T cell maintenance: Care and Feeding

An abstract with the words ‘dynamic’ and ‘interplay’ usually signals a paper to be avoided but this one tells an interesting story about T cell survival. T cells require occasional stimulation through their antigen receptor (TCR) and feeding with interleukin-7 (IL-7), a product of stromal cells, monocytes, and some epithelial cells. T cells seem to share a limited amount of IL-7 through a negative feedback on the receptor gene transcription (IL-7Ra).

Tickling the TCR is trickier: too much could provoke autoimmunity, too little and the T cell dies. Here, Park and colleagues show that IL-7 (and other gamma common chain agonists) also regulates transcription of the CD8 gene encoding the monomorphic co-receptor of the TCR on a large subset of T cells. Moreover, they report that TCR signals from endogenous antigens inhibit CD8 expression, thereby promoting self tolerance.
The balance is illustrated in HY RAG mice, which are are engineered to contain only T cells specific for HY (a tissue antigen encoded on the Y-chromosome and therefore expressed only in males). If no antigen is present (females), then there is no TCR engagement, leading to more CD8 expression and less IL-7Ra expression than normal (Figure, top vs. second line). With more (and more) TCR engagement in males with appropriate H-2b alleles, comes less CD8 and more IL-7Ra expression (bottom 2 lines). They term this dynamic interplay “coreceptor tuning”.
Park JH, et al. Nat Immunol. 2007 Oct;8(10):1049-59. 'Coreceptor tuning': cytokine signals transcriptionally tailor CD8 coreceptor expression to the self-specificity of the TCR.

Adaptive Control of Innate Immunity

Innate immunity controls infections while the adaptive immune response develops. In the absence of T cells, infections that kill the host were thought to result from an uncontrolled infection. However, Kim and colleagues demonstrate here that the adaptive immune response is also necessary to control the innate immune response. They show that host death can result from an uncontrolled innate response that occurs in the absence of an adaptive immune response.
Triggering the innate immune receptor TLR3 with poly(I:C), a double stranded RNA that mimics viruses, produced a destructive and potentially lethal “cytokine storm”. This fatal reaction required Natural Killer (NK) cells and TNF. The figure is a portion of figure 4 showing the response of immune deficient animals: panel e shows that abrogating TNF signaling but not immunoglobulin signaling is protective and panel f shows that NK cell depletion is protective. As the authors note, these findings might be also important for understanding the responses of “individuals with congenital or acquired immune deficiency”.
Kim et al. Nat Med. 2007 Nov;13(10):1248. "Adaptive immune cells temper initial innate responses".

Shear forces trigger T cell adhesion

At sites of inflammation, T lymphocytes bind to activated endothelium and move from the bloodstream into lymph nodes. Recruited T cells remain highly motile within the lymph node, scanning many dendritic cells and other antigen-presenting cells for cognate antigen. T cell motility is stimulated by chemokines, which can also promote adhesion by activating integrins.

Woolf and colleagues observed that many cells, including T cells themselves, express the same adhesion molecules and asked how premature arrest and clumping was avoided. To delineate the contributions of chemokines and adhesion molecules, they coated surfaces with CCL21, ICAM-1, or VCAM-1 and observed T cells moving on these surfaces. They found that surface-bound CCL21 stimulated T cell motility but soluble CCL21 did not. Although CCL21 also induced clustering of LFA-1 and VLA-4, these integrins did not mediate adhesion to their ligands ICAM-1 and VCAM-1. However, when T cells were exposed to shear stress, they rapidly developed strong adhesion to ICAM-1 or VCAM-1 (Figure). Video microscopy of T cells and lymph nodes cells obtained from mice deficient in adhesion molecules provided additional support for their conclusion that integrin-mediated adhesion, which is crucial to T cell recruitment from the bloodstream, is 'silenced' within the lymph node due to the absence of shear force.

Woolf et al. Nat Immunol. 2007 Oct;8(10):1076-85. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces

TNF-R family member 4-1BBL keeps TNF production “on”

The bacterial cell wall component LPS (lipopolysaccharide) binds to Toll-like receptor 4 (TLR4) on macrophages and stimulates them to produce the strong pro-inflammatory cytokine TNF (tumor necrosis factor). To identify potential signaling proteins, Kang and colleagues used the intracellular domain of TLR4 in a two-hybrid screen of binding proteins. They isolated 4-1BBL (CD137L), a transmembrane protein previously best known as a T cell costimulator, and the well-known TLR signaling protein MyD88. 4-1BBL binding was not abrogated by a P712H substitution in the TLR4 TIR (Toll-IL-R) motif that is required for MyD88 binding, indicating that they bind to different parts of TLR4.

LPS induced less TNF production at 24 h from 4-1BBL “knockout” (4-1BBL KO) macrophages than from wild-type (WT) macrophages, though equivalent amounts of IL-1beta were produced. Moreover, kinetic analysis demonstrated that TNF production was equivalent soon after stimulation but ceased after about 6 hours in 4-1BBL-KO macrophages while continuing in WT macrophages (Figure, panel b), accounting for the difference at 24 h. Although 4-1BBL was cloned as the ligand for 4-1BB, which is also expressed by macrophages, it does not contribute to this response (Figure, panel c).

