Granzyme B deficient NOD mice have reduced insulitis at 70 days of age, which is not maintained at 100 days.

<p>(A) Pancreata from female NOD and granzyme B^−/− (GrzB) mice were harvested at 70 days of age (n = 10, total islets scored: NOD = 1004 islets, Granzyme B^−/− = 1443 islets), 100 days of age (n = 5–7, total islets scored: NOD = 572 islets, Granzyme B^−/− = 548 islets) and 150 days of age (n = 10, total islets scored: NOD = 688 islets, Granzyme B^−/− = 1079 islets) and scored for insulitis on frozen sections stained for insulin. The data are the mean of the percentage of islets with each score. (B) For each mouse the insulitis score was calculated as described in material and methods, and data are the mean±SEM insulitis score. Insulitis is significantly reduced in 70 day old granzyme B^−/− mice compared to NOD (*p = 0.0274), but not at 100 days or 150 days. Data analyzed by unpaired Student’s t test. ns = not significant. (C) Flow cytometric analysis of islets from female NOD and granzyme B^−/− (GrzB) mice at 70 days of age. Live cells were gated using propidium iodide exclusion. CD45 is the proportion of live cells expressing CD45. CD3 is the proportion of live CD45⁺ cells expressing CD3. The proportion of CD4⁺ and CD8⁺ cells was determined as the percentage of live CD45⁺ and CD3⁺ cells. The percentage of CD45⁺ cells (*p = 0.0461), CD3⁺ cells (**p = 0.0087) and CD8⁺ cells (*p = 0.0141) but not CD4⁺ cells was significantly reduced in granzyme B^−/− mice. Mean±SEM for n = 10–15 NOD mice and n = 5–9 GrzB mice is shown. 150 islets were used for each sample. Data analyzed by unpaired Student’s t test. (D) The proportion of live CD45⁺ cells in the islets from 70 day old female mice that were CD11c⁺ or F4/80⁺. The percentage of CD11c⁺ cells (*p = 0.0286), but not F4/80⁺ cells (p = 0.5189) were increased in granzyme B^−/− mice. Data analyzed by unpaired Student’s t test.</p

Antigen specific proliferation of granzyme B-deficient CD8⁺ T cells is reduced.

<p>(A, B) NOD8.3, GrzB^−/−NOD8.3 or Pfp^−/−NOD8.3 splenic CD8⁺ T cells were labeled with CFSE and transferred into 70- or 100-day old female NOD or granzyme B^−/− (GrzB) mice. Six days later ILN and PLN were harvested and analyzed for the proliferation of CFSE labeled cells. Data represent mean±SEM of n = 5–10 recipients for NOD8.3 or GrzB^−/−NOD8.3, n = 4 for Pfp^−/−NOD8.3, performed as individual mice over at least three independent experiments. Statistical significance, *p = 0.0183 (NOD8.3 vs GrzB^−/−NOD8.3 in granzyme B^−/− PLN) and **p = 0.0315 (GrzB^−/−NOD8.3 in NOD vs granzyme B^−/− PLN). ns = not significant. Data analyzed by unpaired Student’s t test. (C) Dot plots showing proliferation of CFSE labeled NOD8.3 (left panels) or GrzB^−/−NOD8.3 (right panels) T cells in the islets of 70 day-old female NOD (top panels) or granzyme B^−/− (bottom panels) mice. The box in each dot plot shows the number of CD8⁺CFSE⁺ cells that has migrated into the islets. Data are representative of n = 3 experiments. (D) Percentage of CFSE labeled CD8⁺ NOD8.3 or GrzB^−/−NOD8.3 T cells that have undergone division in the islets of 70 day old NOD or granzyme B^−/− recipients (n = 3). No significant difference was observed.</p

Reduced proportion of CD8⁺ T cells in granzyme B-deficient pancreatic lymph nodes.

<p>(A) Total cell counts in ILN, PLN or spleens (n = 4–7, age 70–100 days) from NOD or granzyme B^−/− (GrzB) mice. ns = not significant. (B) Pancreatic lymph node cells from 70–100 day old NOD or granzyme B^−/− (GrzB) mice were stained with antibodies to CD3 (n = 18), CD4 and CD8 (n = 13). CD3⁺ T cells were gated on CD4 and CD8 to determine the percentage of cells. No difference was observed in the overall percentage of CD3⁺ T cells. The percentage of CD8⁺ T cells was significantly reduced in granzyme B^−/− mice compared to NOD (***p = <0.0001), and the percentage of CD4⁺ T cells was increased (***p = <0.0001). Data analyzed by unpaired Student’s t test. (C) Absolute numbers of cells were determined. There was no difference in total CD3⁺ T cells in the PLN of 70–100 day old NOD or granzyme B^−/− mice (n = 7 mice). The number of CD4⁺ T cells was increased (n = 7 mice, *p = 0.0442) and the number of CD8⁺ T cells was reduced (n = 7 mice, *p = 0.0421) in the PLN of granzyme B^−/− mice. ns = not significant. Data analyzed by unpaired Student’s t test. (D) Percentage of CD4⁺CD8⁻ and CD8⁺CD4⁻ T cells in CD3 gated thymocytes in 70 day old female NOD or granzyme B^−/− mice (n = 6 mice performed over 3 independent experiments). ns = not significant. (E) The percentage of CD8⁺ T cells expressing CD44 was determined by flow cytometry at 100 days of age (n = 5). ns = not significant. (F) BrdU incorporation into thymic CD4⁺CD8⁻ and CD8⁺CD4⁻ T cells, and CD4⁺ and CD8⁺ T cells from PLN of 4-week-old NOD and granzyme B^−/− mice (n = 3 mice). Percentage of CD8⁺BrdU⁺ cells in PLN was significantly reduced in granzyme B^−/− compared to NOD (**p = 0.0012). ns = not significant. Data analyzed by unpaired Student’s t test. Horizontal bars in A-F show mean±SEM.</p

