Beyond TNF: TNF superfamily cytokines as targets for the
treatment of rheumatic diseases
Michael Croft
1
and Richard M. Siegel
2
1
Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, and Department of
Medicine, University of California San Diego, La Jolla, California 92037, USA
2
Immunoregulation Section, Autoimmunity Branch, NIAMS, NIH, Bethesda, Maryland, USA
Abstract
TNF blockers are highly efficacious at dampening inflammation and reducing symptoms in
rheumatic diseases such as rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis, and
also in nonrheumatic syndromes such as inflammatory bowel disease. As TNF belongs to a
superfamily of 19 structurally related proteins that have both proinflammatory and anti-
inflammatory activity, reagents that disrupt the interaction between proinflammatory TNF family
cytokines and their receptors, or agonize the anti-inflammatory receptors, are being considered for
the treatment of rheumatic diseases. Biologic agents that block B cell activating factor (BAFF) and
receptor activator of nuclear factor-κB ligand (RANKL) have been approved for the treatment of
systemic lupus erythematosus and osteoporosis, respectively. In this Review, we focus on
additional members of the TNF superfamily that could be relevant for the pathogenesis of
rheumatic disease, including those that can strongly promote activity of immune cells or increase
activity of tissue cells, as well as those that promote death pathways and might limit inflammation.
We examine preclinical mouse and human data linking these molecules to the control of damage in
the joints, muscle, bone or other tissues, and discuss their potential as targets for future therapy of
rheumatic diseases.
Over 30 years have passed since the molecular identification of TNF as a mediator of fever
and cachexia
1
, and approximately 20 years since the first introduction of TNF inhibitors into
clinical practice for the treatment of rheumatoid arthritis (RA)
2
. During this time, much has
been learned about the basic biology of the 19 structurally related cytokines of the TNF
superfamily (TNFSF), their receptors (TNF receptor superfamily, TNFRSF), the intracellular
signalling pathways activated by these receptors, as well as the unique and overlapping roles
of TNFSF cytokines in a number of inflammatory and autoimmune diseases. TNFSF
proteins organize lymphoid tissue development, co-stimulate lymphocyte activation and can
Correspondence to: M.C. and R.M.S. mick@lji.org; siegelr@mail.nih.gov.
Author contributions
Both authors researched data for the article and made a substantial contribution to discussion of content, writing, reviewing and editing
of the manuscript before submission.
Competing interests statement
M.C. has licensed patents on several TNF superfamily molecules. R.S. has issued patents on antibodies against the TNF superfamily
molecule TL1A.
HHS Public Access
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either increase lymphocyte survival and function or induce cell death
3–6
. Outside the
immune system, TNFSF cytokines can promote the development and survival of osteoclasts,
as well as cells in the mammary glands, hair follicles and sweat glands. TNFSF cytokines
can also regulate neuronal activity and drive inflammatory responses in a range of tissue
structural cells, including epithelial cells and fibroblasts. These insights have led to intensive
efforts to treat other inflammatory diseases through TNF neutralization, and multiple TNF-
blocking agents (such as adalimumab, certolizumab pegol, etanercept, golimumab and
infliximab) are now approved for diseases such as juvenile idiopathic arthritis, psoriasis,
psoriatic arthritis, spondylarthropathies, inflammatory bowel disease and uveitis
7,8
(TABLE
1). Investigations into the targeting of other TNFSF members have led to a number of
clinical trials in different diseases and resulted in the successful development of belimumab,
an antibody against B cell activating factor (BAFF, also known as TNFSF13B), and
denosumab, an antibody targeting receptor activator of nuclear factor-κB (NF-κB) ligand
(RANKL, also known as TNFSF11), for the treatment of systemic lupus erythematosus
(SLE) and osteoporosis, respectively
9–11
.
