HLA is the Major HIV Controller (MHC)

Upon infection by HIV, there follows a massive but transient increase in viruses in the blood followed by a period of years during which CD4+ T lymphocytes slowly decline. However, after the initial spike, some people have far fewer viruses in their blood and their CD4 counts do not decline. They remain apparently healthy, asymptomatic, for a very long time. These people are called “controllers” because their immune system controls virus replication without medication. The authors of this paper – with an impressive number of collaborators – looked for genetic variations in the controllers that could underlie the differences.

A genome wide association study (GWAS) performed with 1, 974 controllers (cases) and 2,648 progressors revealed a strong link with the HLA (the real human MHC, or major histocompatibility complex) on the short arm of chromosome 6 (figure shown). A weaker correlation with a chemokine receptor CCR5delta32 polymorphism previously identified with HIV resistance was also identified. HLA association is not surprising because practically everything immunological is strongly influenced by the HLA. What the investigators did next, however, was novel, imaginative, and highly illuminating.

From the SNP data, they were able to impute the controllers’ HLA type. Of the hundreds of HLA alleles, controllers tended to possess a remarkably narrow number of particular HLA-B and, to a lesser degree, HLA-A and -C alleles. They identified HLA-B*57:01, B*27:05, B*14/Cw*08:02, B*52, A*25 as protective alleles, and B*35 and Cw*07 as risk alleles. Moreover, they identified specific amino acids within the peptide-binding cleft as key variables in HIV control. These HLA amino acids define the ability of a pathogen to stimulate the cellular immune response.

These results provide a solid, well understood albeit complex mechanistic understanding to HIV progression and control. The next step could identify those HIV peptides that are bound preferentially by the protective and risk allele proteins.

The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation. The International HIV Controllers Study. Science. 2010 Nov 4.

Why Lupus can be Unresponsive to Glucocorticoid Therapy

Glucocoriticoids, including the natural hormone cortisol, are powerful immune suppressants. Synthetic glucocorticoids, such as dexamethasone and prednisone, are often taken by mouth to control autoimmune diseases. However, controlling the autoimmune disease systemic lupus erythematosus (SLE, lupus) often requires more aggressive treatments with high doses of more potent glucocorticoids, such as methylpredisolone, given intravenously.

Lupus is characterized by serum antibodies to nucleic acids (anti-nuclear antibodies, ANA), by a pattern of interferon-alpha-induced genes transcribed ("IFN signature”), and by an increase in plasmacytoid dendritic cells (PDC). PDC help produce antibodies and IFN. The IFN signature and PDC levels in patients are reduced to normal levels by high doses of intravenous glucocorticoids (see Figure 1c).

The authors noted that serum nucleic acids, which trigger the production of ANA, also stimulate PDC though specific receptors called toll-like receptors-7 and -9 (TLR-7 & -9) expressed on many cells of the immune system. Further, they hypothesized that TLR stimulation renders the PDC resistant to the suppressive effects of glucocorticoids.

Indeed, purified PDC treated with glucocorticoids (1-10 uM) do not survive overnight in a flask unless they are also treated with a nucleic acid (CpG) that stimulates TLRs (Figure 2a, first panel shown here). Nucleic acid-mediated protection is partially reversed by IRS, a synthetic oligonucleotide inhibitor of TLRs (IRS 954). Nucleic acid-containing immune complexes, isolated from the sera of lupus patients, also protected PDC by triggering TLRs.

Similar results were obtained with PDC from healthy people, suggesting that the PDC of lupus patients are not different but instead PDC are made glucocorticoid resistant by chronic stimulation of TLRs by nucleic acids. The authors conclude that “inhibitors of TLR7 and 9 signaling could prove to be effective corticosteroid-sparing drugs.”

Guiducci C, Gong M, Xu Z, Gill M, Chaussabel D, Meeker T, Chan JH, Wright T, Punaro M, Bolland S, Soumelis V, Banchereau J, Coffman RL, Pascual V, Barrat FJ. “TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus.” Nature. 2010 Jun 17;465(7300):937-41.

