Immunology

1

Immunology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in immunology. Current Opinion in Immunology 2001, 13:1–8 Contents (chosen by) 1 Antigen processing and recognition (Elliott) 2 Innate immunity (Bonneville) 2 Lymphocyte development (Kruisbeek) 3 Tumour immunology (Walker) 3 Lymphocyte activation and effector functions (Casolaro) 3 Immunity to infection (Glaichenhaus and Rowland-Jones) 6 Immunogenetics (Casanova) 6 Immunotherapy (Liu) 6 Transplantation (Wood and Bushell) 7 Allergy and hypersensitivity (Gorham) • ••

of special interest of outstanding interest

Antigen processing and recognition Selected by Tim Elliott University of Southampton, Southampton, UK

Impaired assembly yet normal trafficking of MHC class I molecules in tapasin mutant mice. Grandea AG III, Golovina TN, Hamilton SE, Sriram V, Spies T, Brutkiewicz RR, Harty JT, Eiseniohr LC, Kaer LV: Immunity 2000, 13:213-222. AND

Impaired immune responses and altered peptide repertoire in tapasin-deficient mice. Garbi N, Tan P, Diehl AD, Chambers BJ, Ljunggren H, Momburg F, Hammerling GJ: Nat Immunol 2000, 1:234-238. • Significance: The molecular details of how MHC class I molecules select and present peptides for recognition by cytotoxic T lymphocytes (CTLs) are still unclear. Evidence from tapasin deficient cell lines indicates that this molecule plays a key role. By generating mice that lack a gene for a functional tapasin molecule, these groups have confirmed studies performed in vitro and have evaluated the effects of tapasin loss on immune responses in vivo. The results clearly show that genetic defects in the tapasin gene could profoundly affect immune responses to tumours, infectious agents and self antigens. Findings: Knockout mice were generated by inserting the neomycin resistance gene into exon 4 of tapasin. Both groups show that splenocytes of the resulting mice express low cellsurface levels of MHC class I molecules. Grandea et al. show reduced ability of TAP (transporter associated with antigen processing) to transport peptides across the endoplasmic reticulum (ER) membrane in the tapasin knockout cells as well as the failure of MHC class I molecules to bind to TAP. Assembled MHC class I molecules are exported from the ER at a normal rate but these are more unstable, suggesting poor loading with peptide antigen. Both studies show that tapasin deficient splenocytes present immunodominant CTL epitopes poorly and Garbi et al. demonstrate an antigen-presentation defect in dendritic cells derived from the tapasin knockout mice. Garbi et al. showed that tapasin knockouts have slightly fewer CD8+ T cells in the thymus than wild-type but considerably

more than in TAP knockout mice, suggesting a defect in positive selection. Grandea et al., however, show normal positive selection of a transgenic TCR that recognises an immunodominant epitope derived from ovalbumin. The same study shows defective negative selection of an H-Y-specific, transgenic TCR. Nevertheless the study by Garbi et al. shows that CTLs from tapasin knockout mice cannot be primed to splenocytes from wild-type mice, indicating that they are tolerant. Grandea et al. explore in vivo responses to infectious agents in the tapasin deficient mice. They find that, although the mice do not respond to influenza virus, CTL responses to vaccinia virus and lymphocytic choriomeningitis virus (LCMV) are normal and that the mice are able to clear an infection of Listeria monocytogenes. The study by Garbi et al. explores NK cell activity in the tapasin deficient mice. They find that NK cells from wild-type mice but not from tapasin deficient mice will kill tapasin deficient target cells although the latter will kill the standard NK target cell line, YAC1. These results indicate that the tapasin knockout mice have an altered NK cell repertoire. Bone marrow-derived antigen-presenting cells are required for the generation of cytotoxic T lymphocyte responses to viruses and use transporter associated with antigen presentation (TAP)-dependent and -independent pathways of antigen presentation. Sigal LJ, Rock KL: J Exp Med 2000, 192:1143-1150. AND

Requirements for bone marrow-derived antigen-presenting cells in priming cytotoxic T cell responses to intracellular pathogens. Lenz LL, Butz EA, Bevan MJ: J Exp Med 2000, 192:1135-1142. • Significance:The role of bone marrow derived, professional antigen-presenting cells (BMAPCs) in priming CTL responses to nonreplicating antigens, such as naked DNA and tumour cells, is becoming well established. It is not clear whether the same route for priming CTL responses is used for intracellular pathogens, such as viruses and bacteria. These papers indicate that they are but that some intracellular pathogens may utilise a different priming pathway. Findings: Using radiation-treated bone marrow chimeras reconstituted with T cell depleted bone marrow from MHC identical, MHC disparate or F1 donors, these investigations explore the requirement for BMAPCs in priming CTL responses to influenza virus (Sigal and Rock) vaccina virus (Lenz et al.), L. monocytogenes (Lenz et al.) or LCMV (both). They show that the CTL responses to flu, vaccina, L. monocytogenes and the immunodominant H2-Db-restricted response to LCMV require donor BMAPCs bearing the correct MHC class I molecule. Sigal and Rock, using MHC-identical bone marrow from TAP-knockout mice, showed that that the induction of a CTL response to three H2-Db-restricted LCMV epitopes was TAP independent. For a fourth H2-Ld-restricted LCMV epitope, both groups demonstrated induction of a strong CTL response even when irradiated mice were reconstituted with MHC disparate (H2-Ld-negative) bone marrow. Both groups attribute this result to priming by a small residual pool of radioresistant host antigen-presenting cells and Sigal and Rock go on to show that the response disappears in supra-irradiated recipient mice. Interestingly, Lenz et al. shows

2

Paper alert

that the presentation of this epitope depends on the viral vector used to infect mice. Although the epitope is presented when delivered via wild-type LCMV, it does not elicit a CTL response when delivered by recombinant vaccinia virus encoding the LCMV nucleoprotein from which the epitope is derived.

