and CSF neutrophils from patients 1, 2 and 3, respectively, during acute-phase meningitis.
RT-PCR expression profiling was performed for all 25 known human TCR Vβ chains (Vβ1-25). The scattergrams (top) document that the CD15 + neutrophils are free of T lymphoid cells (CD2). For each patient, expressed TCR Vβ chains are represented by filled boxes and their respective designations are indicated. Empty boxes represent nonexpressed Vβ chains. Note the marked increase in expressed TCR Vβ chain genes in CD15 + CSF neutrophils relative to CD15 + PB neutrophils. The detailed repertoires assessed by length variant analysis of the antigen-binding CDR3β region ("CDR3β spectratyping") in each of the expressed Vβ chains are shown.

Figures S10
Fuchs et al.     Repertoire analyses by CDR3β spectratyping in all three patients reveal that the total numbers of the expressed TCRβ CDR3 length variants are consistently higher in CD15 + CSF neutrophils than in CD15 + PB neutrophils indicative of repertoire broadening at the site of inflammation.

CSF PB
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Figure S12
CDR3β repertoire broadening in CSF neutrophils (bottom) relative to PB neutrophils (top) shown on the detailed clonotype level. Amino acid (aa) sequences of expressed TRBV5-4 chain variants are representatively shown for patient 1. Identified CDR3β segments (shaded area) span 7-10 aa displaying a similar length variation as those expressed by T cells. The sequences of the flanking V-and J-chain segments are indicated. Note expression of three additional TRBV5-4 clonotypes in CSF neutrophils (white) that were not detected in PB neutrophils. The TRBV5-4 variants were identified by cloning and sequencing.
Following pages: NGS TCRβ transcriptome analyses reveal induction of the TCR in CSF neutrophils during the acute phase of bacterial meningitis.
(top) Repertoire diversity tree plots visualize the relative abundance of the TCRβ CDR3 transcript variants that are expressed by CD15 + neutrophils in CSF and in peripheral blood of patient 2 ( Fig S13a) and patient 3 (Fig. S13b). Each spot represents a rearranged TCRβ transcript that encodes a unique CDR3β sequence. It is defined by a unique color and its area is proportional to the relative transcript frequency. The position of each spot within the plot area is defined according to its Vβ usage (x-axis: Vβ1→Vβi) and Jβ usage (y-axis: Jβ1→Jβi). Each plot has a distinct color code. Total numbers of identified nonredundant ("unique") TCRβ CDR3 sequence variants are indicated for each diversity tree plot. CDR3, complementarity determining region 3.
(bottom) Detailed list of the 10 most frequently expressed TCRβ CDR3 variants in each neutrophil population. Transcript copy numbers (f)     Quantitative length variant analysis of the antigen-binding CDR3β region in patients 1, 2 and 3 shows a dramatic increase in TCR Vβ repertoire gene expression levels in CSF neutrophils compared to PB neutrophils as exemplified for three TCR variable chains (TRBV7, TRBV14 and TRBV12). Peak heights are indicated as fluorescence units.

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Vβ gene usage of CD15 + neutrophils and CD3 + lymphocytes, respectively, in peripheral blood and CSF of patient 1 (A) and patient 2 (B,C). The 2D-plots demonstrate the relative usage of the Vβ genes for each cell compartment. The results are shown as regular and normalized distributions. The normalized distribution counts the value (for V Gene usage) of each distinct CDR3 as one, no matter how many of the particular CDR3s are observed. In short, each CDR3-VDJ combination is treated as a quantity of 1 regardless of read count, and then analyzed for V usage. This allows for a view of the repertoire removing the skewing which may occur due to one or just a few highly dominant clones. The regular distribution is based on the number directly observed from the read count data. Note the markedly restricted Vβ gene usage in CD15 + cells relative to CD3 + cells. X-axis: Vβ gene; y-axis: percentage of used Vβ genes.   Patient 2-normalized distribution Following pages: (top) Relative abundance of TCRβ CDR3 transcript variants expressed by CD15 + neutrophils and CD3 + lymphocytes in CSF (patient 1). Total numbers of identified nonredundant TCRβ CDR3 sequence variants are indicated for each leukocyte subpopulation.
(bottom) Comparison of the 10 most frequently expressed TCRβ CDR3 variants reveals that neutrophils and lymphocytes in CSF share common repertoires (arrows), but also express a similar proportion of distinct TCRβ variants. Numbers designate transcript copy numbers. Note that two of the shared TCRβ CR3 sequence variants exhibit higher expression rates in CSF neutrophils than in CSF lymphocytes (blue).

Figure S16
Ex vivo exposure to bacterial pathogens that cause meningitis triggers dynamic TCR Vβ repertoire changes. Figure S9 shows the TCR Vβ repertoires that are expressed by purified peripheral blood CD15 + neutrophils from two healthy subjects (2,3) after 9 h incubation with N. meningitidis (orange), S. pneumoniae (green) and in the absence of a bacterial pathogen, respectively (Ø, control, black). The detailed neutrophil-TCR Vβ repertoires are shown for each subject in the right panels. The purity of the CD15+ neutrophils is shown in the scattergrams (left). Note that N. meningitidis and S. pneumoniae induce distinct neutrophil-TCR Vβ chain usage (center) and CDR3β repertoires (right) in each subject.

Figures S17
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PB neutrophils (donor 2)
TCRαβ-directed bead uptake in intact neutrophils and during cytochalasin D-induced blockade of phagocytosis.
(A) Representative unstained cytospin preparations (20x) of purified CD15+ PB neutrophils from a normal individual (donor 2) that were co-incubated with bead baits targeted to the TCRαβ or untargeted beads (nonspecific IgG) for 3 hours. Cytochalasin D (5 μg/ml) treated neutrophils are shown in the bottom panel (+ CytoD). Right panel: untargeted control beads (nonspecific IgG). The red arrow highlights internalized beads at a higher magnification, the white arrow an external bead during inhibition of phagocytosis.

Figure S20
Enhanced TCRαβ-directed phagocytosis in neutrophils. Quantitative analysis of phagocytosis and respiratory burst (percentage of phagocytosing cells,top; oxidizing cells, bottom) assessed by flow cytometry. Note that neutrophil phagocytosis is enhanced when cells are stimulated with anti-CD3ε/anti-CD28 antibodies whereas the number of oxidizing cells is not affected. Mean number of phagocytosing/oxidizing cells from five (top) and three (bottom) independent healthy donors are shown, respectively.
nonspecific IgG