Kill your ECTV!

It’s a mystery why mice resist the Ectromelia poxvirus (ECTV) better than humans resist smallpox, a similar infection. Previous studies demonstrated a role for natural killer (NK) cells in ECTV resistance. Fang and colleagues show here that in addition to 'opportunity', actual NK killing with perforin is essential to the resolution. Many T cells armed with IFN-g were also found in the spleens of infected mice, suggesting that NK cells involve T cells in the murderous conspiracy.

The proximal motive for NK action is NKG2D (AKA KLRK1), which can be triggered by viruses that increase MICA expression. NK cell depletion or NKG2D blockade, but not the depletion of T cells, stop virus clearance (Figure 4B shown). Fang makes a good case for poxvirus clearance triggered by NKG2D/KLRK1 on NK cells.

Fang M, Lanier LL, Sigal LJ. A role for NKG2D in NK cell-mediated resistance to poxvirus disease. PLoS Pathog. 2008 Feb 8;4(2):e30

Genome Modules Track Disease

The idea is alluring: take a little blood, measure some gene transcripts, and diagnose disease. The problems have been (1) identifying the genes that signal disease, and (2) overcoming natural and laboratory variations. Chaussabel and colleagues approached these problems with reliable microarray measurements of samples from 239 people and by identifying small sets, “modules”, of genes that are coordinately-expressed across a “wide range” of conditions. These modules are likely to be more reproducible than measurements of individual genes. The (seemingly arbitrary) conditions included juvenile idiopathic arthritis (47 patients), lupus (40), and type I diabetes (20), melanoma (39), immune-suppression after liver transplantation (37), and infections with E. coli (22), Staph aureus (18), and influenza (16). Genes from all 239 samples were clustered using a “K-means algorithm” with K=30 (which yields up to 30 groups) without regard to the magnitude of change in expression. The first round grouped all 8 conditions, the second round, 7, and the third, 6 (procedure). A total of nearly 5,000 transcripts in 28 modules were identified using the sample data. When the data were randomized, no modules were identified in 200 trial clusterings, suggesting that the modules reflect states of health and are not statistical artifacts. Modules range from 22 to 325 transcripts. Genes with known relationships, e.g. particular cell types or pathways, constitute about half the modules, underscoring the functional coherence.

Two examples of how health conditions change these 28 modules are shown: healthy vs. melanoma (top) and healthy vs. lupus (bottom) (from fig. 1B, red=overexpressed, blue=underexpressed). All 8 conditions are clearly distinct from healthy and distinguished from each other. The authors also identified 'biomarker' modules, e.g. M1.2 & M1.8 in melanoma or M1.7 & M3.1 in lupus in the examples shown, and made circular ('spider') graphs that can display several patients or one patient over a course of treatment.

In contrast to typical repository-biomarker analyses, very few patient samples were used to generate these modules. It will be crucial to see how they accommodate more, different samples. Also, I'm curious whether rational groupings of 'conditions', e.g., cancers or infections or autoimmune diseases, might further improve module definition. Finally, in addition to improving patient care, this information should provide invaluable insights into disease origin and progression.
Chaussabel et al. A modular analysis framework for blood genomics studies: application to systemic lupus erythematosus. Immunity. 2008 Jul;29(1):150-64.

Tolerating Aire - Out of the Thymus

The immune system protects us against infections but it must also tolerate the many different proteins that constitute our bodies. T lymphocytes of the immune system that react against the body’s own proteins could trigger autoimmunity, so they are usually deleted during their maturation in the thymus. A protein called Aire (Autoimmune regulator) turns on expression in the thymus of nearly 2,000 genes that are otherwise expressed in only a few organs and tissues, thereby exposing new T cells to these proteins and avoiding autoimmune disease. Indeed, mutations in Aire result in a broad autoimmunity against many organs and tissues. Previously, Aire was thought by many to be expressed only in the thymus.

Gardner and colleagues detected Aire in rare stromal (non-lymphoid) cells they called extrathymic Aire-expressing cells (eTACs) within the spleen, lymph nodes, and Peyer’s patches (nodes associated with the gut). These cells are located between the T and B cell areas of these organs but are highly mobile. Surprisingly, Aire turned on different genes in eTACs than were turned on in the thymus, suggesting that Aire expression in eTACs is not a ‘safety net’ for T cells that accidentally escaped the thymus but rather acts to delete additional autoreactive T cells. In eTACS, Aire turned on fewer than 200 genes, but several of these are suspected to be involved in autoimmunity, including desmoglein 1a (in pemphigous foliaceus) and the N-methyl-D-aspartate receptor 2C (in lupus). In an animal model of type 1 diabetes, eTACs expressing glucose-6-phosphate-related protein deleted T cells specific for this protein and prevented onset of this autoimmune disease (Fig. 2f shown, open squares represent mice expressing glucose-6-phosphatase-related Adig in eTACs).