Clinical targeting of TNF, BAFF and RANKL has been reviewed elsewhere
7–10,12–17
, as has
the targeting of all the TNF and TNFRSF members in both immune and nonimmune
disorders
11
. In this Review, we focus on TNF family proteins that are produced by the
immune system but are not yet targets of approved drugs. These molecules might be crucial
to the immune response underlying rheumatic diseases and are promising future targets for
intervention and therapy in diseases such as RA and SLE (FIG. 1). Although blocking nerve
growth factor binding to its receptor TNFRSF16 (also known as nerve growth factor
receptor) is of primary interest for the treatment of pain associated with osteoarthritis,
TNFRSF16 is not an immune-system-related molecule and so we do not present a discussion
here but refer readers to several other published articles
11,18–21
.
TNF superfamily
Multiple functional polymorphisms in the genes encoding TNFSF cytokines, their receptors
and their signalling proteins are associated with susceptibility to autoimmune diseases
11,22
.
Yet, many functions of TNFSF proteins remain poorly understood. TNFSF and TNFRSF
proteins have many structural and biological similarities (FIG. 1). TNFSF molecules are
trimeric type II transmembrane proteins characterized by C-terminal TNF homology
domains that can be cleaved from cells to form soluble ‘cytokine-like’ molecules
23
. Their
receptors are type I transmembrane proteins that have varying numbers of extracellular
ligand-binding cysteine-rich domains
23
. The extracellular domains of the TNFRSF can also
be cleaved to form soluble molecules, which might be useful as biomarkers for
inflammation, although their exact function is not clear. Engagement of receptors by their
cognate ligands is thought to primarily lead to trimerization of the receptors, which can
further form higher-order oligomers on a cell’s surface. Although overall sequence similarity
between TNFRSF molecules is low (20–30%), once engaged by their cognate ligands they
can drive common or overlapping signalling pathways
24–26
(FIG. 2). Moreover, membrane-
bound TNF family ligands can also signal through themselves when engaged to their
cognate receptors (a process known as reverse signalling), which might contribute to their
function. When crosslinked on the surface of cells, various consequences of reverse
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signalling have been described, such as proinflammatory cytokine production (for example
IL-1 and IL-6) and cell maturation, which depend on the cell type that receives the TNFSF
ligand signal
27
.
The expression of TNF family proteins is quite broad and dynamically regulated (FIG. 1).
Many ligand–receptor pairs are constitutive or inducible on lymphocytes, including antigen
presenting cells (APCs; such as dendritic cells, macrophages and B cells) and T cells, and
normally participate in promoting T and B cell responses, which are central to most
autoimmune and rheumatic diseases. Similarly, death-inducing molecules can also be
expressed by lymphocytes, and participate in maintaining self-tolerance and limiting
adaptive immune responses. Additionally, a number of TNF family ligands and/or receptors
are constitutive or inducible in non-lymphoid cells including epithelial cells, fibroblasts,
smooth muscle cells, and endothelial cells. These molecules participate in the
proinflammatory and anti-inflammatory crosstalk that occurs between tissue structural cells
and the immune system, which might either contribute to autoimmune tissue pathology or
limit damage.
Below, we discuss the biological activities of TNFSF members and their potential
involvement in rheumatic diseases. For simplicity, we have grouped TNFSF proteins, as
described above, into immune cell activators, tissue inflammatory proteins and molecules
that induce cell death or immune suppression. This classification is not absolute and the
reader should be aware that molecules such as TNF, CD40 ligand (CD40L, also known as
TNFSF5), LIGHT (also known as TNFSF14), TNF-like ligand 1A (TL1A, also known as
TNFSF15), and TNF-related apoptosis inducing ligand (TRAIL, also known as TNFSF10)
can exert functions on both immune cells and tissue cells (FIG. 1). Moreover, a number of
proteins, including TNF and Fas ligand (FasL, also known as TNFSF6), are able to promote
cell death as well as being proinflammatory, depending on the target cell type and the
context in which they are active.
