Richard Flavell, PhD, FRS

The innate immune system contains genome-encoded receptors that
provide a first line of defense to infection. Activation of innate
immunity triggers adaptive immunity. There are three classes of innate
immune receptors:

• Toll-like receptors (TLRs), which sense agents in the extracellular/vesicular space;
• Nod-like receptors (NLRs), which sense microorganisms that penetrate the cytoplasmic space; and

• RIG-like receptors (RLRs), which recognize viral infection and trigger type 1 interferon production.

We identified the TLRs for double-strand RNA
(TLR3), single-stranded RNA (TLR7), flagellar protein (TLR5), and
lipoprotein (TLR1/2).
Upon penetration of the cytoplasm, NLRs trigger
NF?B activation, interleukin-1 (IL-1) production, or apoptosis. In
humans, NLR mutation correlates with inflammatory disease. Nod2 carries
a leucine-rich repeat region probably recognizing bacterial muramyl
dipeptide, a nucleotide-binding domain mediating conformational change,
thereby enabling oligomerization between CARD domains of Nod2 and
downstream receptor-interacting protein (RIP) kinase, causing
activation of NF?B and antimicrobial peptides. NOD2
is mutated in Crohn's disease (CD), an inflammatory bowel disease
(IBD). Our Nod2-deficient mice were more susceptible to infection with
pathogens delivered to the gut, because of reduced production of
antimicrobial peptides. Thus, patients with CD may be unable to develop
an effective antimicrobial response, causing enhanced infection and
severe inflammation.

The Nalp proteins comprise a second arm
of the NLR family. We study several of these, including Nalp3 (NLRP3),
which senses infection or other stress that leads to K+
efflux and activates the "inflammasome" through oligomerization with
the adaptor apoptosis-associated speck-like protein (ASC), enabling ASC
to bind to and activate caspase-1 to process pro-IL-1ß and other
substrates.

We found that multiple stimuli activate the Nalp3 inflammasome, including Listeria
infection. Jürg Tschopp (University of Lausanne) showed that this
inflammasome recognizes uric acid crystals, explaining the inflammatory
properties of uric acid in gout. We found that alum, a crystalline
immune adjuvant and the only USA-approved human adjuvant, activates the
NALP3 inflammasome, which triggers macrophage inflammatory cytokine
production and adaptive immunity in vivo.

Disruption of the pathway
eliminates alum's adjuvant capacity. Likewise, particulate
environmental pollutants, including silica and asbestos, also activate
the Nalp3 inflammasome to cause devastating chronic inflammatory
disease. Thus, inflammasomes mediate anti-infective immunity,
immunopathology to environmental pollutants, and adaptive immunity.
The immune response sometimes reacts to
self-tissues, causing autoimmunity. How can antigenic stimulation of a
lymphocyte lead to such different outcomes? During an immune response
or in autoimmunity, the lymphocytes divide and differentiate into
effector cells. However, when immune tolerance occurs, the cell is
either inactivated or dies.

How are the decisions made to proliferate,
differentiate, be tolerized, or die, and how is this controlled?