Note: Similar results were reported by Lepelletier and colleagues at the Hopital Necker.

Old news: Targeting the Target of Rapamycin (TOR) to Stay Youngish

There are many strategies to remain healthy and reduce the effects of aging: healthy diet and exercise, yoga, drastically reducing calories, etc... all disappointingly slow and indirect. On the other hand, simply feeding mice the anti-rejection drug rapamycin has been shown to dramatically increase both the average and maximum life span, even when started late in life, probably by postponing death from cancer, by retarding mechanisms of aging, or both. Unfortunately, rapamycin also strongly suppresses the immune system, increasing the risk of a dangerous infection. How can we derive the benefits of rapamycin while avoiding the risks?
An endogenous inhibitor of the “Target Of Rapamycin” (TOR), and thus a candidate for the physiological rapamycin-like agent, are sestrins (Sesns), which are conserved proteins that are induced by cellular stress including by elevated adenosine monophosphate kinase (AMPK) or DNA damage.

Drosophila (fruit flies) have a single form of sestrin (dSesn). To define the activities of dSesn, these authors created flies with either gain- or loss-of-function dSesn mutations. Clever sleuthing with agonists and employing other mutant signaling molecules showed that TOR induces dSesn through reactive oxygen species (ROS, quenched by antioxidants such as vitamin E), jun-N-terminal kinase (JNK), and a transcription factor (FoxO). p53 was implicated but narrowly escaped the round-up of usual suspects. dSesn repaid TOR's attention by suppressing its stimulation of tissue growth. Thus dSesn is induced by TOR and suppress TOR activities, constituting a feedback inhibition system.
These authors go on to show that flies lacking dSesn suffer multiple age-related pathologies, including muscle degeneration and heart malfunction. These pathologies are prevented by activating AMPK with AICAR (5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside) or inhibiting TOR with rapamycin (Figure 5C shows that heart arrythmias suffered by dSesn-deficient flies are treated by feeding them AICAR, rapamycin, or vitamin E).

Forget caloric restriction, I'll take my ice cream with a statin mixin and Rapa sprinkles. Or a burger with heaping side order of sestrins.

JLee JH, Budanov AV, Park EJ, Birse R, Kim TE, Perkins GA, Ocorr K, Ellisman MH, Bodmer R, Bier E, Karin M. “Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies” Science 2010 Mar 5;327(5970):1210-1.

Cholesterol-retentive Leukocytes

Elevated white cells in the bloodstream (leukocytosis) was correlated with cardiovascular disease and atherosclerosis nearly a century ago but the cause remains unclear. Elevated high-density lipoprotein (HDL), in contrast, is correlated with protection from cardiovascular disease, in part because HDL carries cholesterol away from macrophages in atherosclerotic plaques. Here, Yvan-Charvet and colleagues report that leukocytosis develops in mice lacking the membrane proteins ABCA1 and ABCG1 that normally transport cholesterol out of macrophages to lipoproteins. These ABCA1/G1-deficient mice suffer a myeloproliferative disorder and display an expansion of particular blood-forming (hematopoietic) stem cells. Although lymphoid (B, T, NK cells) precursor cells were unchanged, myeloid precursor cells, which give rise to granulocytes, macrophages, etc, were doubled. Similar findings in MyD88-knockout mice ruled out innate inflammation as a cause of leukocytosis. Transplantation of bone marrow from ABCA1/G1 transporter-deficient mice into (apoA1-transgenic) mice with elevated HDL blocked or slowed the development of leukocytosis, myeloproliferation, the particular stem cell population, and atherosclerosis. The figure shows that elevated HDL in apoA1-tg mice protected against heart disease (Fig 4B, left side shows the entire hearts and, right side, tissue sections from hosts transplanted with ABCA1/G1-deficient bone marrow; upper panel: diseased heart from a normal recipient mouse showing leukocyte infiltration and, lower panel, healthy heart from a HDL-elevated recipient).