LPS induces a gradual appearance of 4-1BBL on the cell surface, with marked accumulation after 2 hours, which accounts for the delayed influence on TNF production. Indeed, surface 4-1BBL alone at high levels of expression, or cross-linked at lower levels of expression, is sufficient induce TNF expression. Finally, in the ultimate test of relevance, they showed that 4-1BBL-KO mice survived a dose of LPS that killed all WT mice. Their discovery of 4-1BBL's role in sustained TNF production provides a new target for therapeutic intervention in the development of inflammatory diseases.

Young Jun Kang et al. Nat Immunol. 2007 Jun;8(6):601-9 "Cell surface 4-1BBL mediates sequential signaling pathways ‘downstream’ of TLR and is required for sustained TNF production in macrophages"

Micro RNA sets sensitivity of T cell

MicroRNAs are single-stranded RNA molecules, averaging 22 nucleotides in length and encoded in the genome, that regulate the expression of other genes. MicroRNA and the process of “RNA interference” were discovered only a few years ago. The miRNA miR-181a was known to be hightly expressed in the thymus and to function in T cell development. Li and colleagues investigated the function of this miRNA by manipulating its expression in T cells. Forced (ectopic) expression makes T cells more sensitive to stimulatory peptides, generating a stronger calcium flux with about half as many peptides (Figure, left panel). Even an antagonist peptide, which usually turns off T cells, triggers calcium flux (right panel) and growth factor (IL-2) secretion from T cells overexpressing miR-181a. This apparently occurs by miR-181a repressing transcripts encoding phosphatases, including DUSP5 and 6, SHP-2 (but not SHP-1), and PTPN22. For example, phosphorylated ERK (the substrate for DUSP6) remains high longer after activation of T cells expressing more miR-181a. Knocking down individual phosphatases did not overcome miR-181a-enhanced sensitivity, suggesting that multiple pathways are targeted. However, overexpression of DUSP6 or DUSP5 or SHP-2 (using modified sequences that are not recognized by miR-181a) restored repression in T cells expressing miR-181a. The authors designed an “antagomir” to block miR-181a and found it disrupted selection of antigen-specific T cells in the thymus. This new and unexpected mechanism of regulation may be important in understanding T cell development and activation.
Cell. 2007 Apr 6;129(1):147-61. “miR-181a Is an Intrinsic Modulator of T Cell Sensitivity and Selection.” Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, Liu G, Braich R, Manoharan M, Soutschek J, Skare P, Klein LO, Davis MM, Chen CZ.

Interferon type I mediates resistance to Trypanosomes

The intracellular parasite Trypanosoma cruzi causes Chagas disease in over 16 million people worldwide, mostly in poor and rural areas in Central and South America. Chagas disease has a variety of debilitating symptoms and kills about 50,000 people annually. Prevention focuses on killing the insect vectors; treatment is expensive and not entirely effective. Resistance to T. cruzi in humans is thought to be initiated by TLRs and effected by interferon (IFN)-gamma and nitric oxide (NO), produced by host T cells and NK cells, which kill the parasite. Here, Koga and colleagues investigated the pathway between TRL activation by the parasite and effector mechanisms. They found that resistance requires TLR signaling because infected macrophages and dendritic cells cultured from mice without the TLR adapter proteins MyD88 and TRIFF support T. cruzi proliferation. Since TRIFF signals the production of type I interferons, namely the IFN-alphas and IFN-beta, the investigators tested cells from mice without a receptor for these IFNs: IFNAR1-/-. As expected, these cells were unable to control T. cruzi in culture (Figure, panel A). Moreover, these mice quickly succumb to infection (panel B). A gene expression analysis found that IRG47 was strongly induced and previous reports had implicated this GTPase in the IFN response. When IRG47 expression was “knocked down” with RNAi, even wild type cells were rendered susceptible to T. cruzi, demonstrating the critical role of type I IFNs and induced IRG47 in resistance to this trypanosome.
"TLR-Dependent Induction of IFN-beta Mediates Host Defense against Trypanosoma cruzi." Koga et al. J Immunol. 2006, 177: 7059

TLR2 Helps Bacteria Weaken Gums

The bacterium Porphyromonas gingivalis gets most of the blame for periodontal disease, which erodes bone that supports teeth and is the leading cause of tooth loss. P gingivalis adheres to oral surfaces using molecules such as lipopolysaccharides (LPS) and lipoproteins. These molecules are recognized by Toll-like receptors (TLRs) that are expressed on cells of the immune system: TLR2 binds bacterial lipopeptides and TLR4 binds LPS. Burns and colleagues tested how these TLRs are involved in the host mouse response against P gingivalis. They implanted metal coils subcutaneously and injected bacteria into the lumen, and then sampled the space at later times to monitor the immune response in wild-type (WT), TLR2, and -4 knockout (-/-) mice. They observed differences in the induction of cytokines, most dramatically a reduced accumulation of TNF, interferon-gamma, and interleukin-10 in TLR2-/- mice. Surprisingly, TLR2-/- mice cleared the bacteria from the injection site and blood within a day. In contrast, bacteria remained in the blood of WT and TLR4-/- mice for at least 4 days. Moreover, when mice were orally challenged with P. gingivalis, significant bone loss resulted within 6 weeks in WT but not TLR2-/- mice (Figure). Would blocking TLR2 with lipopeptides in a mouthwash or chewing gum help protect teeth?
J Immunol. 2006 Dec 15;177(12):8296-300. "TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption." Burns E, Bachrach G, Shapira L, Nussbaum G.

