Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

Abstract

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.

Opposing Development of Cytotoxic and Follicular Helper CD4 T Cells Controlled by the TCF-1-Bcl6 Nexus

“…Notably, GzmB protein expression was detected in env-reactive effector CD4 + T cells even without in vitro restimulation (Figure S1A), suggesting that it reflected in-vivo-induced production. Moreover, EF4.1 env-reactive CD4 + T cells, additionally carrying an allele encoding a fusion of GzmB and tdTomato fluorescent protein (Mouchacca et al., 2013), contained a significantly higher frequency of GzmB-tdTomato + cells when primed by Ad5.pIX-gp70 than when primed by FV (Figure S1B). Together, these data support the idea that GzmB production was induced in vivo in splenic CD4 + T cells during Ad5.pIX-gp70 immunization.…”

“…Inbred B6 and CD45.1 + congenic B6 (B6.SJL- Ptprc a Pep3 b /BoyJ) mice, TCRβ-transgenic EF4.1 mice (Antunes et al., 2008), TCRαβ doubly transgenic EVα2 mice (Merkenschlager et al., 2016), Rag1-deficient ( Rag1 −/− ) mice (Mombaerts et al., 1992), B cell-receptor-deficient ( Ighm −/− ) mice (Kitamura et al., 1991), mice with an activatable YFP gene targeted into the Gt(ROSA)26Sor ( R26 ) locus (Srinivas et al., 2001), mice with a targeted insertion of Cre recombinase into the Tnfrsf4 locus (Klinger et al., 2009) ( Tnfrsf4 Cre ), mice with a conditional Bcl6 allele (Kaji et al., 2012) ( Bcl6 fl ), endogenous ecotropic murine-leukemia-virus-deficient ( Emv2 −/− ) mice (Young et al., 2012), mice with a targeted insertion of GFP into the Prdm1 locus (Kallies et al., 2009) (Blimp1-GPF), and mice with a targeted insertion of tdTomato fluorescent protein into the Gzmb locus (Mouchacca et al., 2013) (GzmB-tdTomato) were all on the B6 genetic background and were maintained at the Francis Crick Institute’s animal facilities. All animal experiments were approved by the ethical committee of the Francis Crick Institute and were conducted according to local guidelines and UK Home Office regulations under the Animals (Scientific Procedures Act) 1986 (ASPA).…”

Visualization of granzyme B‐expressing CD8 T cells during primary and secondary immune responses to Listeria monocytogenes

“…Mice were housed under specific pathogen‐free conditions in the Centre d'Immunologie de Marseille‐Luminy facility. The GZMB‐Tom KI mice that express the fusion protein GZMB‐tdTomato in place of GZMB have been described . They were established on the C57BL/6 (CD45.2) background.…”

“…The perforin/granzyme (GZM)‐dependent cytolytic function is acquired during differentiation of naive CD8 T cells into cytolytic T lymphocytes (CTL) in response to antigenic stimulation. Perforin and GZMs are localized in cytoplasmic granules of CTL (and natural killer cells) and are released at the immune synapse by degranulation upon CTL interaction with antigen‐presenting target cells . Through a mechanism dependent on perforin, a pore‐forming protein, GZMs (of which GZMs A and B are the most abundant) enter target cells and induce their apoptosis …”

Assessing T lymphocyte function and differentiation by genetically encoded reporter systems

“…While yellow and green FPs are particularly sensitive to low pH, many blue, red, and far-red variants are less pH dependent and, thus, can be used for this purpose [81]. Recently, a transgenic mouse expressing a GrzB-tdTomato fusion protein was developed (GZMB-Tom-KI) that enabled the analysis of the distribution of GrzB-containing vesicles at different steps of CTL-target cell interactions [82]. Alternatively, combinations of pH-stable and pHsensitive FP-cytotoxin-fusion proteins (which only become fluorescent once lytic granules fuse with the plasma membrane) have been used in in vitro studies to trace lytic granules continuously throughout exocytosis [83][84][85].…”