These findings suggest that manipulating eTACs is a promising approach to therapy for autoimmune disease. Comparing eTACs in healthy people and autoimmune patients might also help identify deficiencies that can be corrected to prevent or ameliorate disease.

Gardner JM, Devoss JJ, Friedman RS, Wong DJ, Tan YX, Zhou X, Johannes KP, Su MA, Chang HY, Krummel MF, Anderson MS. Deletional tolerance mediated by extrathymic Aire-expressing cells. Science. 2008 Aug 8;321(5890):843-7.

Homozygosity mapping: Autism in the family

Most genes identified in genome-wide analyses contribute only modestly to disease risk or protection, with already low relative risks changed only ~20%, and taken together probably contribute to a minority of disease cases. Morrow and colleagues took a different approach, believing that analyzing individual families might reveal higher-risk genes for autism spectrum disorders (ASDs). They noted that offspring of first-cousins have twice as many neurological birth defects, including ASDs.

They also noted that a significant involvement of autosomal recessive genes would be 'signaled' by change in the male-to-female ratio (because the M:F ratio is typically 1:1 for autosomal recessive traits vs. male-dominant ASD). Indeed, among the 104 families recruited for the study, the M:F ratio was 2.6 for the offspring of the 88 consanguineous families (cousins) vs. 7.4 for the non-consanguineous.

They genotyped using arrays of SNPs and BACs, the latter validated for comparative genomic hybridization (CGH) detection of copy number variation (CNV) due to deletions, etc. Few de novo (not inherited) CNVs were detected. Large deletions on both DNA strands of the affected child (homozygous), and one strand of each parent (hemizygous), were found in 5 of the 78 consanguineous families but not among any of the other ~400 ASD cases or ~2,400 controls. In the figure (from Figure 1C), the SNPs spanning a large deletion in the AU-3100 pedigree is shown: the parents 3103 & 3104 (top and second) are hemizygous as is one child (3102, third down) but the other child, 3101 (bottom), who is homozygous for the deleted chromosome, has autism and seizures.

The investigators were surprised to find 3 genes linked to the 2 largest deletions were previously identified as being regulated by neural activity, and thus candidates for involvement with learning. Mutations in one of these genes, NHE9, were detected in non-consanguineous families and associated with additional neurological disorders (epilepsy) and learning delays (language acquisition).

EM Morrow et al. Identifying autism loci and genes by tracing recent shared ancestry. Science. 2008 Jul 11;321(5886):218-23.

Therapeutic Interruption of Protein Degradation

Some antibodies that bind ‘self’ molecules (autoantibodies) can signal and even cause autoimmune diseases. Antibodies are produced by B cells, largely by a differentiated, typically short-lived variety called plasma cells (PC), which dedicate 10-20% of their protein-synthesis capacity to immunoglobulin. PC develop in the germinal centers of lymph nodes and spleen and then migrate to the bone marrow, where a subset endures. Treatments that target B cells, such as Rituxan, might often fail to substantially reduce autoantibodies because PC are resistant.

Neubert and colleagues reasoned that precisely the hallmark of PC – their high rate of protein synthesis – could sensitize them to bortezomib (Bz, Velcade), a proteasome inhibitor that is therapeutic in some cancers. Bz interferes with the ubiquitin protein degradation pathway, thereby blocking NF-kB release and promoting the unfolded protein ‘stress’ response that induces apoptosis. Here, they show that Bz reduced short- and long-lived PC (CD138+ CD25- cells with cytoplasmic immunoglobulin light or heavy chains) ~90% in the bone marrow and spleens of mice after just 48 h treatment. Antibody-secreting cells remained decreased during 8 weeks of treatment. Bz had little or no effect on total B cell numbers or many B cell subsets, although germinal center B cells were reduced (Fig. 1). Cyclophosphamide or dexamethasone were less effective in reducing the numbers of long-lived (BrdU-) PC, suggesting a cellular basis for the failure of these current therapeutics in reducing autoantibodies (Fig. 2). In bone marrow and splenic PC, Bz rapidly induces transient expression of CHOP (20-40 fold within 4 h), a signaling protein previously implicated in the apoptotic response to the stress of protease inhibitors.