Innate immunity Selected by Marc Bonneville Institut de Biologie, Nantes, France

CD95/CD95 ligand interactions on epithelial cells in host defense to Pseudomonas aeruginosa. Grassmé H, Kirschneck S, Riethmueller J, Riehle A, von Kurthy G, Lang F, Weller M, Gulbins E: Science 2000, 290:527-530. • Significance: This study provides for the first time a molecular mechanism for the lung epithelial cell apoptosis observed after infection by P. aeruginosa, an intracellular bacteria responsible for severe lung infections in cystic fibrosis and immunosuppressed patients. It also demonstrates that bacterium-induced epithelium apoptosis has a pivotal role in host defense against P. aeruginosa infection, presumably by activating early innate immune mechanisms that prevent bacterial replication and spreading. Findings: The CD95/CD95-ligand system is one of the major mechanisms of mammalian cell apoptosis. It is shown here that in vivo and in vitro infection of fibroblasts and epithelial cells by P. aeruginosa leads to rapid cell apoptosis. Deficiency of CD95 or CD95-ligand on epithelial cells prevents P.-aeruginosainduced apoptosis. Furthermore the relevance of this phenomenon to host defense against infection is demonstrated by the rapid development of sepsis following P. aeruginosa infection in mice deficient for CD95 or CD95-ligand but not in wild-type mice.

Lymphocyte development Selected by Ada Kruisbeek The Netherlands Cancer Institute, Amsterdam, The Netherlands

BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Shaffer AL, Yu X, He Y, Boldrick J, Chan EP, Staudt LM: Immunity 2000, 13:199-212. • Significance: Gene expression profiling is going to cause great strides forward in our understanding of the genetic programs underlying numerous biological events, including those in the immunological arena. This study uses this powerful new technology to address how the transcriptional repressor BCL-6 regulates lymphocyte differentiation. A considerable proportion of B cell non-Hodgkin lymphomas has BCL-6 translocations and BCL-6 mutant mice have a dramatic phenotype (i.e. absence of germinal-center formation upon antigen encounter and a fatal inflammatory disease) but the molecular pathways regulated by BCL-6 are unknown. Findings: To prepare the search for BCL-6 target genes, the authors first prepared gain-of-function and loss-of-function cell systems in which BCL-6 expression is manipulated. They then went on to compare relative gene expression on lymphochip microarrays in two cell samples in each experiment and selected for genes whose mRNA levels were changed in more than one cell type when BCL-6 was modulated. This led to selection of 14 genes, validated in northern blots and through semiquantitative RT-PCR. Many of these represent B cell activation genes and BCL-6 also represses two chemokine genes (MIP-1α and IP-10). BCL-6 repression of Blimp-1 may help to keep plasmacell differentiation under control. These and other data presented

in this study indicate that BCL-6 contributes to lymphomagenesis by inhibiting differentiation and promoting proliferation. Control of pre-T cell proliferation and differentiation by the GTPase Rac-I. Gomez M, Tybulewicz V, Cantrell DA: Nat Immunol 2000, 1:348-352. • Significance: Transition of T cell precursors from the CD4–CD8– (DN [double negative]) to the CD4+CD8+ (DP [double positive]) stage of thymic development is driven by the pre-TCR. This receptor triggers several functions (survival, expansion and differentiation) but it remains to be determined which cell-fate decision is orchestrated by which pre-TCR dependent signaling event. Several cytoplasmic mediators have already been defined and the present study explores whether the GTPase, Rac-1, is involved in proper pre-TCR functioning. Rac GTPases organize filamentous actin structures and modulate multiple other pathways. Findings: Transgenic mice in which Rac-1 is expressed in the T cell lineage were produced. Rac-1 appears to accelerate preTCR driven proliferation and differentiation but in the absence of a pre-TCR (i.e. in Rag-1 deficient mice) it only stimulates differentiation. Rac-1 therefore appears to function in conjunction with the pre-TCR and, indeed, in Vav-1 deficient mice (which have a defect in actin polymerization but possess a functional pre-TCR), transgenic Rac-1 does rescue the DN to DP transition and thymic cellularity. Furthermore, the activity of Rac-1 in pre-TCR function maps to its actin-cytoskeleton-organizing domain; its other effector functions are not required for its potentiating effects on pre-TCR. Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex. Guo B, Kato RM, Garcia-Lloret M, Wahl MI, Rawlings DJ: Immunity 2000, 13:243-253. • Significance: B cell development can only occur when the pre-BCR triggers prerequisite survival, expansion and differentiation signals. How the pre-BCR (and the pre-TCR, for that matter; see above) initiates those signals remains to be defined. Both receptors are present at extremely low levels and appear to signal in the absence of extracellular ligand. This has led to the hypothesis that pre-TCRs and pre-BCRs form constitutively active signaling complexes; the present study provides support for that view, as far as the pre-BCR is concerned. Findings: Multichain immunoreceptors are, after ligand binding, organized into lipid rafts, which are enriched for glycolipids and signaling molecules. Upon pre-BCR engagement, formation of a raft-associated signaling module occurs, resulting in redistribution of multiple signaling molecules. Importantly, this study shows that a significant fraction of the pre-BCR (in both pre-B cell lines and in primary pre-B cells) is constitutively associated with lipid rafts and this may generate low-level signaling through a local increase in kinases and other signaling molecules. These results are reminiscent of those recently reported for the pre-TCR (see ‘Paper alert’ in this journal, October 2000: C Saint-Ruf et al. Nature 2000, 406:524-527.). Hedgehog signaling regulates differentiation from doublenegative to double-positive thymocytes. Outram SV, Varas A, Pepicelli CV, Crompton T: Immunity 2000, 13:187-197. • Significance: The hedgehog (Hh) family of secreted proteins, first identified in Drosophila, plays a major role in pattern formation in early tissue development. Mammalian homologs have been identified but their role in mammalian development is unclear.

