TGFßR signalling controls CD103+CD11b+ dendritic cell development in the intestine.

CD103+CD11b+ dendritic cells (DCs) are unique to the intestine, but the factors governing their differentiation are unclear. Here we show that transforming growth factor receptor 1 (TGFßR1) has an indispensable, cell intrinsic role in the development of these cells. Deletion of Tgfbr1 results in markedly fewer intestinal CD103+CD11b+ DCs and a reciprocal increase in the CD103-CD11b+ dendritic cell subset. Transcriptional profiling identifies markers that define the CD103+CD11b+ DC lineage, including CD101, TREM1 and Siglec-F, and shows that the absence of CD103+CD11b+ DCs in CD11c-Cre.Tgfbr1 fl/fl mice reflects defective differentiation from CD103-CD11b+ intermediaries, rather than an isolated loss of CD103 expression. The defect in CD103+CD11b+ DCs is accompanied by reduced generation of antigen-specific, inducible FoxP3+ regulatory T cells in vitro and in vivo, and by reduced numbers of endogenous Th17 cells in the intestinal mucosa. Thus, TGFßR1-mediated signalling may explain the tissue-specific development of these unique DCs.Developmental cues for the different dendritic cell (DC) subsets in the intestine are yet to be defined. Here the authors show that TGFßR1 signalling is needed for development of CD103+CD11b+ intestinal DCs from CD103-CD11b+ cells and that they contribute to the generation of Th17 and regulatory T cells.

Distribution, location, and transcriptional profile of Peyer's patch conventional DC subsets at steady state and under TLR7 ligand stimulation.

The initiation of the mucosal immune response in Peyer's patch (PP) relies on the sampling, processing, and efficient presentation of foreign antigens by dendritic cells (DCs). Among PP DCs, CD11b+ conventional DCs (cDCs) and lysozyme-expressing DCs (LysoDCs) have distinct progenitors and functions but share many cell surface markers. This has previously led to confusion between these two subsets. In addition, another PP DC subset, termed double-negative (DN), remains poorly characterized. Here we show that both DN and CD11b+ cDCs belong to a unique SIRPα+ cDC subset. At steady state, cDCs and TIM-4+ macrophages are mainly located in T-cell zones, i.e., interfollicular regions, whereas a majority of subepithelial phagocytes are monocyte-derived cells, namely, LysoDCs and TIM-4- macrophages. Finally, oral administration of a Toll-like receptor 7 ligand induces at least three TNF-dependent events: (i) migration of dome-associated villus cDCs in interfollicular regions, (ii) increase of CD8α+ interfollicular cDC number, and (iii) activation of both CD11b+ and CD8α+ interfollicular cDCs. The latter is marked by a genetic reprograming leading to the upregulation of type I interferon-stimulated and of both immuno-stimulatory and -inhibitory gene expression.

Tissue-specific differentiation of colonic macrophages requires TGFß receptor-mediated signaling.

Intestinal macrophages (mφ) form one of the largest populations of mφ in the body and are vital for the maintenance of gut homeostasis. They have several unique properties and are derived from local differentiation of classical Ly6Chi monocytes, but the factors driving this tissue-specific process are not understood. Here we have used global transcriptomic analysis to identify a unique homeostatic signature of mature colonic mφ that is acquired as they differentiate in the mucosa. By comparing the analogous monocyte differentiation process found in the dermis, we identify TGFß as an indispensable part of monocyte differentiation in the intestine and show that it enables mφ to adapt precisely to the requirements of their environment. Importantly, TGFßR signaling on mφ has a crucial role in regulating the accumulation of monocytes in the mucosa, via mechanisms that are distinct from those used by IL10.

Study of antigen-processing steps reveals preferences explaining differential biological outcomes of two HLA-A2-restricted immunodominant epitopes from human immunodeficiency virus type 1.