Lastly, they treated lupus-prone mice (NZB/W F1) with Bz and found it prevented onset of kidney disease (measured by proteinuria) and death. Remarkably, Bz was therapeutic in early disease, reducing serum levels of the autoantibody associated with kidney disease and preventing proteinuria in this lupus model as well as another (MRL/lpr: shown, dotted line Bz treated, solid line control treated, from Fig. 5a).

As Bz is already approved for use in humans, it should not be long before we know its therapeutic efficacy in lupus and other autoimmune diseases. Also, a new generation of proteasome inihibitors that target specific steps in the process are ready to be tested.

Neubert K, Meister S, Moser K, Weisel F, Maseda D, Amann K, Wiethe C, Winkler TH, Kalden JR, Manz RA, Voll RE. “The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis” Nat Med. 2008 Jun 8.

Adjuvants: a little less ‘dirty’, less secret

How do adjuvants – mixtures that promote immunization/vaccination – work? Malherbe and colleagues applied modern tools and ideas to answer this old question. They analyzed the immune responses of mice (B10.BR strain) to the protein PCC (pigeon cytochrome C), which they had previously shown to be dominated by helper T cells (Th) responding to a single PCC fragment (peptide ‘epitope’ associated with I-Ek) and expressing T cell receptors (TCR) encoded by Valpha11Vbeta3 (a11b3). They concluded from those previous studies that TCR affinity, above a threshold level, does not drive clonal diversity.

Here, they compared Th numbers and diversity after subcutaneous immunization in an aluminum precipitate (Alum), or in an emulsion known as “incomplete Freund’s adjuvant” (IFA), or complete Freund’s adjuvant (CFA = IFA + dead mycobacterium), or with aqueous oligodeoxynucleotide (CpG) that stimulates the innate immunity Toll Like Receptor-9 (TLR-9), or monophosphoryl lipid A (MPL) that stimulates TLR-4.

First, they compared the numbers of TCRa11b3+ Th in draining lymph nodes on day 7 after immunization with or without PCC antigen and found a ~30 fold range among the adjuvants. MPL induced the largest number of PCC-specific Th but also many nonspecific Th. CpG induced the largest differential between PCC-specific and nonspecific Th whereas Alum induced nearly as many nonspecific as specific Th. The response kinetics were all similar, peaking around day 7. Second, they analyzed the TCR sequence “features…that assort with PCC-specificity” in dozens of single Th cells, concluding that clonal dominance occurred with or without TLR agonists or antigen ‘depots’ (IFA, CFA, and Alum). Third, they found lower affinity TCR Jbeta2.5 regions predominated among Th responding to depot-forming adjuvants, whereas higher affinity Jbeta1.2 predominated among Th responding to the non-depot forming adjuvants (CpG and MPL). Also, Vbeta usage depended upon the adjuvant.
Fourth, in affinity tests using PCC-I-Ek tetramers, they found a range of responses similar to those they had found in TCRa11b3+ cells (from Figure 4C, open circles are immunizations without PCC, bars are immunizations with PCC). Also consistent with the sequence analysis, CpG and MPL induced higher levels of binding as measured by mean fluorescence intensity (MFI).
Fifth, upon PCC immunization of mice hosting Th with transgenic high- or low-affinity TCRs, they found that IFA stimulated both populations equally whereas MPL stimulated Th with the high-affinity TCR but not those with the low-affinity TCR. Sixth, they found MPL as effective as the depot-forming IFA in activating transgenic Th transferred 5 days after immunization. Finally, 400 ug PCC in MPL induced as much Th proliferation as did 40 ug without changing the ratio of high- and low-affinity TCR, though 4 ug was suboptimal.

These parameters will be useful guides in determining how adjuvants improve immune protection, the goal of vaccination, especially in humans.

Malherbe L, Mark L, Fazilleau N, McHeyzer-Williams LJ, McHeyzer-Williams MG. “Vaccine adjuvants alter TCR-based selection thresholds”. Immunity. 2008 May;28(5):698-709.

The 3% (sialylated) Solution

Intravenous Immunoglobulin (IVIG) is an immune modulator and anti-inflammatory agent used to treat a wide range of diseases, including rheumatoid arthritis (see also: 1990 NIH consensus panel report). IVIG contains antibodies (immunoglobulins, Ig) pooled from the blood of hundreds or even thousands of donors; pooling raises the risks of infection and complicates standardization.