Paper alert

Here, it is suggested that one Hh family member (sonic hedgehog [Shh]) and its receptors Patched (Ptc) and Smooth (Smo) are expressed in the thymus and regulate thymocyte development. Findings: First, a combination of RT-PCR, in situ hybridization and immunofluorescence shows Shh in the thymic stroma, and Ptc and Smo receptors for Shh on DN thymocytes. Furthermore, blocking of Shh signaling promotes DN to DP differentiation and Shh treatment arrests thymocyte development at the DN stage. Finally, pre-TCR signaling downregulates Smo expression. Together, these findings suggest a model in which termination of Shh signaling is required for DN to DP transition during thymocyte development.

Tumour immunology Selected by Paul R Walker University Hospital Geneva, Geneva, Switzerland

Perforin-mediated cytotoxicity is critical for surveillance of spontaneous lymphoma. Smyth MJ, Thia KY, Street SE, MacGregor D, Godfrey DI, Trapani JA: J Exp Med 2000, 192:755-760. • Significance: The concept of immune surveillance against cancer has persisted for decades but with limited supportive evidence. Information regarding the relative importance of specific effector mechanisms is even more scanty. This study is the first to implicate perforin mediated cytotoxicity in protecting against spontaneous lymphoma. Findings: Perforin knockout mice were previously shown not to be abnormally prone to spontaneous malignancies but these studies only followed mice for 12 months. Here, Smyth et al. had the patience to wait 436–635 days for the eventual appearance of tumours, generally disseminated lymphomas. Using intercrossed knockout mice deficient in perforin and/or p53, other spontaneous tumours also occurred (thymomas and sarcomas) but perforin status of the mice only influenced the apparition of lymphomas. Furthermore, although p53 status influenced the time of onset of lymphomas, their frequency was not augmented, underlining the key role of perforin. Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells. Schuler-Thurner B, Dieckmann D, Keikavoussi P, Bender A, Maczek C, Jonuleit H, Roder C, Haendle I, Leisgang W, Dunbar R et al.: J Immunol 2000, 165:3492-3496. • Significance: DC vaccines can efficiently elicit specific effector T cell responses in mice and healthy human subjects but their application for tumour immunotherapy requires that they are also efficient in advanced cancer patients. The results reported here suggest that this may be the case and indeed melanoma-peptide-specific T cells were induced in such patients; the cells exhibited specific effector function (IFN-γ release) ex vivo. Findings: This study reports the findings of a clinical trial in which advanced melanoma patients were vaccinated with mature, monocyte-derived DCs pulsed with Mage-3A2.1 peptide, influenza matrix A2.1 peptide and in some cases with tetanus toxoid. Specific T cells were assessed by proliferation (in response to tetanus toxoid), ex vivo IFN-γ ELISPOT, HLA–peptide-tetramer flow cytometric analysis and cytotoxic T cell precursor analysis after culture. Vaccination induced Mage-3- and influenza-matrix-specific T cells in all patients but there were no significant clinical responses, perhaps related to poor expression of the Mage-3

3

epitope on tumour cells in vivo. Nevertheless, these are encouraging results to build upon for developing future vaccines targeting better tumour-rejection antigens. Enhanced antigen-specific antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex. Slansky JE, Rattis FM, Boyd LF, Fahmy T, Jaffee EM, Schneck JP, Margulies DH, Pardoll DM: Immunity 2000, 13:529-538. • Significance: Naturally expressed tumour antigens frequently elicit only weak T cell responses. In previous studies, modified epitopes used in peptide vaccines have improved on the natural situation by augmenting peptide affinity for MHC. However, a problem for efficient T cell antitumour responses is that the available repertoire may consist principally of T cells with lowaffinity TCRs. This issue is directly addressed in this study, in which antitumour T cell responses are ameliorated by employing a peptide vaccine with a modified epitope that stabilises MHC–peptide–TCR interactions. Findings: Slansky et al. performed detailed analyses of the MHCand TCR-binding characteristics of a natural epitope (AH1) expressed by mouse colorectal cancer CT26 and of alanine-substituted variants derived therefrom. Peptide AH1-A5 was selected; it had similar H-2Ld-binding affinity to the wild-type (wt) peptide. However, the H-2Ld–peptide complex bound TCR with higher affinity when complexed with the variant peptide rather than the wt peptide. In vitro, an AH1-specific T cell clone lysed variant peptide AH1-A5-sensitised target cells more efficiently than targets pulsed with wt peptide. Furthermore, evidence was also presented for enhanced in vivo immunogenicity of AH1-A5, using peptide-loaded dendritic cells. The AH1-A5 peptide vaccine expanded higher numbers of AH1-specific T cells and afforded a slight delay in tumour appearance after CT26 challenge. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Wei Yq, Wang Qr, Zhao X, Yang L, Tian L, Lu Y, Kang B, Lu Cj, Huang Mj, Lou Yy et al.: Nat Med 2000, 6:1160-1166. • Significance: Inhibiting angiogenesis is an attractive therapeutic strategy for blocking the progression of solid tumours. This study uses a novel approach to achieve this objective: a xenogeneic vaccine that induces antibodies cross-reacting with conserved structures on endothelial cells. Findings: Human and bovine proliferative endothelial cells from in vitro culture were used as a vaccine to protect mice against challenge with a variety of syngeneic tumours; impressive regression of pre-established tumours was also achieved. Vaccines consisting of mouse endothelial cells or non-endothelial xeneogeneic cell lines had no protective effect. Successful vaccination required CD4+ T cells and correlated with IgG antibody deposition on endothelial cells of tumour microvessels. Protective antibodies were partially characterised and included reactivity to vascular endothelial growth factor receptor II and αv integrin, known angiogenesis-associated molecules.