Flu Vaccine - wheeze now or sick later?

The prestigious New England Journal of Medicine just published a paper claiming to demonstrate a superiority of attenuated live flu vaccine over the conventional, dead flu vaccine for young children. About half as many kids in the attenuated vaccine group went on to contract flu (figure). USA Today and other publications trumpeted the results with headlines such as "FluMist spray protects kids better than flu shots". They downplayed the concern raised by independent scientists that the live vaccine "comes with a significant risk for medically important wheezing".
This story illustrates some of the problems with reporting such clinical trials. First, it was "sponsored" (paid for) by MedImmune, the producer of the live vaccine. Sponsors have been known to not publish disappointing results, producing a publication bias. Although we can welcome these results, the arrangement raises questions about the "spin". How should parents and pediatricians balance the decrease in disease (from 9% to 4%) with the increase in wheezing (from 3% to 6%)? Second, the description of the trial is misleading. The Methods section begins "children ... were randomly assigned ...to receive either ... live attenuated influenza vaccine ... or inactivated vaccine in a double-blind manner" (emphasis added). This use of the term "double-blind" is nonsensical because random is completely "blind". The wording seems intended to make the reader think the trial was double-blind. Double-blind is the gold standard for trials because it reduces bias by keeping the both the doctor and patient ignorant of which treatment they are assigned. In this trial, however, the treatments could not be "blinded" because one involves a shot and the other involves sniffing the virus, treatments that are clearly distinguishable even by a patient. Finally, journals and journalists like to report positive, exciting news. These results merit a more sober discussion.
N Engl J Med. 2007 Feb 15;356(7):685-96. "Live attenuated versus inactivated influenza vaccine in infants and young children." Belshe RB, Edwards KM, Vesikari T, Black SV, Walker RE, Hultquist M, Kemble G, Connor EM

Tuberculosis Traffic Arrest

Mycobacterium tuberculosis is a life-threatening pathogen that persists in infected cells after being phagocytosed (eaten). Phagocytosed matter is usually digested but M. tuberculosis avoids this fate by blocking the acidification of the its compartment. The process can be studied in U937 cells because they behave like macrophages ("big eaters") and grow well in culture. Rab proteins form a large family of Ras-like GTPases that mediate vesicle trafficking, budding, etc. A recent report showed that Rab14 participates in trafficking to the early endosome, a stage just after the phagosome. Kyei and colleagues found that Rab14 associated with phagosomes containing live tuberculosis mycobacteria and investigated whether Rab14 was required for the arrested development of the phagosome. Phagosomes of U937 cells contain live tuberculosis bacilli of the strain H37Rv (green label, left panels). The middle panels show the same cells stained with a dye that detects endosomes that have undergone acidification, a step toward digestion and marker of the early endosome. A "knock-down" of Rab14 with siRNA permits the maturation of the phagosome, as demonstrated by the overlap of the acidified (red), bacillus-containing (green) phagosomes, (yellow color in the lower right panel). These findings suggest that inhibiting Rab14 may permit the infected cell to digest M. tuberculosis, thereby offering a new target for therapeutic intervention and potentially reducing the burden of this disease.
Kyei GB, Vergne I, Chua J, Roberts E, Harris J, Junutula JR, Deretic V. "Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest". EMBO J. 2006 Nov 15;25(22):5250-9.

Infectious Cancer Cells

Some cancers are caused by infectious agents, such as viruses that cause cancer by infecting and transforming host cells. For example, Epstein-Barr virus (EBV) is implicated in Burkitt's lymphoma, nasopharyngeal carcinoma, and other cancers (review). For canine transmissible venereal tumor (CTVT), however, the tumor cell itself was thought to be the agent. To test this hypothesis, Murgia and colleagues isolated CTVT cells and non-cancerous blood cells from 40 different dogs on 5 continents, and determined their relationships by comparing nuclear and mitochondrial genetic markers. They conclude that all the tumors are clones of a single tumor that arose between 200 and 2,500 years ago. Although the tumor has a very unusual chromosome arrangement (aneuploid), it is stable. They also studied the highly polymorphic histocompatibility genes. Expression of histocompatibility proteins, which would block transplantation between different animals, is reduced but not extinguished on CTVT cells. Another study demonstrated that CTVT cells make TGF-beta1, which could also reduce the immune response. This surprising cell could provide ideas for inducing tolerance of allografts.
Claudio Murgia, Jonathan K. Pritchard, Su Yeon Kim, Ariberto Fassati, and Robin A. Weiss "Clonal Origin and Evolution of a Transmissible Cancer" Cell 126, 477–487, August 11, 2006