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

Abstract

SummaryCD8 T cells contribute to long-term protection against Listeria monocytogenes infection by differentiating into memory T cells. These rapidly respond to antigen or inflammation upon secondary infection. In this study we used CD8 T cells from OT1 mice and CD4 T cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing L. monocytogenes (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as 2 days after infection. FACS analysis showed GZMB-Tom expression in undivided CD8 T cells, with its level increasing over one to four divisions. OT1 T cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII-positive cells. Twenty hours later, most OT1 T cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom. Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T cells responding either to Lm-OVA or L. monocytogenes. Differences in the activation phenotype and in the splenic distribution of OT1 T cells were observed, depending on the challenge. Notably, OTI T cells with polarized granules were only observed after challenge with cognate antigen. This work showed that the GZMB-Tom knock-in mice in which GZMB-Tom faithfully reproduced GZMB expression, provide useful tools to dissect mechanisms leading to the development of anti-bacterial effector and memory CD8 T cells and reactivation of the memory response to cognate antigen or inflammatory signals.

Development of an anti-ferret CD4 monoclonal antibody for the characterisation of ferret T lymphocytes

“…3b). Similar proportions of CD4 + cells are reported in mouse lymph node (Mouchacca et al, 2015). The proportion of cellular expression of CD4 in primary and secondary lymphoid organs is similar to human and mouse (Lagresle-Peyrou et al, 2006).…”

Phenotypic and functional characterization of T cells in white matter lesions of multiple sclerosis patients

“…In addition to the Fas/FasL-pathway, T-cell cytotoxicity involves exocytosis of granules containing perforin and granzymes [ 38 ]. Previous studies showed lowered [ 56 ] or absent [ 30 ] intrathecal expression of perforin and grB by CD8 + T cells in non-inflammatory CNS conditions, suggesting a functionally repressed phenotype of CNS surveilling T cells.…”

“…4 c). Collectively, the data indicate that CD8 + T cells in WML of MS patients analyzed express a cytotoxic phenotype indicative for T cells recognizing their cognate antigen in situ [ 38 ].

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“…In comparison, CSF-derived CD8 + T cells had increased expression of both co-inhibitory markers, which refutes active involvement in CNS pathology [ 47 ]. Contrary to normal CNS conditions [ 56 ], CD8 + T cells in MS lesions expressed grB, suggesting that they encountered their cognate antigen locally and are potentially cytotoxic T cells [ 30 , 38 ]. Indeed, occasionally CD8 + T cells with polarized grB expression were localized adjacent to cells showing signs of early apoptosis.…”

“…Overall, the activated and cytotoxic effector memory phenotype of CD8 + T cells in WML, along with overlapping TCRVβ repertoires between paired WML-TCL of the same patient, implicate clonally expanded T cells recognizing their cognate antigen locally. These CD8 + T cells are potentially involvement in the deleterious CNS inflammation that contributes to MS pathogenesis [ 38 ].…”