Lymphocyte activation and effector functions Selected by Vincenzo Casolaro The Johns Hopkins School of Medicine, Baltimore, MD, USA

Functional flexibility in T cells: independent regulation of CD4+ T cell proliferation and effector function in vivo. Laouar Y, Crispe N: Immunity 2000, 13:291-301. •• Significance: As the relative frequency of antigen-specific effector T cells predicts the strength of recall responses, clonal

4

Paper alert

expansion of these cells is viewed as a critical step in adaptive immunity. Although antigen-dependent T cell proliferation is associated with the acquisition of an effector phenotype, a significant proportion of T cells do not cycle following exposure to the relevant antigen in vitro. Are these cells activated at all? Do they contribute to the development of effector responses? A conceptually sound approach is followed in this study to elucidate these issues and determine whether a mechanistic link exists between T cell proliferation and function. Findings: Irradiated mice with T-cell-depleted bone marrow (BM) were reconstituted with a BM chimera containing increasing proportions (0%, 1%, 10%, 50%, 90% and 100%) of cells from pigeon cytochrome c (PCC)-specific, TCR-transgenic Thy-1.2+ mice and correspondingly decreasing amounts of cells from Thy-1.1+ nontransgenic mice. The frequency of cycling T cells following injection of PCC peptide, determined by in vivo incorporation of 5-bromo-2-deoxyuridine, was inversely correlated to the relative proportion of TCR-transgenic BM cells, indicating that the frequency of antigen-specific naive T cells can impose a constraint on their proliferation. However, after in vivo stimulation, both cycling and noncycling T cells (these latter accounting for up to 60% of TCR-transgenic cells) exhibited phenotypic changes characteristic of TCR engagement (i.e. CD69+CD44highCD62Llow) and produced at least comparable amounts of IFN-γ. In contrast to cycling T cells, upon TCR ligation noncycling cells retained expression of the naive T cell marker CCR7, a ligand for chemokines directing T cell homing to lymphoid tissues that is typically lost in effector T cells. Thus, although proliferation is not a requirement for T cell effector function, it may be associated with increased T cell trafficking concomitant to reduced expression of CCR7. Interferon γ signaling alters the function of T helper type 1 cells. Tau GZ, von der Weid T, Lu B, Cowan S, Kvatyuk M, Pernis A, Cattoretti G, Braunstein NS, Coffman RL, Rothman PB: J Exp Med 2000, 192:977-986. • Significance: The polarization of T helper cell responses is controlled in part by cytokines produced by antigen-presenting cells and developing T helper cells. Among these, the Th1 cytokine IFN-γ contributes to Th1 cell differentiation while antagonizing the Th2-polarizing effect of IL-4. Earlier studies by Rothman and co-investigators showed that responsiveness to IFN-γ is lost in Th1 cells subsequent to reduced expression of the second chain of the IFN-γ receptor (IFN-γR2). The significance of this finding has been now determined by the same group, in this study examining the development of polarized T helper cell responses in transgenic mice constitutively expressing IFN-γR2 in all T cells. Findings: The gene encoding IFN-γR2 was placed under the control of the human CD2 promoter and enhancer to allow constitutive, nonregulated T cell expression of this molecule in transgenic mice. Th1 and Th2 clones generated from these mice expressed similar levels of IFN-γR2 (interestingly, from both the endogenous and transgenic loci) and of IFN-γ-induced nuclear STAT1, in contrast to cells from wild-type (WT) littermates. T helper cells from transgenic mice produced consistently lower amounts of IFN-γ but similar amounts of IL-4 as WT cells regardless of whether Th1 polarization was induced using IFN-γ or IL-12. This defect was associated with a reduced recall expression of Th1 cytokines (IFN-γ and IL-2), a reduced Th1-driven delayed-type hypersensitivity in response to antigenic rechallenge in mice sensitized to Listeria monocytogenes and an increased susceptibility to chronic infection with Leishmania major. In addition, transgenic mice produced similar

