Copy number variation of KIR genes influences HIV-1 control.

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

One naive T cell, multiple fates in CD8+ T cell differentiation.

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR-pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.

Extrafollicular plasmablast B cells play a key role in carrying retroviral infection to peripheral organs.

B cells can either differentiate in germinal centers or in extrafollicular compartments of secondary lymphoid organs. Here we show the migration properties of B cells after differentiation in murine peripheral lymph node infected with mouse mammary tumor virus. Naive B cells become activated, infected, and carry integrated retroviral DNA sequences. After production of a retroviral superantigen, the infected B cells receive cognate T cell help and differentiate along the two main differentiation pathways analogous to classical Ag responses. The extrafollicular differentiation peaks on day 6 of mouse mammary tumor virus infection, and the follicular one becomes detectable after day 10. B cells participating in this immune response carry a retroviral DNA marker that can be detected by using semiquantitative PCR. We determined the migration patterns of B cells having taken part in the T cell-B cell interaction from the draining lymph node to different tissues. Waves of immigration and retention of infected cells in secondary lymphoid organs, mammary gland, salivary gland, skin, lung, and liver were observed correlating with the two peaks of B cell differentiation in the draining lymph node. Other organs revealed immigration of infected cells at later time points. The migration properties were correlated with a strong up-regulation of alpha(4)beta(1) integrin expression. These results show the migration properties of B cells during an immune response and demonstrate that a large proportion of extrafolliculary differentiating plasmablasts can escape local cell death and carry the retroviral infection to peripheral organs.

The parallel lives of angiogenesis and immunosuppression: cancer and other tales.

Emerging evidence indicates that angiogenesis and immunosuppression frequently occur simultaneously in response to diverse stimuli. Here, we describe a fundamental biological programme that involves the activation of both angiogenesis and immunosuppressive responses, often through the same cell types or soluble factors. We suggest that the initiation of these responses is part of a physiological and homeostatic tissue repair programme, which can be co-opted in pathological states, notably by tumours. This view can help to devise new cancer therapies and may have implications for aseptic tissue injury, pathogen-mediated tissue destruction, chronic inflammation and even reproduction.

Development and selection of Valpha l4i NKT cells.

Valpha14 invariant natural killer T (Valpha14i NKT) cells are a unique lineage of mouse T cells that share properties with both NK cells and memory T cells. Valpha14i NKT cells recognize CDld-associated glycolipids via a semi-invariant T cell receptor (TCR) composed of an invariant Valpha14-Jalpha 18 chain paired preferentially with a restricted set of TCRbeta chains. During development in the thymus, rare CD4+ CD8+ (DP) cortical thymocytes that successfully rearrange the semi-invariant TCR are directed to the Valpha14i NKT cell lineage via interactions with CD d-associated endogenous glycolipids expressed by other DP thymocytes. As they mature, Valphal4i NKT lineage cells upregulate activation markers such as CD44 and subsequently express NK-related molecules such as NKI.1 and members of the Ly-49 inhibitory receptor family. The developmental program of Valpha l4i NKT cells is critically regulated by a number of signaling cues that have little or no effect on conventional T cell development, such as the Fyn/SAP/SLAM pathway, NFkappaB and T-bet transcription factors, and the cytokine IL-15. The unique developmental requirements of Valphal4i NKT cells may represent a paradigm for other unconventional T cell subsets that are positively selected by agonist ligands expressed on hematopoietic cells.

c-Myc controls the development of CD8alphaalpha TCRalphabeta intestinal intraepithelial lymphocytes from thymic precursors by regulating IL-15-dependent survival.

The murine gut epithelium contains a large population of thymus-derived intraepithelial lymphocytes (IELs), including both conventional CD4(+) and CD8alphabeta(+) T cells (expressing T-cell receptor alphabeta [TCRalphabeta]) and unconventional CD8alphaalpha(+) T cells (expressing either TCRalphabeta or TCRgammadelta). Whereas conventional IELs are widely accepted to arise from recirculation of activated CD4(+) and CD8alphabeta(+) T cells from the secondary lymphoid organs to the gut, the origin and developmental pathway of unconventional CD8alphaalpha IELs remain controversial. We show here that CD4-Cre-mediated inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biologic activities, selectively impairs the development of CD8alphaalpha TCRalphabeta IELs. In the absence of c-Myc, CD4(-) CD8(-) TCRalphabeta(+) thymic precursors of CD8alphaalpha TCRalphabeta IELs are present but fail to develop on adoptive transfer in immunoincompetent hosts. Residual c-Myc-deficient CD8alphaalpha TCRalphabeta IEL display reduced proliferation and increased apoptosis, which correlate with significantly decreased expression of interleukin-15 receptor subunits and lower levels of the antiapoptotic protein Bcl-2. Transgenic overexpression of human BCL-2 resulted in a pronounced rescue of CD8alphaalpha TCRalphabeta IEL in c-Myc-deficient mice. Taken together, our data support a model in which c-Myc controls the development of CD8alphaalpha TCRalphabeta IELs from thymic precursors by regulating interleukin-15 receptor expression and consequently Bcl-2-dependent survival.