Regulatory cells producing inhibitory cytokines are critical to prevent
autoimmunity. Of these, the CD4+CD25+Foxp3+Treg
is the most studied. The functioning, generation, and maintenance of
regulatory T cells (Treg) are controlled by cytokines. Both
transforming growth factor-ß (TGFß) and IL-10-family cytokines are
important. Mice lacking TGFß develop autoimmunity to several tissues.
To elucidate upon which cells TGFß acts, we expressed a
dominant-negative TGFß receptor (dnTGFßRII) on either T cells or
antigen-presenting cells (APCs). Mice displaying the dnTGFßRII on T
cells recapitulate the diseases of TGFß-knockout mice: autoimmunity and
IBD. In addition to autoimmunity, such animals have an enhanced
anti-infective response, better resistance to infection. Finally, mice
carrying the dnTGFßRII on their T cells are resistant to tumors. Thus,
tumors use TGFß to inhibit the antitumor T cell response; but if TGFß
cannot act, immune clearance of tumors occurs.
To determine whether TGFß controls innate immunity,
we expressed dnTGFßRII using the CD11c promoter, which expresses in
dendritic (DC) and natural killer (NK) cells, both key mediators of
innate immunity. When innate immune cells cannot be inhibited by TGFß,
both NK and DC innate, as well as adaptive, immune responses are
enhanced. CD11c dnTGFßRII mice are also more susceptible to
autoimmunity, because TGFß fails to control APC function. Thus, TGFß
controls T cells, APCs, and NK cells.
We revealed additional mechanisms of TGFß function
by studying conditional-knockout mice lacking TGFßRII on T cells. TGFß
is required for Treg homeostasis and function and TGFßRII must be
present on a target cell for a Treg to be suppressed. We also found
that TGFß controls the magnitude of T helper 1 cell (Th1) response by
setting the level of CD122 ß chain of the IL-15 receptor, which
controls the pool size of Th1 cells. Many cells make TGFß. To determine
which TGFß source is important, we first eliminated TGFß on T cells,
using conditional targeting. Mice with T cells that cannot make TGFß
also developed autoimmune disease and IBD, albeit slower than mice
lacking the receptor on all T cells. Thus, T cell–produced TGFß is
important in immune response, but other sources must play a role.
Regulatory T cells that cannot produce TGFß poorly control IBD, and T
cell–produced TGFß is essential to generate Th17 cells, which mediate
disease in experimental autoimmune encephalomyelitis.
T cells are activated and differentiate into
specialized effector cells.

How is the effector pathway triggered that
is appropriate to the class of infection? We found the Th2 response is
activated when parasite antigen induces Notch ligand expression on
dendritic cells. This activates Notch in naïve T cells, which in turn
induces GATA3, the key Th2 transcription factor, by a Notch-responsive
promoter. Thus is a pathogenic signal converted to a signal for T cell
differentiation through Notch.

We identified cis-regulatory elements that are the targets of transcription factors, such as GATA3. In the interleukin-4 (IL-4) locus, the IL-4, IL-13, and IL-5 genes are clustered, and several DNA elements within that region are important for gene expression. IL-4 gene regulation occurs through epigenetic mechanisms that target regulatory elements distal from the IL-4
gene. One of these elements is a previously unrecognized locus control
region (LCR) that is found embedded in the introns of the RAD50
gene in the cluster. This LCR, together with these respective promoters
and other cis elements of the locus, is in a preassembled complex in
naïve T cells that serves as a hub from which epigenetic changes in
histone acetylation and DNA methylation occur and enables rapid
response of the loci.

When naïve T cells are activated, both the IL-4 locus on chromosome 11 and the interferon-? (IFN-?)
locus on chromosome 10 are expressed almost immediately, despite the
fact that following differentiation these loci are never coexpressed
but instead are alternatively expressed in the Th2 and Th1 lineages,
respectively. To investigate this rapid coexpression, we examined the
physical relationship between these two loci on the different
chromosomes. The LCR of the IL-4 locus on chromosome 11 and the IFN-?
gene region on chromosome 10 are associated in the interphase nucleus
of the precursor cells but separate upon differentiation into effector
cells. Mutation in the LCR on chromosome 11 delays expression of the IFN-?
gene on chromosome 10. We find other such associations and further
evidence for their functional roles. Thus, regulatory sequences on one
chromosome likely control "in trans" gene expression on other
chromosomes.
Our laboratory retains a long-standing interest in
the underlying mechanisms of apoptosis. The program of cell death is
triggered through the activation of cysteine proteases called caspases.
Caspase-3 and -7 cleave similar substrates. Caspase-7–knockout mice
have only a mild phenotype, but the combination with caspase-3
deficiency results in embryonic lethality. Caspase-3 and -7 are also
required for upstream mitochondrial functions in apoptosis, via a
positive-feedback loop, in addition to their roles as effector
caspases.

• TGF-b in autoimmune diabetes

• TGF-b in memory T cell development

• The role of BCL2 in aging of the immune system

• AMCase in lung inflammation

• The role of TGF-b in the immune response to melanoma

• Generation and analysis of mice with human immune systems

• Developing immune therapies for Type 1 diabetes

• Genetic approaches to immune function and tolerance