Nicholas R. English

Increased Production of Immature Myeloid Cells in Cancer Patients: A Mechanism of Immunosuppression in Cancer

Defective dendritic cell (DC) function caused by abnormal differentiation of these cells is an important mechanism of tumor escape from immune system control. Previously, we have demonstrated that the number and function of DC were dramatically reduced in cancer patients. This effect was closely associated with accumulation of immature cells (ImC) in peripheral blood. In this study, we investigated the nature and functional role of those ImC. Using flow cytometry, electron microscopy, colony formation assays, and cell differentiation in the presence of different cell growth factors, we have determined that the population of ImC is composed of a small percentage (<2%) of hemopoietic progenitor cells, with all other cells being represented by MHC class I-positive myeloid cells. About one-third of ImC were immature macrophages and DC, and the remaining cells were immature myeloid cells at earlier stages of differentiation. These cells were differentiated into mature DC in the presence of 1 μM all-trans-retinoic acid. Removal of ImC from DC fractions completely restored the ability of the DC to stimulate allogeneic T cells. In two different experimental systems ImC inhibited Ag-specific T cell responses. Thus, immature myeloid cells generated in large numbers in cancer patients are able to directly inhibit Ag-specific T cell responses. This may represent a new mechanism of immune suppression in cancer and may suggest a new approach to cancer treatment.

Human peripheral blood contains two distinct lineages of dendritic cells.

Human peripheral blood contains two populations of dendritic cells (DC) but their developmental relationship has not been established. Freshly isolated CD11c– DC possessed a lymphoid morphology, lacked myeloid markers but expressed lymphoid markers (CD4+ CD10+) whilst the CD11c+ DC were monocytoid in appearance and expressed myeloid markers. Although both populations were allostimulatory, only the CD11c+ DC were able to take up antigen. Irrespective of the culture conditions the CD11c– cells developed into CD11c– CD13– CD33– CD4+ CD1a– CD83+/– DC. In contrast, cultured CD11c+ cells developed the phenotype CD11c+ CD13+ CD33+/– CD4– CD1a+ CD83+ CD9+. Only the CD11c+ DC expressed macrophage colony‐stimulating factor (M‐CSF) receptor and gave rise to CD14+, esterase+, phagocytic macrophages when cultured in M‐CSF. These data suggest that these two populations of DC represent distinct lineages of antigen‐presenting DC.

Migration and Maturation of Human Colonic Dendritic Cells

Dendritic cells (DC) in the colon may regulate intestinal immunity but remain poorly characterized. In this study a CD11c+HLA-DR+lin− (CD3−CD14−CD16−CD19−CD34−) population has been identified by flow cytometry in cells obtained by rapid collagenase digestion of human colonic and rectal biopsies. These day 0 (d0) CD11c+HLA-DR+lin− cells comprised ∼0.6% of the mononuclear cells obtained from the lamina propria, were endocytically active, and had the phenotype of immature DC; they were CD40+ and expressed low levels of CD83 and CD86, but little or no CD80 or CD25. Similar d0 DC populations were isolated from the colonic mucosa of healthy controls and from both inflamed and noninflamed tissue from patients with Crohn’s disease. The lamina propria also contained a population of cells capable of migrating out of biopsies during an overnight culture and differentiating into mature DC with lower levels of endocytic activity and high cell surface expression of CD40, CD80, CD86, CD83, and CD25. This mature DC population was a potent stimulator of an allogeneic mixed leukocyte (MLR). Overnight culture of cells isolated by enzymatic digestion on d0 yielded DC with a phenotype intermediate between that of the d0 cells and that of the cells migrating out overnight. Overnight culture of colonic cells in which DC and HLA-DR+lin+ cells were differentially labeled with FITC-dextran suggested that some of the maturing DC might differentiate from HLA-DR+lin+ progenitors. This study presents the first analysis of the phenotype, maturational status, and migratory activity of human gut DC.