The authors state that “these results suggest that HDL suppresses the proliferation of myeloid progenitor cells by promoting cholesterol efflux”. Thus, these transporters (intrinsically) or elevated HDL (extrinsically) can regulate hematopoiesis and atherosclerosis. They also reported that stimulating ABCA1/G1 expression above normal levels using a transcriptional activator (TO901317) increased cholesterol efflux and suppressed myeloid cell proliferation, suggesting a new therapeutic rationale.

Statins, such as atorvastatin (Lipitor) and rosuvastatin (Crestor), were designed and are prescribed to reduce serum cholesterol, which is associated with cardiovascular disease. Surprisingly, statins were also found to be anti-inflammatory. Could these results help explain statins' anti-inflammatory effects?

Laurent Yvan-Charvet, Tamara Pagler, Emmanuel L. Gautier, Serine Avagyan, Read L. Siry, Seongah Han, Carrie L. Welch, Nan Wang, Gwendalyn J. Randolph, Hans W. Snoeck, Alan R. Tall, "ATP-Binding Cassette Transporters and HDL Suppress Hematopoietic Stem Cell Proliferation" Science 25 June 2010: Vol. 328. no. 5986, pp. 1689 - 1693

Rapamycin-induced Stress Stimulates anti-Tuberculosis Immunity

Tuberculosis (Tb) is an intracellular mycobacterium that causes 9 million illnesses and nearly 2 million deaths annually world-wide. One third of humans are chronically infected and about 9 million are infected every year, which a vaccine could prevent.

Stressing cells infected by Tb has been shown to induce autophagy ("self-eating", free review), increase presentation of Tb antigens on the infected cell surface, and stimulate specific T lymphocytes. This group found that rapamycin, a pharmaceutical drug used to suppress immunity after solid organ transplantation, induces autophagy and improves immune recognition of Tb. This figure shows that rapamycin treatment of dendritic cells causes a >10-fold increase in their ability to induce anti-Tb immunity in mice (Fig. 5e). Most of the cell culture experiments aimed at dissecting this effect show a much more modest effect of extremely high doses of rapamycin (1 mM!). Oddly, many of the early figures measure IL-2, a hallmark of the T lymphocyte response, even though rapamycin's mode of action inhibiting IL-2 activity would seem to confound the interpretation of the results.

Could this adjuvant effect be translated into a vaccine strategy?

Jagannath C, Lindsey DR, Dhandayuthapani S, Xu Y, Hunter RL Jr, Eissa NT. Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nat Med. 2009 Mar;15(3):267-76.

Infectious Fatigue

Between 1 and 4 million Americans suffer from Chronic Fatigue Syndrome (CFS), which is a “complicated disorder characterized by extreme fatigue that may worsen with physical or mental activity, but doesn't improve with rest.” (Mayo Clinic). The cause is unknown and there is no known cure. An infectious cause has been suspected and sought for decades without success. The recent discovery of XMRV, new retrovirus found in some prostate cancer patients (but not others) prompted Lombardi and colleagues to test for its involvement with CFS. They performed PCR for XMRV gag, which encodes structural viral proteins, on peripheral blood mononuclear cells from CFS patients in a repository at their Whittemore Peterson Institute. Of 101 CFS samples tested, 68 (67%) were positive in contrast to only 8 out of 218 samples (4%) from healthy donors. Another viral gene, env, was also detected in most CFS patients positive for gag as were the proteins encoded by these genes. Oddly, patient sample WPI-1118 is negative for gag and env (Fig 1) but weakly positive by cytometry (Fig 2 A & D) and clearly positive by Western protein blot (Fig 4 A). Both B and T lymphocytes express XMRV proteins. And, for what it's worth, some CFS patient cells make virus that can productively infect other cells (shown, from Figure 3 B & C, electron micrographs of budding virus particles).

