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Project 2 AG Prof. Jahrsdörfer:
Perforin as switch between cytotoxicity and immune regulation
Summary: A multitude of immune cells is capable of producing the serine protease granzyme
B (GzmB). One group of cells is formed by cytotoxic cells such as NK cells and cytotoxic T
lymphocytes (CTL), which after activation secrete both GzmB and Perforin (Pfn). Their
cytotoxic function results from the fact that after exocytosis GzmB reaches the target cell
cytosol in a Pfn-dependent manner. The cytosol contains certain enzymes which are sensitive
to GzmB cleavage and which induce apoptosis after activation. In contrast, a different group
of immune cells including regulatory T cells, certain regulatory B cells (GraB cells) and
tolerogenic plasmacytoid dendritic cells (pDC) secrete GzmB in the absence Pfn. This results
in GzmB primarily reaching extracellular and membrane-close localisations after its secretion.
In this case, GzmB encounters different substrates than in the cytosol, such as the ζ-chain of
the T cell receptor. Consequently, the cleavage of such alternative substrates has different
effects, such as immune regulation. Pfn therefore is involved in determining whether GzmB
acts primarily in a cytotoxic or in an immunoregulatory fashion. Our project is designed to test
this hypothesis using various kinds of cytotoxic and regulatory cell types. On the one hand,
targeted transfer of Pfn-cDNA into the GzmB locus of cells, which normally produce GzmB
only, but not Pfn, may convert their immunoregulatory function into a cytotoxic one. Besides
regulatory T cells, certain regulatory B cells and tolerogenic pDCs may thereby acquire a
cytotoxic function, which may possibly open completely novel approaches for the
immunotherapy of tumors and viral infections. On the other hand, suppression of Pfn in
classical cytotoxic cells such as NK cells and CTL may convert their cytotoxic function into an
immunosuppressive one. This may be of interest for the treatment of inflammatory diseases
including GvHD and autoimmune diseases.
Detailed description: Granzyme B (GzmB) represents one of the major constituents of the
1,2
granules of Cytotoxic T Lymphocytes (CTL) and Natural Killer Cells (NK cells) . CTL and
NK cells are able to transfer active GzmB along with perforin into the cytoplasm of target cells
3
such as virally infected or tumor cells, where it activates apoptosis-inducing substrates . The
recognition of target cells by CTL requires that professional antigen-presenting cells (APC)
such as dedritic cells (DCs) take up antigens, process them, load them onto MHC molecules
and express them on their cell surface. Subsequently, these DCs migrate to local lymph
nodes, where they can eventually encounter and activate antigen-specific T cells. The
involved processes are complex and absorb a significant amount of time. Therefore, more
direct cytotoxic immune mechanisms exist, which are independent of MHC presentation and
which bride the gap between the first recognition of danger signals and the establishment of
an efficient CTL response. A series of immune cell populations including NK and NKT cells,
all of them belonging to the innate arm of the immune system, can play a role in such early
4-6
and MHC-independent immune responses . B cells recognize antigens via their antigenspecific immunoglobulin-based B cell receptors (BCRs), which also bind antigens in an MHC7
independent manner . Moreover, the spectrum of antigens, which are recognized by BCRs,
is broader than that recognized by the MHC-restricted T cell receptors (TCRs), and not only
8
involves peptide antigens, but also nucleic acid, glycolipid and carbohydrate antigens .
Therefore, from a teleogical point of view, it appears plausible that B cells may be involved in
early cytotoxic immune responses against tumors and infectious agents, before their terminal
9
differentiation into plasma cells is initiated .
The acute phase cytokine interleukin 21 (IL-21) is considered the key cytokine for the
10,11
. IL-21 is secreted by NKT cells as well as
differentiation of B cells into plasma cells
+
12,13
+
. Complete activation of CD4 T cells requires stimulation of both
activated CD4 T cells
the TCR and the co-stimulatory molecule CD28, which is not only associated with secretion of
IL-21, but also a strong upregulation of the co-stimulatory molecule CD40 ligand (CD40L,
CD154). We demonstrated that exclusive activation of the TCR in the absence of CD28 co9,14
.