Immune cell activation
CD40L
CD40 (also known as TNFRSF5) is a stimulatory receptor expressed on dendritic cells,
macrophages and B cells, whose signals drive activation, maturation, survival and
inflammatory cytokine production
28,29
(FIG. 3). CD40 is crucial in both inducing IgG
autoantibodies and driving immunoglobulin class switching
30,31
, and is also a primary driver
of T cell immunity. Its ligand, CD40L, is induced in T cells shortly after activation and, via
ligation of CD40 on professional APCs, can lead to an increase in antigen presentation and
activation of T cells by upregulating MHC molecules and inducing expression of stimulatory
ligands such as CD86 and those belonging to the TNF superfamily, which are described
below (for example OX40 ligand (OX40L))
32,33
.
Studies have long linked the interaction between CD40L and CD40 to rheumatic disease
pathogenesis. In the early 1990s, studies of multiple autoimmune models, including
collagen-induced arthritis and lupus-like disease in NZB/SWR or NZB/NZW F1 mice
34–36
,
demonstrated markedly reduced signs of inflammation in mice lacking either CD40 or
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CD40L, or in wildtype mice treated with CD40L blocking reagents. Similar to other
molecules discussed below, the idea that the CD40L–CD40 axis is also active in human
disease largely derives from expression studies in patients. The caveat with expression
studies is that detection of the molecules in serum or tissues does not automatically imply
they are functional or important, but could simply reflect the presence of activated immune
cells. However, such data, particularly for conventional cytokines such as IL-5, IL-13, and
IL-17, has aided their clinical targeting and enabled patient stratification into those most
likely to respond to biologic agents. Therefore, with TNFSF molecules the expression data
are highly useful regardless of the caveats, especially if linked to either other disease
markers or the magnitude of clinical symptoms. Soluble CD40L in serum, or CD40L
expression in inflamed tissue, epithelial cells, endothelium or T cells, is upregulated in
patients with RA, psoriatic arthritis, ankylosing spondylitis, SLE, Sjögren syndrome and
systemic sclerosis (Ssc), often correlating with disease severity or levels of
autoantibodies
28,29
. Additionally, polymorphisms near the genes encoding CD40L or CD40,
which are thought to lead to elevated or prolonged expression, have been associated with
susceptibility to SLE, RA and other rheumatic disorders (such as Behçet disease)
37–44
.
Animal studies have shown that the neutralization of CD40L has a strong suppressive effect
on pathogenic T cell development and antibody responses. These results, together with data
from human expression and association studies, made CD40L an attractive therapeutic target
for rheumatic diseases, particularly SLE and RA. As reviewed elsewhere
11,28,29
, phase I–II
trials in several patient groups, including patients with lupus nephritis, demonstrated some
beneficial activity of antibodies against CD40L (such as ruplizumab, ab1793 and
toralizumab)
45–47
. Unfortunately, the thromboembolic activity of these antibodies, linked to
crosslinking of CD40L expressed by platelets, led to discontinuation of their further
development (TABLE 1). To circumvent the thromboembolic effect, preclinical studies in
mice or nonhuman primates are assessing new biologic agents that block CD40L without
causing aggregation of the molecule; these biologic agents either lack an Fc region or are
mutated to prevent their binding to Fc receptors. Results suggest that they can be as
efficacious as the parent (Fc intact) antibody — without the thromboembolic effect — in
scenarios such as animal models of lupus
48–50
. However, in certain settings Fc effector
function might be necessary for therapeutic activity, as shown by the lack of activity of an
aglycosylated anti-CD40L antibody in nonhuman primate transplantation studies
48
.
MEDI4920, a Tn3-fusion protein with reactivity to CD40L, is currently in phase I safety
trials. Additionally, antagonist and/or depleting antibodies against CD40 have been produced
(ch5D12, chi220–BMS-224819, ASKP1240, FFP104, CFZ533), with encouraging
preclinical results
51
, and some of them are being tested in phase I–II trials in Sjögren
syndrome
52
, RA
53
and other autoimmune conditions (TABLE 1). If these strategies can
overcome the adverse effects associated with agents that block CD40-C40L interactions,
such agents are an attractive avenue, and the possibility for clinical benefit in rheumatic
diseases is high.