Murine dendritic cells internalize Leishmania major promastigotes, produce IL-12 p40 and stimulate primary T cell proliferation in vitro

Metacyclic Leishmania promastigotes (PM), transmitted by sand‐fly bite, are likely to interact initially with cells of the dendritic cell (DC) lineage(s) in the epidermis or dermis. Epidermal Langerhans cells internalize L. major amastigotes (AM) and transport them to draining lymphnodes (Moll, H., Fuchs, H., Blank, C. and Röllinghoff, M., Eur. J. Immunol. 1993. 23: 1595) but little is known about the interaction of DC with PM. The present study demonstrates that DC are able to internalize PM and that the fate of the parasites within DC differs from that within macrophages (Mϕ). DC took up small numbers of PM which did not differentiate into AM but appeared to be degraded; Mϕ internalized large numbers of PM into parasitophorous vacuoles where they differentiated into AM. In response to direct stimulation with PM, DC from both C3H (“resistant” to L. major infection) and BALB/c (“susceptible”) up‐regulated production of IL‐12 p40. In contrast, IL‐12 production by Mϕ was not detected. DC exposed to either metacyclic PM or PM culture supernatants were also able to stimulate proliferative responses in lymph node T cells from naive mice. These data indicate that DC have the capacity to promote protective Th1 immune responses in Leishmania infection and suggest that DC exposed to PM may be useful in immunotherapy and vaccination.

Adipose tissue of human omentum is a major source of dendritic cells, which lose MHC class II and stimulatory function in Crohn's disease

Adipose tissue is reported to contain monocyte‐like pre‐adipocytes, which may mature into macrophages, contributing to local inflammation. Dendritic cells (DC) can be derived from monocytes and initiate and regulate primary immune responses. We hypothesized, therefore, that adipose tissue may provide DC involved in local immune activity. To test this, we studied cells from human omental adipose tissue samples from 17 patients with benign gynecological disease. The hypothesis that adipose tissue DC are involved in inflammatory disease was tested by comparing these cells with those from 18 patients with Crohns disease, where hypertrophy of adipose tissue suggests involvement in disease. A high proportion of the 1.33 ± 0.12 × 105 CD45‐positive cells/mg, obtained from control omenta, expressed CD11c, CD1a, and CD83; costimulatory molecules CD40, CD80, and CD86; and major histocompatibility complex (MHC) Class II but little CD14, CD16, or CD33. Omental cells showing morphological characteristics of DC were also observed. Metrizamide gradient‐enriched DC from these populations were potent stimulators of primary proliferation of allogeneic T cells in mixed leukocyte reactions. Increased numbers of CD45+ cells from omentum of Crohns patients (4.50±1.08×105 CD45+ cells/mg) contained higher percentages of CD11c+ and CD40+ cells (80.8±3.8% vs. 63.4±6, P=0.032; 77.9±4% vs. 58.8±6.5, P=0.029, respectively), but MHC Class II and stimulatory capacity were almost completely lost (P=<0.001), suggesting innate activation but lost capacity to stimulate adaptive immune responses. Granulocytes were also present amongst the omental cells from Crohns patients. Results indicated that omentum may provide DC, which could “police” local infections and contribute to and/or reflect local inflammatory activity.

CD4 expression on dendritic cells and their infection by human immunodeficiency virus.

Infection of dendritic cells (DC) by human immunodeficiency virus (HIV) has been disputed. Employing a fluorescence-activated cell sorter, DC, identified by the absence of membrane markers for T, B, natural killer (NK) and monocytic cells and by high levels of MHC class II DR antigen, were shown to express low levels of CD4. Immunomagnetic beads were used to separate blood low density cells, which are enriched for DC, into CD4-positive and -negative populations. Examination of these cells by electron microscopy showed an increase in the percentage of cells with DC morphology in the CD4-positive fraction and a reduction in the CD4-negative fraction. Electron microscopy of semi-purified DC preparations infected in vitro for 5 days with HIV-1 revealed morphologically distinct veiled DC with mature virions on the cell surface and virus budding through the cell membrane. Further evidence for the growth of HIV in DC was provided by experiments in which DC were extensively depleted of contaminating lymphocytes and monocytes prior to infection. Estimation of provirus load by a nested PCR indicated that after 5 days an infection level of one provirus copy per five cells could be achieved. After 7 days the provirus copy number could exceed the cellular genome copy number, suggesting that some cells had more than one provirus. Infectious virus could not be demonstrated in these cultures after 24 h but was detected after 5 or 7 days. Infection of DC in the presence of antibodies against CD4 was inhibited and suggests infection occurs via a CD4-dependent pathway. These results confirm that DC are susceptible to HIV infection in vitro. The immunological consequences of DC infection in vivo may be significant in the pathogenesis of AIDS.

Rapid dendritic cell mobilization to the large intestinal epithelium is associated with resistance to Trichuris muris infection.