stimulation results in continued secretion of IL-21, but lacking upregulation of CD40L
Physiologically, such situations are conceivable during acute inflammatory reactions in the
course of anti-viral or anti-neoplasic immune responses, when cellular disintegration releases
high amounts of intracellular self antigens. Despite established self tolerance such self
+
antigens may keep a variety of low-affinity T cells in an incomplete (IL-21 CD40L ) activation
15-17
. In contrast, highly antigen-specific and completely acitvated T cells (ILstage
+
+
21 CD40L ) are not yet present at the site of inflammation. We demonstrated that
incompletely activated T cells induce strong expression of GzmB in B cells, instead of
9,14
. Beyond that, we showed that GzmBtriggering their full differentiation into plasma cells
expressing B cells are able to induce apoptosis in certain tumor cell types, although they do
9
not express perforin . This is not surprising, since apart from perforin, other molecules
18,19
20,21
, viral and bacterial proteins
as well as
including mannose-6-phosphate receptors
22
heat shock proteins are also able to transfer limited amounts of GzmB into the cytoplasm of
23,24
. This shows that GzmB-secreting B cells may indeed play a role in early
target cells
cytotoxic immune defense mechanisms against viral and bacterial infections as well as in
tumor immunosurveillance, before antigen-specific CTL are taking over and B cells continue
9
their full differentiation into plasma cells (Fig. 1) .
Figure 1. Example of a biphasic antiviral cytotoxic immune response involving both B cells and T cells. In the
+
early phase (phase I, upper panel) of a viral infection, a broad spectrum of low-affinity CD4 T cells are present that
are in a pre-activated state secreting IL-21, but not CD40 ligand. B cells are activated by recognizing antigen in an
MHC-independent manner. T cell-derived IL-21 in the absence of CD40 ligation may trigger differentiation of these
activated B cells into GzmB-secreting cytotoxic B lymphocytes (CBL). Using various routes such as the endosomal
infection pathway engaged by a series of viruses, GzmB may reach the target cell cytoplasm, where it may then
induce apoptosis, thereby slowing down virus replication. At the same time, premature activation of non-specific
peripheral T cells may be suppressed by extracellularly secreted GzmB to prevent the development of cellular
autoimmune responses. In a later phase of viral infection (phase II, lower panel), after co-stimulation by professional
+
antigen-presenting cells (APC), antigen-specific T cells arrive at the site of infection in a fully activated state. CD4 T
helper cells now express both IL-21 and CD40 ligand, thereby abrogating GzmB secretion and triggering
differentiation of activated B cells into plasma cells that secrete antibodies against viral antigens. Simultaneously,
+
antigen-specific CD8 T cells arriving from the draining lymph nodes recognize antigens in an MHC-restricted manner
and induce target cell apoptosis using the classical granule exocytosis pathway.
During classical cytotoxic granule exocytosis by CTL, GzmB is co-secreted together with Pfn
24
25
(Fig. 1, lower panel) . Pfn in high (lytic) concentrations can directly induce cell lysis . Such
cell lysis however may be associated with uncontrolled autoantigen release in vivo, posing a
significant risk of autoimmune reactions. Therefore, CTL do not normally induce lytic necrosis,
1,24
. This
but rather apoptosis of target cells in vivo, which depends on both GzmB and Pfn
programmed cell death involves controlled degradation of various cellular components by
proteases and normally suppresses extensive presentation of autoantigenic material.
Although endosomal release of GzmB (endosomolysis) is supported by Pfn, its presence is
23,24
. Instead,
not considered a sine qua non for GzmB to reach the target cell cytosol
microbial proteins like bacterial lysins and viral transport proteins as well as stress- or tumor-
associated molecules including heat shock proteins can allow for cellular uptake of GzmB in
18-22
. Therefore, the fact that GzmB-secreting B cells do not express Pfn,
the absence of Pfn
does not preclude their cytotoxic function, but may rather limit such an immune response to
target cells, which are clearly associated with microbial infections or neoplastic transformation
(Fig. 1, upper panel). B cells are able to recognize antigens and antigen-bearing cells in an
MHC-unrestricted manner. Insofar, B cells may contribute to the first line of antigen-specific
defense against cells in malignant transformation. A limited cytotoxic response of such B cells
may therefore also represent an early contribution to immunosurveillance. In two recent
26
studies we demonstrated that GzmB-secreting B cells are not only infiltrating solid tumors ,
but are indeed able to transfer active GzmB to tumor cells, thereby inducing apoptosis in
9
these cells (Fig. 2) .