The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells.

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.

T and B lymphocytes exert distinct effects on the homeostasis of NK cells.

There is growing evidence that lymphocytes impact the development and/or function of other lymphocyte populations. Based on such observations we have tested whether the NK cell compartment was phenotypically and functionally altered in the absence of B and/or T cells. Here we show that T cell deficiency significantly accelerates BM NK cell production and the subsequent seeding of splenic and liver NK cell compartments. In contrast, B cell deficiency reduces splenic NK cell survival. In the absence of T and B cells, the size of the NK cell compartments is determined by the combination of these positive and negative effects. Even though NK cell homeostasis is significantly altered, NK cells from T and/or B cell-deficient mice show a normal capacity to kill a susceptible target cell line and to produce IFN. Nevertheless, we noted that the usage of MHC class I-specific Ly49 family receptors was significantly altered in the absence of T and/or B cells. In general, B cell deficiency expanded Ly49 receptor usage, while T cell deficiency exerted both positive and negative effects. These findings show that B and T cells significantly and differentially influence the homeostasis and the phenotype of NK cells.

IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells.

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

Cutting edge: cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo.

Anergic T cells display a marked decrease in their ability to produce IL-2 and to proliferate in the presence of an appropriate antigenic signal. Two nonmutually exclusive classes of models have been proposed to explain the persistence of T cell anergy in vivo. While some reports indicate that anergic T cells have intrinsic defects in signaling pathways or transcriptional activities, other studies suggest that anergy is maintained by environmental "suppressor" factors such as cytokines or Abs. To distinguish between these conflicting hypotheses, we employed the well-characterized bacterial superantigen model system to evaluate in vivo the ability of a trace population of adoptively transferred naive or anergized T cells to proliferate in a naive vs anergic environment upon subsequent challenge. Our data clearly demonstrate that bacterial superantigen-induced T cell anergy is cell autonomous and independent of environmental factors.

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.

Medicinal application of long synthetic peptide technology.

This review covers the latest developments of long synthetic peptide technology for the rapid identification and development of malaria vaccine candidates and immunological modulators. A brief description of the two most common solid-phase synthetic procedures, together with the latest advances in optimisation of peptide chain assembly and analytical instrumentation, is given, with special attention to non-specialists. Several examples of vaccine candidates developed in the authors' or their collaborators' laboratories are also provided.

IL-7/anti-IL-7 mAb complexes restore T cell development and induce homeostatic T Cell expansion without lymphopenia.

IL-7, a member of the common gamma-chain family of cytokines, is essential for B and T lymphocyte development and homeostasis of mature T cell subsets. Thus, naive and memory T cells are both dependent on IL-7 for survival and homeostatic proliferation under lymphopenic conditions. In line with prior findings with IL-2, we show in this study that the biological activity of IL-7 in vivo is greatly increased by association with anti-IL-7 mAb. Under in vivo conditions, IL-7/mAb complexes displayed 50- to 100-fold higher activity than free IL-7 and induced massive expansion of pre-B cells. IL-7/mAb complexes also increased thymopoiesis in normal mice and restored thymopoeisis in IL-7-deficient mice. For mature T cells, IL-7/mAb complexes induced marked homeostatic proliferation of both naive and memory CD4(+) and CD8(+) cell subsets even under normal T cell-replete conditions. Finally, IL-7/mAb complexes were able to enhance the magnitude of the primary response of Ag-specific naive CD8(+) cells. The strong stimulatory activity of IL-7/mAb complexes could be useful for treatment of immunodeficiency and cancer.

Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses.

Correlates of immune-mediated protection to most viral and cancer vaccines are still unknown. This impedes the development of novel vaccines to incurable diseases such as HIV and cancer. In this study, we have used functional genomics and polychromatic flow cytometry to define the signature of the immune response to the yellow fever (YF) vaccine 17D (YF17D) in a cohort of 40 volunteers followed for up to 1 yr after vaccination. We show that immunization with YF17D leads to an integrated immune response that includes several effector arms of innate immunity, including complement, the inflammasome, and interferons, as well as adaptive immunity as shown by an early T cell response followed by a brisk and variable B cell response. Development of these responses is preceded, as demonstrated in three independent vaccination trials and in a novel in vitro system of primary immune responses (modular immune in vitro construct [MIMIC] system), by the coordinated up-regulation of transcripts for specific transcription factors, including STAT1, IRF7, and ETS2, which are upstream of the different effector arms of the immune response. These results clearly show that the immune response to a strong vaccine is preceded by coordinated induction of master transcription factors that lead to the development of a broad, polyfunctional, and persistent immune response that integrates all effector cells of the immune system.