The authors note in their introduction that “patients with CFS often have active β herpesvirus infections, suggesting an underlying immune deficiency”. This increases the odds that XMRV is an opportunistic infection. The authors return to this question in their closing discussion, asking “Is XMRV infection a causal factor in the pathogenesis of CFS or a passenger virus in the immunosuppressed CFS patient population?” Stand by.

Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. "Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome". Science. 2009 Oct 8.

Infecting the Protectors (fluNK)

Influenza (flu) is typically transmitted through the air, infecting lung epithelial cells. Natural Killer (NK) cells are lymphocytes that help protect from flu, especially early after infection, by killing infected cells and secreting cytokines and chemokines that inhibit virus replication. Here, Guo and colleagues ask whether NK might themselves be infected by flu. They confirmed that NK express on their surface sialic acids, which serve as flu receptors by binding hemagluttinin (HA). By microscopy, they examined mice infected with PR8, a strain originating from the human flu virus A/PR/8/34 and detected flu virus protein M2 inside of NK cells within the lungs of infected mice. NK possess several activating receptors including NCR1, NKG2D, Ly49D and inhibiting receptors including NKG2A, Ly49A. Flow cytometry demonstrated that PR8 did not modulate these receptors or developmental markers.
However, infection did alter the effector functions of NK cells. Infected NK cells were less capable of killing cells from 3 target lines, cells susceptible because they either express ligands for NK activating receptors or fail to express ligands for NK inhibiting receptors (Figure 5, shown). EL4 cells express a transfected H60, which binds the activating receptor NKG2D; YAC-1 cells express H60 constitutively, and RMA-S cells express very little HLA class I protein, which binds the inhibitory Ly49 receptors. The authors and others had previously shown that the 85 kDa subunit of phosphotidyl inositol 3-kinase (PI3K-p85) was critical for NK effector functions, including killing and production of cytokines/chemokines, and that flu NS1 protein interacted with this kinase. Surprisingly, a PR8 virus with a mutated NS1 protein was even more effective in suppressing NK effector functions, leaving the mechanism of inhibition to be determined.
Guo H, Kumar P, Moran TM, Garcia-Sastre A, Zhou Y, Malarkannan S. “The functional impairment of natural killer cells during influenza virus infection.” Immunol Cell Biol. 2009 Sep 1.

Overreacting to Infection is Dangerous to Your Health

Contributed by DP "Following soon after a similar paper on anthrax pathogenesis, this paper suggests that the downregulation of a lymphocyte surface protein can single-handedly stem the destruction by the Ebola virus. The authors identified mice expressing about half (62%) the "wild type" levels of the leukocyte membrane tyrosine kinase CD45 are protected from the lethal effects of Ebola infections (compare CD45-62% to control 100% in the top panel), with an overall survival rate of 90% and a complete clearance of the virus 10 days after challenge. CD45-62% mice expressing CD45 without phosphatase activity did not survive viral challenge, highlighting the importance of CD45’s enzymatic function.

The proposed mechanism is via constitutively high levels of activated CD8 T cells and IFN-gamma because antibodies that kill CD8 cells or block IFN-gamma render CD45-62% mice susceptible to the virus (fig 4, panels B & C). Can this protection translate to other infectious pathogens? Can this information be used clinically? Should we worry about modulating surface CD45 or just give IFN-gamma to affected individuals? I like the former, if one can come up with inexpensive small molecules, rather than the huge expense for IFN-gamma. These molecules might be used at higher doses for GVHD." And what is the evolutionary value of having wt levels of CD45 if reduced levels are in fact protective?

Panchal RG, Bradfute SB, Peyser BD, Warfield KL, Ruthel G, Lane D, Kenny TA, Anderson AO, Raschke WC, Bavari S. Reduced levels of protein tyrosine phosphatase CD45 protect mice from the lethal effects of Ebola virus infection. Cell Host Microbe. 2009 Aug 20;6(2):162-73.