Here, Anthony and colleagues tested their idea that sialylation is required for the therapeutic activity of the invariant region of Ig called Fc (fragment crystallizable). Sialic acid can be linked through alpha 2,3 or alpha 2,6, which are distinguished by the location of the hydroxyl and methyl groups on the 6 member ring.
First, they looked for these linkages in a preparation of IVIG by comparing its mass spectrum with standards. Nearly all the sialylated IVIG was alpha 2,6 linked and treatment with an enzyme that selectively removed this linkage abolished the anti-inflammatory activity of IVIG. Of potentially enormous significance was their success in making Fc derived from cloned human IgG therapeutic by alpha 2,6 sialylation. The figure shows the clinical score of mice made arthritic by administration of K/BxN sera alone (top line), or also treated therapeutically with IVIG (second line), 1/30 as much sialylated-enriched Fc from IVIG (0.033 g/kg SNA IVIG, third line), or an equivalent amount of recombinant, sialylated Fc (bottom line). This finding could lead directly to the manufacture of much safer, much more consistent IVIG preparations.
Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV. Science. 2008 Apr 18;320(5874):373-6. "Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc".

Microbe cheats death by misdirecting killer enzyme chaperone

Two million people die each year from tuberculosis and about a third of all humans are infected with the intracellular parasite responsible: Mycobacterium tuberculosis. This microbe overcomes the defenses of the macrophages it infects by inhibiting the fusion of phagosomes with lysosomes and avoiding the activity of reactive oxygen and nitrogen intermediates, including nitric oxide (NO). NO is extremely reactive and consequently short-lived, so the microbe misdirects the enzyme that produces NO, inducible NO synthase (iNOS), away from the phagosome. Davis and colleagues guessed that EBP50, a protein previously implicated in anchoring iNOS and other cellular proteins to the actin cytoskeleton, might be responsible.

They first showed that EBP50 is colocated with iNOS in the phagosomes of activated macrophages but less so after infection by M. tuberculosis. Reducing EBP50 with siRNA also reduced colocalization. Moreover, siRNA knockdown of EBP50 increased the viability of M. tuberculosis in infected macrophages, demonstrating its involvement in controlling this microbe (Figure, coronin is a control cytoskeletal chaperone that does not bind EBP50). However, since EBP50 is involved in many cellular processes, the specificity of the inhibition remains unproven.

Davis AS, Vergne I, Master SS, Kyei GB, Chua J, Deretic V. “Mechanism of Inducible Nitric Oxide Synthase Exclusion from Mycobacterial Phagosomes”. PLoS Pathog. 2007 Dec 7;3(12):e186

Identifying Friend or Foe - Mreg?

How do the immune cells within the gut distinguish between potentially dangerous pathogens and harmless or beneficial commensal organisms? [Hint: It’s not through PAMPs (pathogen-associated molecular patterns.)]

These investigators identified in mice a novel population of macrophages within the lamina propria, the layer of mucosa beneath the epithelium on the lumenal side of the intestine wall. These macrophages secrete IL-10 and other anti-inflammatory cytokines but no pro-inflammatory cytokines (even when stimulated through their Toll-like receptors by PAMPs). They also stimulate the development of regulatory T cells (Tregs) more potently than splenic macrophages (figure, CD4+ FoxP3+ Treg) and suppress the secretion of IL-17 by DC within the lamina propria. The authors suggest that a “dynamic interaction between these subsets may influence the balance between immune activation and tolerance”. They need to abbreviate the idea... how about 'Mregs'?

Denning et al. "Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses". Nat Immunol. 2007 Oct;8(10):1086-94.

Targeting the Plant Inside

Toxoplasm gondii are intracellular parasites that cause toxoplasmosis, the “third leading cause of death attributed to foodborne illness in the United States. More that 60 million [Americans] carry the Toxoplasma parasite, but very few have symptoms because the immune system usually keeps the parasite from causing illness” (CDC). T.gondii contain organelles or plastids called “apicoplasts” that were probably acquired in evolution along with an algal endosymbiont.

Apicoplasts contain unique proteins that may be effectively targeted by therapeutic agents. These researchers previously demonstrated a calcium control of protein secretion and identified conserved Ca-responsive proteins. Here they show that calcium-dependent development in T. gondii is controlled by a plant hormone produced by the apicoplast, abscisic acid (ABA, named for its role in abscission, the shedding of plant leaves, fruit, etc.). They found that fluridone, an herbicide that specifically inhibits an enzyme on the synthetic pathway producing ABA, blocked T gondii maturation. Finally, they demonstrated that the herbicide also worked as a therapeutic, protecting mice from a lethal dose of T gondii (shown, from Figure 4). Another infamous apicoplast-containing parasite is Plasmodium falciparum, which causes severe malaria, suggesting an exciting new approach to treating this scourge.

Nagamune et al. "Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii" Nature. 2008 Jan 10;451(7175):207-10.