data_sheet_1.PDF

<p>The ability of cytotoxic lymphocytes (CL) to eliminate virus-infected or cancerous target cells through the granule exocytosis death pathway is critical to immune homeostasis. Congenital loss of CL function due to bi-allelic mutations in PRF1, UNC13D, STX11, or STXBP2 leads to a potentially fatal immune dysregulation, familial haemophagocytic lymphohistiocytosis (FHL). This occurs due to the failure of CLs to release functional pore-forming protein perforin and, therefore, inability to kill the target cell. Bi-allelic mutations in partner proteins STXBP2 or STX11 impair CL cytotoxicity due to failed docking/fusion of cytotoxic secretory granules with the plasma membrane. One unique feature of STXBP2- and STX11-deficient patient CLs is that their short-term in vitro treatment with a low concentration of IL-2 partially or completely restores natural killer (NK) cell degranulation and cytotoxicity, suggesting the existence of a secondary, yet unknown, pathway for secretory granule exocytosis. In the current report, we studied NK and T-cell function in an individual with late presentation of FHL due to hypomorphic bi-allelic mutations in STXBP2. Intriguingly, in addition to the expected alterations in the STXBP2 and STX11 proteins, we also observed a concomitant significant reduction in the expression of homologous STXBP1 protein and its partner STX1, which had never been implicated in CL function. Further analysis of human NK and T cells demonstrated a functional role for the STXBP1/STX1 axis in NK and CD8+ T-cell cytotoxicity, where it appears to be responsible for as much as 50% of their cytotoxic activity. This discovery suggests a unique and previously unappreciated interplay between STXBP/Munc proteins regulating the same essential granule exocytosis pathway.</p

The structural basis for membrane binding and pore formation by lymphocyte perforin

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin ‘key-shaped’ molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, cryo-electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores

GzmB^W and GzmB^P have equivalent cytotoxic potential <i>in vitro</i>.

<p>(<b>A</b>) Mouse P815, (<b>B</b>) mouse EL-4, (<b>C</b>) human Jurkat and (<b>D</b>) HeLa cells were labeled with ⁵¹Cr, and exposed to sub-lytic concentrations of recombinant perforin in combination with either mouse p (black bars) or w (white bars) recombinant GzmB. Specific ⁵¹Cr release is shown as the mean of individual experiments (each performed in triplicate) ± SEM. The number of experiments performed were: P815 (n = 5), EL-4 (n = 2), Jurkat (n = 3) and HeLa (n = 4).</p

GzmB^W/W mice are sensitive to infection with Δm157 MCMV.

<p>(<b>A</b>) B6 mice (black square) or GzmB^W/W mice (white square) were infected with 2×10⁴ pfu of MCMV Δm157 and survival monitored over the indicated time course (n = 5 for each group). ***P&lt;0.0001. (<b>B</b>) The indicated mouse strains were infected with 2×10⁴ pfu of MCMV Δm157, the indicated organs were removed at day 7 post-infection, and viral load quantified by plaque assay. Data are pooled from 2 independent experiments, mean ± SEM are plotted, where n≥8. *P&lt;0.05. (<b>C</b>) Livers from uninfected or MCMV-infected mice were isolated at day 6 post-infection, fixed and tissue sections stained with haematoxylin and eosin. The results are representative of two independent experiments. (<b>D</b>) Liver enzymes in the serum of B6 mice (black bar) or GzmB^W/W mice (white bar) were measured at day 6 post-infection. Data are pooled from two independent experiments where n≥5. *P&lt;0.05.</p

GzmB^W/W CD8 T cells are unable to lyse MCMV infected cells.

<p>(<b>A</b>) The numbers of CD8 and CD4 T cells localizing to the liver of B6 mice (black bar) or GzmB^w/w mice (white bar) after infection with MCMV Δm157 are shown. (<b>B</b>) At the indicated times post-infection, splenocytes were stained with anti-CD8, anti-TCRβ, and M45 tetramers. Representative FACS plots showing the percentage of M45-specific CD8 T cells are shown, and (<b>C</b>) the total numbers of M45-specific CD8 T cells are plotted. Data are pooled from 2 independent experiments, where n≥5. (<b>D</b>) Splenocytes were isolated from MCMV Δm157 infected B6 (black square) and GzmB^w/w mice (white circle), or from uninfected B6 mice (white square) and GzmB^w/w mice (black circle). Splenocytes were cultured with ⁵¹Cr-labeled M45 pulsed EL4 cells for 4 h and specific lysis determined. n = 6 for each data point. (<b>E</b>) CD8 T cells were purified from B6 mice (black square) and GzmB^w/w mice (open circle) and co-cultured with MCMV infected IC-21 macrophages for 18 h at the indicated E∶T ratios. n = 5 for each data point.</p

Exploration of a Series of 5‑Arylidene-2-thioxoimidazolidin-4-ones as Inhibitors of the Cytolytic Protein Perforin

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure–activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 μM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed <i>T</i>_1/2 values of 1.1–1.2 h (dose of 5 mg/kg iv) and MTDs of 60–80 mg/kg (ip)

GzmB^W has a substrate preference similar to human GzmB.

<p>GzmB^W has a substrate preference similar to human GzmB.</p