Th17 cells have issues with commitment

Th17 cells, so named because they secrete the protein hormone interleukin-17 (IL-17), belong to a recently-described subset of helper T cells responsible for regulating helper subsets Th1 (cellular) and Th2 (allergic).

Naïve T cells develop into different T helper subsets depending upon culture conditions. Lee and colleagues used an IL-17F reporter mouse (IL17F promoter driving expression of a surface protein Thy1.1) to test the stability of the Th17 phenotype in serial cultures. First, they caused Th17 to develop from naïve CD4+ T cells (OT-II transgene receptors specific for ovalbumin (ova) + MHC class II) by treating them in culture with TGF-beta, IL-6, anti-IFNgamma, and anti-IL-4 in the presence of IL-12p40-deficient antigen-presenting cells (APC). (Culture systems don't get much more manipulated than that!) Then, the surviving cells were phenotyped or cultured with TGNbeta or with IL-23, which stimulates the development of Th1 cells.

Several other groups had reported that Th17 cells required IL-23 for pathogenic autoimmunity. Here, Lee et al. showed that TGFbeta, but not IL-23, is essential for maintaining Th17 commitment (IL-17F expression). However, even after two cultures under Th17 conditions, IL-12 added to subsequent cultures 3 and 4 drove Th1 development and expression of interferon-gamma (IFNg, on the x-axis of figure 2, shown below).

Moreover, this final conversion by Th17 required Th1 factors STAT4 and T-bet, suggesting that Th17 might constitute a prolonged adolescence for adult Th1 cells.

Immunity 30:92-107. “Late developmental plasticity in the T helper 17 lineage”. Lee YK, Turner H, Maynard CL, Oliver JR, Chen D, Elson CO, Weaver CT.

IRF4 etc Required for Th17 Development

Interleukin-17 (IL-17A) is a proinflammatory protein hormone produced by activated T lymphocytes that binds to a ubiquitous low-affinity receptor IL17RA. A subset of T helper (Th) cells that produce IL17 were dubbed Th17 and shown to be induced by IL23 and suppressed by IFNg or IL4, which support the development of Th1 or Th2 cells, respectively.

Brüstle and colleagues tested the ability of lymphocytes to develop into Th17 cells by treating naïve (CD4+CD62L+) Th in culture for 3 days with anti-CD3 and anti-CD28 to stimulate Th0 differentiation. Some cultures also received (1) IL12 and anti-IL4 to stimulate Th1, or (2) IL4 and anti-IFNgamma to stimulate Th2, or (3) anti-IL4, anti-IFNgamma, TGFbeta, IL6, IL1beta, TNF, and IL23 to stimulate Th17. Cells were then restimulated and analyzed by intracellular staining. They found that IRF4-deficient cells differentiate normally into Th1 cells, contradicting an earlier report by the senior author [they attribute the difference to using specific-pathogen-free mice], but not into Th2 cells, as previously reported. They also found that IRF4-deficient cells did not differentiate into Th17 cells. IRF4 is from a family of transcription factors that are required for the production of interferons alpha and beta as well as innate immunity (TLR) signaling and T helper cell differentiation. Mixing cells from normal 'wild-type' (WT) and deficient mice (IRF4-/- also CD45.2+) demonstrated that the defect was intrinsic to the Th17 cell lineage and not due to suppression (figure 1c shown).
They made several other, interesting observations and pursue the IRF4 pathway, finding that the transcription factor RORgamma, also previously reported to be required for Th17 cells, is largely dependent on IRF4. They also showed that TGFbeta-induced FoxP3, a marker of regulatory T cells, is only weakly suppressed by IL6 in IRF4-deficient cells, suggesting that this alternative differentiation pathway may explain the failure of Th17 to develop.
Brüstle A, Heink S, Huber M, Rosenplänter C, Stadelmann C, Yu P, Arpaia E, Mak TW, Kamradt T, Lohoff M. “The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4” Nat Immunol. 2007 Sep;8(9):958-66