levels of antigen-specific IgG1 but drastically reduced levels of IgG2a following immunization to protein antigen. Together, these findings show that abnormal expression of IFN-γR2 in Th1 cells is associated with a unique phenotype, characterized by impaired Th1 immunity with conserved Th2 responses. Therefore, its downregulation appears to be a prerequisite for the function of Th1 cells but not for their differentiation. Th1 and Th2 CD4+ T cells provide help for B cell clonal expansion and antibody synthesis in a similar manner in vivo. Smith KM, Pottage L, Thomas ER, Leishman AJ, Doig TN, Xu D, Liew FY, Garside P: J Immunol 2000, 165:3136-3144. • Significance: An established paradigm implies that, whereas Th1 cells play a crucial role in cell-mediated responses, help for B cell dependent humoral responses is provided by Th2 cells. However, studies in knockout mice prove that Th2 cells may not be essential for B cell proliferation and differentiation, and indeed the Th1 cytokine IFN-γ does promote IgG2a isotype switching. This study is a welcome attempt to clarify the relative roles of the two T helper cell subsets in B cell responses. Findings: The authors studied B cell clonal expansion and antibody production in mice adoptively transferred with in-vitro-polarized ovalbumin (OVA)-specific, TCR-transgenic T helper cells and hen egg lysozyme (HEL)-specific, BCR-transgenic B cells. HEL-specific B cells underwent a similar degree of clonal expansion and anti-HEL IgM production, both peaking on day five after immunization with coupled OVA–HEL, in mice receiving Th1- or Th2-polarized OVA-specific cells. These changes were preceded by a similar pattern of lymph node redistribution of unpolarized or polarized, TCR-transgenic T helper cells, whereby transferred cells invariably migrated from the paracortical to follicular areas within three days of immunization. The administration of anti-CD154 antibody, previously shown to interfere with cognate interaction between B and T cells, reduced T and B cell expansion and antigen-specific IgM production in animals receiving either T helper cell subset but in neither case prevented T cell follicular migration. Taken together, these findings provide formal in vivo evidence that both Th1 and Th2 cells can support antigen-specific B cell expansion and antibody production in a CD154-dependent manner.

Immunity to infection Selected by Nicolas Glaichenhaus* and Sarah Rowland-Jones† *Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France †John Radcliffe Hospital, Oxford, UK

Pattern recognition receptors TLR14 and CD14 mediate response to respiratory syncitial virus. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ, Finberg RW: Nat Immunol 2000, 1:398-401. •• Significance: Although CD14 and Toll-like receptors (TLRs) are critical in initiating innate immune responses to bacteria, mycobacteria, spirochetes and yeast, it was not clear whether these receptors also play a role in antiviral responses. In this elegant study, the authors show that a protein of RSV can stimulate the innate antiviral immune response through CD14 and TLR4. Thus, targeting the expression or the signaling of these receptors may be useful in altering the course of infection with RSV and possibly other viruses. Findings: The authors found that both purified human monocytes and mouse peritoneal macrophages secreted high

Paper alert

amounts of IL-6, IL-8, TNF-α and IL-1β when incubated with purified RSV fusion (F) protein. Cytokine-stimulating activity of the RSV F preparation was abolished by trypsin treatment and was blocked by anti-F monoclonal antibodies, confirming that the cytokine-stimulating activity was due to the F protein itself. Further experiments using macrophages from either CD14- or TLR4-deficient mice showed that the induction of IL-6 by the RSV F protein requires the expression of these two receptors. In agreement with this latter result, TLR4-deficient mice were impaired in their ability to clear RSV infection. CD1d deficiency impairs murine host defense against the spirochete, Borrelia burgdorferi. Kumar H, Belperron A, Barthold SW, Bockenstedt K: J Immunol 2000, 165:4797-4801. • Significance: CD1 molecules have been shown to bind microbial lipid antigens and subsequently present these antigens to T cells. Although a large body of evidence has suggested that CD1-restricted T cells are critical for protection against intracellular pathogens, this study is the first to show a role for CD1 molecules in host defense against an extracellular microbial agent. Findings: Wild-type (C57BL6 [B6] and 129) mice do not show joint or soft-tissue abnormalities upon infection with Borrelia burgdorferi (Bb). In contrast, this study shows that B6 and 129 CD1d-deficient mice develop arthritis upon infection with Bb spirochetes. Interestingly, sera from these CD1d-deficient animals contain Bb-specific IgG2a, a feature that is commonly seen in susceptible mouse strains infected with Bb. Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells. Sevilla N, Kunz S, Holz A, Lewicki H, Homann D, Yamada H, Campbell KP, de la Torre JC, Oldstone MBA: J Exp Med 2000, 192:1249-1260. •• Significance: By studying several variants of the lymphocytic choriomeningitis virus (LCMV), the authors show that interactions between LCMV and DCs are critical for the initiation of virusinduced immunosuppression and persistence. These interactions may result in the in vivo selection of high-affinity viral variants capable of inducing immunosuppression. This type of in vivo selection may also apply to other viruses, such as HIV and measles. Findings: Although most LCMV strains are able to cause immunosuppression in mice and to establish persistent infection, some variants are not immunosuppressive and are rapidly cleared through a cytotoxic T lymphocyte (CTL)-dependent mechanism. Recently, α-dystroglycan (α-DG) was identified as the LCMV receptor. Here, the authors show that the ability of LCMV to cause immunosuppression correlates directly with the ability of the viral GP1 protein to bind to α-DG with high affinity. In agreement with this latter result, high-affinity-binding variants were found to preferentially replicate in CD11c+ and DEC-205+ splenic DCs. Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Barouch DH, Santra S, Schmitz JE, Kuroda MJ, Fu TM, Wagner W, Bilska M, Craiu A, Zheng XX, Krivulka GR et al.: Science 2000, 290:486-492. • Significance: This report describes a vaccine strategy that effectively induces cellular immune responses and control of SIV viraemia by combining a DNA vaccine with co-administration of IL-2. Findings: DNA vaccines have been shown to be potent immunogens in small animals but have rarely had the same