OX40L
OX40L (also known as TNFSF4) is an inducible molecule expressed on several cell types,
although arguably most importantly, on APCs. OX40 (also known as TNFRSF4) is largely
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found on activated T cells as well as natural killer T cells and innate lymphoid cells such as
natural killer cells
5,54
(FIG. 3). OX40L can trigger signalling through its receptor OX40,
resulting in a range of activities including expansion and accumulation of effector T cells
(such as type 1 T helper cells (T
H
1), type 2 T helper cells (T
H
2), type 17 T helper cells
(T
H
17) and cytotoxic T lymphocytes) and their cytokine production
5,6,11,54
. Additionally,
reverse signalling through OX40L can promote expression of inflammatory cytokines (such
as IL-12 or TNF) in APCs
5,54
, although the importance of this activity as compared with
that driven by OX40 is not clear at present.
Data from human and mouse studies suggest that the OX40–OX40L axis has an important
role in rheumatic diseases. Blockade of OX40L reduces bone and cartilage destruction in
mouse models of collagen-induced arthritis
55,56
or autoimmune arthritis
57
, with results from
the former model being attributed to reduced numbers of collagen-specific T cells. Synovial
fluid samples of patients with RA contain elevated numbers OX40-expressing T cells,
suggesting OX40 signalling controls T cell numbers in human RA
56,58
. Targeting of OX40
with cytotoxic drugs to deplete T cells has also shown some therapeutic benefit in an animal
model of adjuvant arthritis
59
. Surprisingly, signalling via OX40L antagonizes the activity of
RANK in promoting osteoclast development from macrophage progenitors. OX40L-
deficient mice are accordingly osteopenic
56
, although the implication of this finding in the
context of therapeutic inhibition of OX40–OX40L interactions in arthritis is not clear.
In patients with SLE who have proliferative glomerulonephritis, OX40L is upregulated in
glomeruli, most likely on endothelial cells
60
and/or dendritic cells
61
. Similarly, studies have
shown that in peripheral blood and renal biopsy samples from patients with lupus nephritis,
OX40 expression by CD4
+
T cells correlates with disease activity, urine proteinuria and
serum creatinine
62–65
. Furthermore, on the basis of an initial report
66
, many studies have
confirmed an association between susceptibility to developing SLE and polymorphisms
upstream of the
OX40L
gene (also known as
TNFSF4
), which probably leads to its
increased expression. The OX40–OX40L axis is also involved in kidney disease, as patients
with Henoch–Schönlein purpura with nephritis have elevated levels of serum OX40L and
OX40
+
T cell numbers compared with patients without nephritis
67
. Surprisingly, no reports
have yet demonstrated a functional role for these molecules in mouse models of nephritis,
even though human studies imply that OX40–OX40L crosstalk between T cells and
endothelial or dendritic cells might contribute to disease.
As well as controlling the accumulation and/or activity of pathogenic effector T cells,
OX40–OX40L interactions have been associated with production of pathogenic antibodies.
Transgenic mice overexpressing OX40L display elevated levels of anti-DNA antibodies
68
.
Furthermore, soluble OX40 and/or OX40L are increased in the plasma of patients with
early-stage RA compared with healthy individuals, and correlate with levels of anti-
citrullinated protein antibodies and IgM rheumatoid factor
69
. Similarly, an association
between OX40L expression on myeloid APCs (dendritic cells and monocytes), SLE disease
activity and anti-ribonucleoprotein (RNP) antibodies has been described
61
. As activated B
cells express OX40L, this ligand could directly signal and contribute to autoantibody
production. However, the primary rationale for the association with anti-RNP antibodies is
that OX40L on dendritic cells can signal via OX40 expressed by T cells and might aid
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