Cytotoxic T-lymphocyte (CTL) responses directed to different human immunodeficiency virus (HIV) epitopes vary in their protective efficacy. In particular, HIV-infected cells are much more sensitive to lysis by anti-Gag/p17(77-85)/HLA-A2 than to that by anti-polymerase/RT(476-484)/HLA-A2 CTL, because of a higher density of p17(77-85) complexes. This report describes multiple processing steps favoring the generation of p17(77-85) complexes: (i) the exact COOH-terminal cleavage of epitopes by cellular proteases occurred faster and more frequently for p17(77-85) than for RT(476-484), and (ii) the binding efficiency of the transporter associated with antigen processing was greater for p17(77-85) precursors than for the RT(476-484) epitope. Surprisingly, these peptides, which differed markedly in their antigenicity, displayed qualitatively and quantitatively similar immunogenicity, suggesting differences in the mechanisms governing these phenomena. Here, we discuss the mechanisms responsible for such differences.

Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology.

We show here that mouse interferon-alpha (IFN-alpha)-producing cells (mIPCs) are a unique subset of immature antigen-presenting cells (APCs) that secrete IFN-alpha upon stimulation with viruses. mIPCs have a plasmacytoid morphology, can be stained with an antibody to Ly6G and Ly6C (anti-Ly6G/C) and are Ly6C+B220+CD11cloCD4+; unlike other dendritic cell subsets, however, they do not express CD8alpha or CD11b. Although mIPCs undergo apoptosis in vitro, stimulation with viruses, IFN-alpha or CpG oligonucleotides enhanced their survival and T cell stimulatory activity. In vivo, mIPCs were the main producers of IFN-alpha in cytomegalovirus-infected mice, as depletion of Ly6G+/C+ cells abrogated IFN-alpha production. mIPCs produced interleukin 12 (IL-12) in response to viruses and CpG oligodeoxynucleotides, but not bacterial products. Although different pathogens can selectively engage various APC subsets for IL-12 production, IFN-alpha production is restricted to mIPCs' response to viral infection.

NK cell functions restrain T cell responses during viral infections.

NK cell functions for regulation of T cell responses were evaluated during acute viral infections. In vivo depletion studies established that the presence of NK cells in murine cytomegalovirus (MCMV)-infected immunocompetent mice negatively affected CD4 and CD8 T cell IFN-gamma expression, bromodeoxyuridine (BrdU) incorporation, and expansion. To evaluate NK cell effects, under conditions when NK cells do not control viral replication, experiments were performed using lymphocytic choriomeningitis virus (LCMV). Depletion of NK cells did not affect LCMV-elicited T cell responses in immunocompetent mice; however, the presence of NK cells did inhibit CD4 T cell IFN-gamma production, BrdU incorporation, and expansion in infected MHC class I- and CD8 T cell-deficient beta2M-/- mice. Together, the results reveal a previously unappreciated immunoregulatory role of NK cells for downstream T cell responses.

Characteristics of HIV-1 Nef regions containing multiple CD8+ T cell epitopes: wealth of HLA-binding motifs and sensitivity to proteasome degradation.

First and foremost among the many factors that influence epitope presentation are the degradation of Ag, which results in peptide liberation, and the presence of HLA class I molecules able to present the peptides to T lymphocytes. To define the regions of HIV-1 Nef that can provide multiple T cell epitopes, we analyzed the Nef sequence and determined that there are 73 peptides containing 81 HLA-binding motifs. We tested the binding of these peptides to six common HLA molecules (HLA-A2, -A3, -A24, -B7, -B8, and -B35), and we showed that most of them were efficient binders (54% of motifs), especially peptides associating with HLA-A3, -B7/35, and -B8 molecules. Nef peptides most frequently recognized by T cells of HIV-1-infected individuals were 90-97, 135-143, 71-81, 77-85, 90-100, 73-82, and 128-137. The frequency of T cell recognition was not directly related to the strength of peptide-HLA binding. The generation of Nef epitopes is crucial; therefore, we investigated the digestion by the 20S proteasome of a large peptide, Nef(66-100). This fragment was efficiently cleaved, and NH(2)-terminally extended precursors of epitope 71-81 were recognized by T cells of an HIV-1-infected individual. These results suggest that a high frequency of T cell recognition may depend on proteasome cleavage.