5

effects in primates, for reasons that are not entirely clear. In this study, 20 rhesus macaques were immunised with a DNA construct expressing SIV gag or appropriate control antigens and 8 of these received the immunogen in combination with either IL-2 expressed as a fusion protein with the Fc portion of IgG or a plasmid expressing the same construct. The animals that received additional IL-2 had significantly better CTL responses (both in magnitude and duration) than those that received DNA alone. Although all the animals were infected following challenge with a virulent SIV strain, the monkeys that received IL-2 had a substantially lower viral load and much-improved clinical course after infection than those that received DNA alone. It is particularly interesting that the IL-2 recipients also maintained SIV-specific CD4+ T cell responses after infection, as these responses are rapidly lost in the majority of SIV-infected animals. Immune control of HIV-1 after early treatment of acute infection. Rosenberg ES, Altfeld M, Poon SH, Phillips MN, Wilkes BM, Eldridge RL, Robbins GK, D’Aquila RT, Goulder PJ, Walker BD: Nature 2000, 407:523-526. • Significance: This paper describes a study in which highly active anti-retroviral therapy (HAART) with structured treatment interruptions significantly augmented the cellular immune response to acute HIV infection and the subsequent control of viraemia. Findings: An important question in HIV management is whether potent anti-retroviral therapy initiated early after infection can alleviate some of the immune impairment associated with HIV infection. In this study, 18 patients with acute or recent HIV infection were started on anti-retroviral therapy within days of diagnosis. HIV-gag-specific CD4+ T cell responses became detectable in all patients in whom viral load fell below detection, and were maintained at levels usually associated only with some rare long-term non-progressors. Treatment was interrupted in a structured fashion in 8 patients, with the plan to reintroduce therapy if viral plasma load rose above a threshold of 50,000 copies/ml. Although CD8+ T cell responses detected by IFN-γ ELISpot assays were relatively modest, the responses became stronger and more broadly directed after subsequent treatment interruptions and were maintained even after therapy was resumed. Five patients were able ultimately to discontinue therapy altogether and remain well, with undetectable viral load. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Allen TM, O’Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel TU, Dunphy E, Liebl ME, Emerson C, Wilson N: Nature 2000, 407:386-390. • Significance: This study clearly demonstrates viral evolution under CTL selection pressure in early SIV infection and shows that this is most marked in the tat gene. Findings: Although viral escape from CTL pressure by mutations in CTL epitopes has been postulated to occur in a number of small studies of HIV-infected individuals, the best way of showing unequivocally that it happens in vivo is to closely follow the evolution of cloned virus in experimentally infected animals. In this study, 18 rhesus macaques were infected with a molecular clone of SIV and their immunodominant CTL responses were mapped in the early weeks after infection. The strongest responses were directed towards tat epitopes and these peaked within three weeks of infection. Subsequent analysis of the virus showed that mutations rapidly accumulated in these epitopes leading to abrogated of CTL recognition. It is suggested that tat may therefore be a useful target for vaccine design although the relative ease with which

6

Paper alert

escape mutations can develop and be tolerated by the virus could undermine this strategy.

Immunogenetics Selected by Jean-Laurent Casanova Laboratory of Human Genetics of Infectious Disease, Necker-Enfants Malades Medical School, Paris, France

Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia. Dale DA, Person RE, Bolyard AA, Aprikyan AG, Bos C, Bonilla MA, Boxer LA, Kannourakis G, Zeidler C, Welte K: Blood 2000, 96:2317-2322. AND

Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC: Nat Genet 1999, 4:433-436. • Significance: The elucidation of a common molecular basis for two human inherited disorders of neutrophils, cyclic neutropenia (cyclic haematopoiesis) and severe congenital neutropenia (Kostmann’s disease). Findings: Cyclic neutropenia is a heritable disease in which blood neutrophils vary between normal numbers and zero with 21-day periodicity. Severe congenital neutropenia is marked by a bone marrow promyelocytic maturation arrest and persistent blood agranulocytosis at least three months after birth. Both conditions are associated with severe bacterial infections but cyclic neutropenia is generally a more benign disorder. Cases are generally sporadic but kindred with autosomal dominant inheritance have been reported. By positional cloning, the gene responsible for cyclic neutropenia was first identified as ELA2, encoding a serine protease of phagocyte granules that is known as neutrophil elastase 2. The hypothesis that the two conditions were allelic was then tested and heterozygous mutations in ELA2 were found in most patients with sporadic or dominant severe congenital neutropenia. Interestingly, in cyclic neutropenia the mutations seem to cluster near the active site of the enzyme whereas the opposite face was predominantly affected by the mutations found in severe neutropenia. Understanding how mutations in neutrophil elastase 2 affect either hematopoiesis timing or myeloid maturation is a challenging and exciting prospect.

Immunotherapy Selected by Yang Liu Ohio State University, Columbus, OH, USA

Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. Kappos L, Comi G, Panitch H, Oger J, Antel J, Conlon P, Steinman L, Comi G, Kappos L, Oger J et al.: Nat Med 2000, 6:1176-1182. AND

Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Bielekova B, Goodwin B, Richert N, Cortese I, Kondo T, Afshar G, Gran B, Eaton J, Antel J, Frank JA et al.: Nat Med 2000, 6:1167-1175. • Significance:These two studies documented apparently conflicting findings in two phase II studies involving therapy of MS patients using an APL. The results raised serious concerns about the potential of APLs in therapy of autoimmune diseases.