The large intestine is a major site of infection and disease, yet little is known about how immunity is initiated within this site and the role of dendritic cells (DCs) in this process. We used the well-established model of Trichuris muris infection to investigate the innate response of colonic DCs in mice that are inherently resistant or susceptible to infection. One day postinfection, there was a significant increase in the number of immature colonic DCs in resistant but not susceptible mice. This increase was sustained at day 7 postinfection in resistant mice when the majority of the DCs were mature. There was no increase in DC numbers in susceptible mice until day 13 postinfection. In resistant mice, most colonic DCs were located in or adjacent to the epithelium postinfection. There were also marked differences in the expression of colonic epithelial chemokines in resistant mice and susceptible mice. Resistant mice had significantly increased levels of epithelium-derived CCL2, CCL3, CCL5, and CCL20 compared with susceptible mice. Furthermore, administering neutralizing CCL5 and CCL20 Abs to resistant mice prevented DC recruitment. This study provides clear evidence of differences in the kinetics of DC responses in hosts inherently resistant and susceptible to infection. DC responses in the colon correlate with resistance to infection. Differences in the production of DC chemotactic chemokines by colonic epithelial cells in response to infection in resistant vs susceptible mice may explain the different kinetics of the DC response.

IL-10-Producing B220+CD11c− APC in Mouse Spleen

APC acting at the early stages of an immune response can shape the nature of that response. Such APC will include dendritic cells (DCs) but may also include populations of B cells such as marginal zone B cells in the spleen. In this study, we analyze APC populations in mouse spleen and compare the phenotype and function of B220+CD11c− populations with those of CD11c+ spleen DC subsets. Low-density B220+ cells had morphology similar to DCs and, like DCs, they could stimulate naive T cells, and expressed high levels of MHC and costimulatory molecules. However, the majority of the B220+ cells appeared to be of B cell lineage as demonstrated by coexpression of CD19 and surface Ig, and by their absence from RAG-2−/− mice. The phenotype of these DC-like B cells was consistent with that of B cells in the marginal zone of the spleen. On bacterial stimulation, they preferentially produced IL-10 in contrast to the DCs, which produced IL-12. Conventional B cells did not produce IL-10. The DC-like B cells could be induced to express low levels of the DC marker CD11c with maturational stimuli. A minority of the B220+CD11c− low-density cells did not express CD19 and surface Ig and may be a DC subset; this population also produced IL-10 on bacterial stimulation. B220+ APC in mouse spleen that stimulate naive T cells and preferentially produce IL-10 may be involved in activating regulatory immune responses.

Analysis of human immunodeficiency virus type 1 (HIV-1) variants and levels of infection in dendritic and T cells from symptomatic HIV-1-infected patients.

Dendritic cells (DC) are required to initiate primary cellular immune responses. Human immunodeficiency virus type 1 (HIV-1) infection of DC may be central to transmission and persistence of virus and in the pathogenesis of AIDS. In symptomatic HIV-1-infected patients the proportion of DC in the mononuclear cell population was reduced. Provirus load in the T cells was 3-100 times higher than in DC and there was no correlation between the levels of infection in the two cell types. Phylogenetic analysis of amino acids in the V3 loop and flanking regions indicated intermingling of sequences and thus provides the first evidence for transfer of virus between DC and T cells in vivo. In one of three patients analysed there were significant differences in amino acid residues in the V3 region. This may reflect reduced interactions between DC and T cells in infected individuals and for the existence of variants with a stronger tropism for DC, which could play a role in transmission by initiating infection in mucosal DC.

Detection of HIV DNA in peripheral blood dendritic cells of HIV-infected individuals

Previous studies from this laboratory indicated infection of dendritic cells (DC) from peripheral blood of individuals infected with HIV1. Here, further evidence for the infection of peripheral blood DC with HIV1 is presented. Low-density cells (LDC) were prepared from blood mononuclear cells of HIV-infected individuals at different clinical stages of disease. These cells are enriched (10-40%) for MHC class-II-bearing DC, while most of the remaining cells are monocytes, and 2-10% are lymphocytes. A quantitative polymerase chain technique (PCR) was used to estimate the HIV provirus load in LDC and lymphocytes of patients in different disease categories. HIV provirus was detected in every LDC preparation, and for many individuals, particularly CDC stage IV patients, the load was higher in the LDC than in the lymphocyte fraction. These findings suggested that patient DC are infected with HIV. In order to provide confirmatory evidence for this conclusion, PCR was performed on DC that were highly purified from LDC by panning to remove contaminating T, B, natural killer and monocytic cells. High levels of HIV provirus were found in these purified DC. These findings suggest that DC provide a reservoir of HIV and that the consequences of such infection may be relevant to the development of disease.