Weak anti-HIV CD8(+) T-cell effector activity in HIV primary infection.

HIV-specific CD8(+) T cells play a major role in the control of virus during HIV primary infection (PI) but do not completely prevent viral replication. We used IFN-gamma enzyme-linked immunospot assay and intracellular staining to characterize the ex vivo CD8(+) T-cell responses to a large variety of HIV epitopic peptides in 24 subjects with early HIV PI. We observed HIV-specific responses in 71% of subjects. Gag and Nef peptides were more frequently recognized than Env and Pol peptides. The number of peptides recognized was low (median 2, range 0-6). In contrast, a much broader response was observed in 30 asymptomatic subjects with chronic infection: all were responders with a median of 5 peptides recognized (range 1-13). The frequency of HIV-specific CD8(+) T cells among PBMC for a given peptide was of the same order of magnitude in both groups. The proportion of HIV-specific CD8(+)CD28(-) terminally differentiated T cells was much lower in PI than at the chronic stage of infection. The weakness of the immune response during HIV PI could partially account for the failure to control HIV. These findings have potential importance for defining immunotherapeutic strategies and establishing the goals for effective vaccination.

Broad, intense anti-human immunodeficiency virus (HIV) ex vivo CD8(+) responses in HIV type 1-infected patients: comparison with anti-Epstein-Barr virus responses and changes during antiretroviral therapy.

The ex vivo antiviral CD8(+) repertoires of 34 human immunodeficiency virus (HIV)-seropositive patients with various CD4(+) T-cell counts and virus loads were analyzed by gamma interferon enzyme-linked immunospot assay, using peptides derived from HIV type 1 and Epstein-Barr virus (EBV). Most patients recognized many HIV peptides, with markedly high frequencies, in association with all the HLA class I molecules tested. We found no correlation between the intensity of anti-HIV CD8(+) responses and the CD4(+) counts or virus load. In contrast, the polyclonality of anti-HIV CD8(+) responses was positively correlated with the CD4(+) counts. The anti-EBV responses were significantly less intense than the anti-HIV responses and were positively correlated with the CD4(+) counts. Longitudinal follow-up of several patients revealed the remarkable stability of the anti-HIV and anti-EBV CD8(+) responses in two patients with stable CD4(+) counts, while both antiviral responses decreased in two patients with obvious progression toward disease. Last, highly active antiretroviral therapy induced marked decreases in the number of anti-HIV CD8(+) T cells, while the anti-EBV responses increased. These findings emphasize the magnitude of the ex vivo HIV-specific CD8(+) responses at all stages of HIV infection and suggest that the CD8(+) hyperlymphocytosis commonly observed in HIV infection is driven mainly by virus replication, through intense, continuous activation of HIV-specific CD8(+) T cells until ultimate progression toward disease. Nevertheless, highly polyclonal anti-HIV CD8(+) responses may be associated with a better clinical status. Our data also suggest that a decrease of anti-EBV CD8(+) responses may occur with depletion of CD4(+) T cells, but this could be restored by highly active antiretroviral treatment.

Altered ex vivo balance between CD28+ and CD28- cells within HIV-specific CD8+ T cells of HIV-seropositive patients.

The CD8+CD28- cell population in the blood of HIV-infected individuals is considerably expanded. Yet the cause of this expansion is not clear. The recent demonstration of identical TCR-rearranged genes in CD8+CD28+ and CD8+CD28- expanded T cells of HIV-seropositive patients supports the hypothesis that these two subpopulations are phenotypic variants of the same lineage. To further elucidate the precise relationship between them, we measured the fraction of CD28+ and CD28- T cell subsets in IFN-gamma-producing CD8+ T cells by intracellular staining and cytofluorometry as a functional test for ex vivo recognition of epitopes derived from HIV-1, Epstein-Barr virus (EBV) and influenza virus. HIV-specific CD8+ T cells were predominantly CD28 in all the eight HIV-seropositive subjects tested. In contrast, the anti-EBV and anti-influenza CD8+ T cells were mainly CD28+ in these patients as well as in HIV-seronegative individuals. This supports the notion that the CD8 CD28 hyperlymphocytosis observed in HIV infection is due mainly to chronic activation and differentiation of HIV-specific memory CD8+CD28+ T cells into terminally differentiated CD8+CD28-lymphocytes, because of intense HIV-1 replication and without any important bystander activation. This clarification of the mechanisms underlying the CD8+CD28- expansion in HIV-induced pathogenesis may have important therapeutic implications.