Findings: Both studies reported significant allergic reactions among patients receiving 50 mg/dose of the APL. More importantly, both studies revealed that the APL induced T cells that cross-react with the endogenous myelin basic protein. Whereas the study of Bielekova et al. suggested that 50 mg/dose of APL treatment can be linked in exacerbations of MS in two patients, that of Kappos et al. — with a larger number of patients — failed to support this. Moreover, Kappos et al. observed preferential induction of Th2-type T cells by the APL whereas Bielekova et al. were unable to substantiate this observation. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Wei Yq, Wang Qr, Zhao X, Yang L, Tian L, Lu Y, Kang B, Lu Cj, Huang Mj, Lou Yy et al.: Nat Med 2000, 6:1160-1166. •• Significance: A simple and novel approach to induce a broad-spectrum protection against tumor cells by breaking immunological tolerance with xenogeneic endothelial cells. Findings: Immunization of multiple sources of fixed human vascular endothelial cells induces protection against challenges by fibrosarcoma Meth A, mammary carcinoma MA782/5S and hepatoma H22 in mice. Part of the immunity can be attributed to antibodies specific for VEGFR II-H and αv integrin-H.

Transplantation Selected by Kathryn Wood and Andrew Bushell John Radcliffe Hospital, Oxford, UK

Fibronectin-mononuclear cell interactions regulate type-1 helper T cell cytokine network in tolerant transplant recipients. Coito AJ, Onodera K, Kato H, Busuttil RW, Kupiek-Weglinski JW: Am J Path 2000, 157:1207-1218. •Significance: It is generally accepted that acute graft rejection is initiated by cellular mechanisms and clinical biopsies from rejecting grafts usually have a massive cellular infiltrate. However, significant cellular infiltration is also often seen in biopsies obtained from patients with little sign of rejection. Although these patients could not be described as tolerant, significant infiltration without graft damage is also seen in many animal models of transplantation tolerance, suggesting a common theme. Clinical and experimental observations imply that the difference in cellular infiltration in rejecting versus nonrejecting grafts is qualitative rather than quantitative. Understanding this difference could help in the design of strategies aimed at delivering transplantation tolerance in man. Findings: Fibronectin (FN) is an extracellular-matrix protein expressed mainly on macrophages/monocytes, vascular endothelium and smooth-muscle cells. Upregulation of β1 integrins (ligands for FN) on T cells is driven by T cell activation, suggesting an important role for these interactions in recruitment of T cells into the graft. In this study, rats were made tolerant by exposure to donor antigen (skin grafts) under the cover of a non-depleting anti-CD4 antibody. Donor heart grafts survived indefinitely and were free of myocardial damage and vasculopathy yet showed a persistent cellular infiltrate, which in terms of cell numbers was at times similar to that in untreated, rejecting grafts. However, macrophages in tolerant grafts remained negative for FN whereas those from rejecting grafts were clearly positive. Although T cells were readily detected in tolerant grafts, very few were activated, as judged by CD25 expression, and this seemed to correlate with an almost complete absence of IL-2 and IFN-γ expression. FN induction was examined in two other models: in one, tolerance was adoptively transferred to secondary recipients then abrogated by

Paper alert

depleting, anti-CD4 antibody; in the other, a chronic rejection model, sub-therapeutic cyclosporin prevented acute but not chronic rejection. In both situations, upregulation of macrophage FN expression was seen. In a previous study from the same group, blockade of fibronectin–integrin interactions using peptides derived from the binding domain of FN prevented acute graft rejection and in the present study this appeared to correlate with a reduced expression of IL-2 and IFN-γ. Overall the data from this group identify FN expression on infiltrating macrophages as an important difference seen between tolerant and rejecting grafts. The implication seems to be that modulation of FN expression alters the phenotype of T cells recruited and retained within the graft and identifies FN as a potential therapeutic target. Anti-CD154 therapeutic antibodies induce infectious transplantation tolerance. Graca L, Honey K, Adams E, Cobbold SP, Waldmann H: J Immunol 2000, 165:4783-4786. •Significance: Anti-CD154 (anti-CD40L) antibodies have been shown to be very effective anti-rejection agents in a number of mouse and primate transplant models and at least one preparation has briefly entered clinical trials. Although these trials were halted prematurely (see T Kawai et al., Nat Med 2000, 6:114), the importance of the CD40–CD154 pathway in T cell and antigen-presenting cell activation suggests that such antibodies remain prime candidates for further clinical evaluation. Though it is well established that blockade of the CD40–CD154 pathway prevents cellular activation and can lead to activation-induced cell death, what is not clear is whether such treatment has the potential to lead to true transplantation tolerance. Findings: In this study, CBA mice transgenic for human CD52 under the control of the CD2 promoter were thymectomised and treated with a depleting anti-CD8 antibody in order to deplete CD8+ T cells (since CD8+ T cells appear to be quite resistant to anti-CD154 therapy) and to stabilize the peripheral T cell compartment. Such mice transplanted with B10.BR skin grafts (H-2b minors on an H-2k background — a multiple minor mismatch) under the cover of anti-CD154 (MR1) treatment accepted their grafts indefinitely and were clearly tolerant, since infused spleen cells from syngeneic mice (CBA) were unable to reconstitute rejection of either the original or a second donor skin graft. This is not too surprising, since tolerance can be demonstrated in many models where long-term graft survival has been facilitated by a wide variety of different therapeutic agents. However, the important observation of this study was made using anti-CD52 antibody to deplete tolerant mice of tolerant T cells at different times relative to adoptive transfer of naive, recipient-type cells. When the tolerant mice were depleted of tolerant T cells 1 day after adoptive transfer, both the original and a second B10.BR skin graft were rejected acutely. However, if the infused naive cells were allowed to coexist in the tolerant host for 42 days before depletion of the original tolerant cells, the primary, a second and a third skin graft were accepted. Thus, tolerance induced by anti-CD154 blockade appears, in this model at least, to involve an infectious mechanism whereby a cohort of tolerant T cells can prevent naive T cells from causing rejection and convert them to a tolerant phenotype. These data are clearly reminiscent of the phenomenon of infectious transplantation tolerance induced by anti-CD4 plus anti-CD8 antibodies and add an important piece of information to the anti-CD154 story. Although additional strategies will almost certainly be necessary to target CD8+

7

T cells, the data confirm the importance of future studies on CD154 blockade in clinical transplantation.