Figure 2. GzmB-secreting B cells transfer active GzmB to tumor cells and induce apoptosis. (A) B cells with
different potential to produce GzmB were incubated for 16 hrs in the presence of IL-2 or IL-21 on a GzmB-specific
ELISpot plate. (B) IL-2- or IL-21-stimulated B cells were co-cultured for 16 hrs with the cervix carcinoma cell line
+
HeLa. Annexin V/PI-staining of HeLa cells demonstrated significantly lower survival in the presence of GzmB (ARH77) versus GzmB (Raji) B cells. (C) Confocal microscopy of such co-cultures in the presence of a GzmB-specific
fluorogenic substrate shows transfer of active GzmB (green) from a B cell (magenta) to a HeLa cell (red). (D) White
+
arrows point to a HeLa cell, which is attacked by a GzmB B cell (green-yellow).
Of note, due to the absence of Pfn in GzmB-secreting B cells, their potential role as cytotoxic
cells is only one side of the same coin. Meanwhile we showed that GzmB-secreting B cells
are also able to suppress the expansion of T cells, thereby regulating T cell-dependent
14,26
. The reason for this dichtomy may be the fact that in the
cytotoxic immune responses
early phase of acute inflammatory responses a large number of peripheral T cells are kept in
15-17
. GzmB-secreting
a pre-activated state due to continuous autoantigen exposition
regulatory B cells may prevent full activation of such pre-activated and potentially
autoreactive T cells in the periphery, thereby ensuring that complete and antigen-specific
activation of T cells occurs in the draining lymph nodes only. We described a similar function
27
for another non-classical GzmB producer, the plasmacytoid dendritic cell (pDC) . pDC are
also capable of producing large amounts of enzymatically active GzmB in the absence of Pfn,
thereby suppressing T cell proliferation in a GzmB-dependent fashion. As in regulatory B
cells, this immunomodulatory activity requires cell contact and active GzmB, but no Pfn. Our
findings point to a general mechanism used by different regulatory cell types including
regulatory T cells (Treg), whose regulatory function is also mediated in a GzmB-dependent,
28
but Pfn-independent manner .
The above-mentioned findings raise the general question whether Pfn may critically
determine the routing of GzmB into different cell compartments, thereby also influencing its
functions. A preferential targeting of GzmB into the cytoplasm of target cells (presence of Pfn)
results in the activation of apoptosis-inducing cytoplasmic substrates including caspases,
1
DNAses and BID . In contrast, a preferential secretion of GzmB into the extracellular space
(absence of Pfn) allows GzmB to cleave different substrates such as membrane-bound
29
receptors or extracellular proteins . For example, cleavage of the T cell receptor (TCR) ζchain by extracellularly secreted GzmB may partially explain its immunomodulatory effects on
30,31
T cells
. The present project is designed to investigate, whether or not cytotoxic and
regulatory effects of GzmB-expressing cells are interchangeable with each other by
manipulating the co-production of Pfn along with GzmB (Fig. 3).
Figure 3. Modulation of the potential function of Pfn to control the cytotoxic and immunoregulatory function
of GzmB-producing cells.
A variety of immune cells is capable of producing the serine protease GzmB. One group of cells consists of classical
cytotoxic cells such as CTL and NK cells, which after activation secrete both GzmB and Pfn. Their cytotoxic function
is a result of the fact that after exocytosis GzmB reaches the cytosol of target cells in a Pfn-mediated fashion, where
it can activate apoptosis-inducing enzymes (left panel side). A different group of immune cells including regulatory T
cells, certain regulatory B cells (CBL, GraB cells) and tolerogenic plasmacytoid dendritic cells (pDC) secrete only
GzmB after activation, but no Pfn. This results in secreted GzmB primarily encountering substrates with an
extracellular or membrane-bound localisation (right panel side). For example, GzmB-dependent cleavage of the T
cell receptor ζ-chain gives rise to an anti-proliferative and thus immunoregulatory effect on T cells. A targeted transfer
of Pfn-cDNA into the GzmB locus of cells, that normally produce GzmB only, may enable these cells to co-produce
Pfn under the control of the GzmB gene. After activation, such cells would produce Pfn along with GzmB, thereby
gaining a cytotoxic function at the cost of their immunoregulatory potential. Apart from regulatory T cells this
modification may also turn regulatory B cells into cytotoxic cells. On the other hand, suppression of Pfn in CTL or NK
cells may reverse these cytoxic cells into cells with immunoregulatory function.
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