Evolution of cytotoxic T lymphocyte responses to human immunodeficiency virus type 1 in patients with symptomatic primary infection receiving antiretroviral triple therapy.

The impact of highly active antiretroviral treatment (HAART) on anti-human immunodeficiency virus (HIV) cytotoxic T lymphocytes (CTL) was studied in 17 patients with recent symptomatic HIV-1 primary infection receiving triple combination therapy. Anti-HIV CTL were initially detected in 15 patients. In 6, CTL disappeared rapidly and persistently after initiation of therapy. Most of them had a rapid and sustained decrease in plasma HIV RNA to undetectable levels. Conversely, in 6 other patients, CTL remained detectable, which was associated with a less efficient control of viral replication. In 3 others, CTL disappeared only transiently, without clear correlation with the virologic profile. Altogether, despite individual variations, there was a positive correlation between viral replication and anti-HIV-1 cytotoxicity in most subjects, suggesting that the persistence of viral antigens is the main determinant for the maintenance of CTL activity. This raises the question of the potential benefit of anti-HIV CTL induction by immunotherapy in acute seroconverters treated by HAART.

Cross-reactions between the cytotoxic T-lymphocyte responses of human immunodeficiency virus-infected African and European patients.

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.

Delayed virus-specific CD8+ cytotoxic T lymphocyte activity in an HIV-infected individual with high CD4+ cell counts: correlations with various parameters of disease progression.

This 4-year longitudinal study monitored the temporal association between the HIV-specific cytotoxic T lymphocyte (CTL) response and the control viremia in an individual infected with human immunodeficiency virus type (HIV-1). At the beginning of the study, this asymptomatic individual with a high CD4+ cell count showed no HIV-specific cytotoxic activity after polyclonal in vitro restimulation with autologous PHA-blasts, unlike most HIV-seropositive individuals. Anti-HIV CTLs were detected only in the last year of the study, both after in vitro restimulation and directly ex vivo. This was correlated with the inversion of the CD4+/CD8+ ratio, essentially due to increased numbers of CD8+CD28- T lymphocytes. The HIV-specific cytolytic activity was mediated by this CD28+CD28- subpopulation. The amount of HIV-1 provirus in peripheral blood mononuclear cells (PBMCs) did not change during the study, but the HIV RNA in plasma increased and virus was isolated from PBMCs only at the time when HIV-specific CTL activity was detected. This suggests overall that the HIV-1 replication was low in this individual, with a transient increase that could have reached the threshold for CTL reactivation, and was perhaps controlled thereby.

Predominant involvement of CD8+CD28- lymphocytes in human immunodeficiency virus-specific cytotoxic activity.

Distinct functional CD8+ T-cell populations have been observed during human immunodeficiency virus (HIV) infection. One of these functions is the inhibition of viral replication by a noncytotoxic mechanism, which was shown to be mediated by the CD8+CD28+ subpopulation. On the other hand, CD8+ T cells exert an HIV-specific cytotoxic activity. The present study shows that CD8+CD28- lymphocytes display this HIV-specific cytotoxic activity, which is detectable immediately after the cells are purified from peripheral blood. The CD28- population is also able to proliferate and to retain its cytotoxic activity after in vitro restimulation with autologous blast cells. Finally, HIV-specific cytotoxic T cells can be obtained in vitro from the CD8+CD28+ population.