Allergy and hypersensitivity Selected by James Gorham Dartmouth Medical School, Lebanon, NH, USA

Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma. Karp CL, Grupe A, Schadt E, Ewart SL, Keane-Moore M, Cuomo PJ, Köhl J, Wahl L, Kuperman D, Germer S et al.: Nat Immunol 2000 1:221-226. • Significance: The authors cleverly combine strain-specific microarray expression-profiling and quantitative trait locus analysis to identify the gene encoding the complement factor C5 as a likely susceptibility locus for the development of airway hyper-responsiveness (AHR) in mice. Findings: Expression profiling of lungs from ovalbumin-challenged mice of strains susceptible (A/J) or resistant (C3H) to allergen-induced AHR led to the identification of over 200 differentially expressed genes. The gene for one of these, C5, was located within a previously identified genetic interval associated with susceptibility to AHR. Analysis of BC1 (F1 x A/J) mice indicated that low C5 mRNA expression was strongly associated with high susceptibility to AHR and vice versa. The mechanism by which the C5 low-expressor allele might contribute to AHR was suggested by in vitro analysis of primary human monocytes; this indicated that blockade of C5a signaling pathways was associated with decreased IL-12 and increased IL-10 production, consistent with a mechanism in which the absence of C5a permits pro-asthmatic Th2 responses. Definitive identification of C5, and not a closely linked gene, as a murine AHR-susceptibility gene will require appropriate knockin or transgenic expression experiments. Combined stimulation with the T helper cell type 2 cytokines interleukin (IL)-4 and IL-10 induces mouse mast cell apoptosis. Yeatman CF II, Jacobs-Helber SM, Mirmonsef P, Gillespie SR, Bouton LA, Collins HA, Sawyer ST, Shelburne CP, Ryan JJ: J Exp Med 2000 192:1093-1103. • Significance: This study shows that Th2 cytokines, infamous for promoting allergy-inducing ‘type 2’ responses, including enhanced IgE production, may also participate in the attenuation of such responses by inducing apoptosis of mast cells. Findings: Murine bone-marrow-derived mast cells (BMMCs) cultured in the presence of IL-4, IL-10 and IL-3 showed enhanced apoptosis compared with BMMCs cultured with IL-3 alone. The two Th2 cytokines were additive, with IL-4 or IL-10 separately (but with IL-3 in each case) inducing less apoptosis than the combination. Apoptosis was dependent upon the IL-4 signaling molecule Stat6 but was not dependent upon the Fas/FasL axis, since BMMCs cultured from lpr, gld or wild-type mice were all driven into apoptosis by IL-4 plus IL-10 (and IL-3). This combination inhibited expression of bcl-2 and of the related bcl-XL in BMMCs, suggesting (but not proving) that apoptosis is mediated by suppression of expression of bcl-2 family members. Intriguingly, IgE cross-linking enhanced apoptosis of BMMCs mediated by IL-4 plus IL-10 (and IL-3), indicating that allergen exposure may prime mast cells to Th2-mediated death. Functional flexibility in T cells: independent regulation of CD4+ T cell proliferation and effector function in vivo. Laouar Y, Crispe IN: Immunity 2000, 13:291-301. •• Significance: This provocative analysis directly challenges prevailing theories concerning the relationship between

8

Paper alert

T helper cell proliferation and effector cell differentiation, by presenting evidence that T cell division per se is not necessary for the development of the effector state or for the expression of effector cytokines such as IFN-γ. These results are inconsistent with recently proposed models, in which effector cytokine expression is regulated by a cell cycle counting mechanism. Findings: Mice harboring varying proportions of antigen-naive mono-specific TCR-transgenic CD4+ T cells were stimulated with antigen. Cycling and non-cycling T cells were distinguished using BrdU uptake. Antigen challenge elicited an activated cell surface phenotype (e.g. CD44high, CD62Llow) and expression of the effector cytokine, IFN-γ, in cycling and non-cycling cells alike. These observations raise the possibility that naive CD4+ T cells can acquire effector functions, such as cytokine production, without necessarily transiting the cell cycle. Interferon γ signaling alters the function of T helper type 1 cells. Tau GZ, von der Weid T, Lu B, Cowan S, Kvatyuk M,

Pernis A, Cattoretti G, Braunstein NS, Coffman RL, Rothman PB: J Exp Med 2000 192:977-986. • Significance: As they differentiate to the Th1 effector state and begin to produce IFN-γ, T helper cells lose expression of one of the subunits of the IFN-γ receptor (IFN-γR2), secondary to ligand-mediated receptor downmodulation. This paper shows that downregulation of IFN-γR2 is not simply an epiphenomenon of Th1 differentiation and IFN-γ production but is actually a functional requirement for effective Th1 immunity. Findings: Transgenic mice expressing IFN-γR2 driven by the CD2 promoter/enhancer were created, allowing for constitutive expression of IFN-γR2 in all T cells, including Th1 cells. T cells from CD2–IFN-γR2 transgenic mice generated poor Th1 responses in skewing-assays in vitro and mice were deficient in Th1 responses in vivo, exhibiting poor delayed-type hypersensitivity responses, low expression of antigen-specific antibodies of the IgG2a isotype and failure to contain infection with the parasite, Leishmania major.