Judith L. McKenzie

Phenotypic Characterization of Five Dendritic Cell Subsets in Human Tonsils

Heterogeneous expression of several antigens on the three currently defined tonsil dendritic cell (DC) subsets encouraged us to re-examine tonsil DCs using a new method that minimized DC differentiation and activation during their preparation. Three-color flow cytometry and dual-color immunohistology was used in conjunction with an extensive panel of antibodies to relevant DC-related antigens to analyze lin(-) HLA-DR(+) tonsil DCs. Here we identify, quantify, and locate five tonsil DC subsets based on their relative expression of the HLA-DR, CD11c, CD13, and CD123 antigens. In situ localization identified four of these DC subsets as distinct interdigitating DC populations. These included three new interdigitating DC subsets defined as HLA-DR(hi) CD11c(+) DCs, HLA-DR(mod) CD11c(+) CD13(+) DCs, and HLA-DR(mod) CD11c(-) CD123(-) DCs, as well as the plasmacytoid DCs (HLA-DR(mod) CD11c(-) CD123(+)). These subsets differed in their expression of DC-associated differentiation/activation antigens and co-stimulator molecules including CD83, CMRF-44, CMRF-56, 2-7, CD86, and 4-1BB ligand. The fifth HLA-DR(mod) CD11c(+) DC subset was identified as germinal center DCs, but contrary to previous reports they are redefined as lacking the CD13 antigen. The definition and extensive phenotypic analysis of these five DC subsets in human tonsil extends our understanding of the complexity of DC biology.

Liposomal delivery of antigen to human dendritic cells

This study investigated whether formulation of antigen in mannosylated liposomes enhanced uptake and activation of dendritic cells (DC) and increased the ability of DC to induce primed T cell proliferation compared to formulation of antigen in unmodified liposomes or in solution. Immature human DC were generated from peripheral blood monocytes cultured with GM-CSF and IL-4. Uptake of antigen by DC and the degree of expression of the cell surface markers MHC class II, CD80, CD86 and the DC maturation marker CD83, was investigated by flow cytometry following incubation with liposomes or solution containing FITC-conjugated antigen. Exposure to liposomes containing FITC-ovalbumin resulted in enhanced expression of cell surface markers when compared to exposure to antigen in solution. Expression was highest following exposure to mannosylated liposomes. Mannosylated liposomes containing tetanus toxoid (TT) stimulated primed T cell proliferation more effectively than TT-neutral liposomes or TT-solution. This work suggests that mannosylated liposomes provide a versatile delivery vehicle for initiating enhanced immune responses to encapsulated peptide or protein vaccines.

Characterization of interstitial dendritic cells in human liver

Sensitive immunofluorescence and immunoperoxidase techniques were used to test an extensive range of monoclonal antibodies for reactivity with Kupffer cells and interstitial dendritic cells (DCs) in cryostat-cut sections of human liver. Leucocytes with a dendritic cell morphology were identified with CD45 (antileucocyte common) reagents in portal tracts, predominantly around bile ducts, and these cells stained strongly for the HLA-DP, DQ, and DR antigens. Kupffer cells stained less intensely with anti-class-II reagents, particularly anti-HLA-DQ. The interstitial DCs expressed the LFA-1 antigen but failed to stain with CD11b, CD11c, and the defined T and B cell CD antibodies; nor did they stain with antibodies to FcR1, FcR11, FcRIII, or the C3b receptor. Of the myeloid monoclonal antibodies available from the 3rd Leucocyte Differentiation Antigen Workshop, only Y2/131, Ki-M7, Ki-M8, and a minority of CD14 antibodies stained DCs, whereas Kupffer cells showed a wider reactivity with antimacrophage antibodies including those of workshop groups 11, 15, 16, and other unique antibodies. A 2nd probable DC population was identified in the liver capsule that had a similar phenotype to portal interstitial DCs. Although some minor phenotypic differences between liver portal DCs and the phenotypes of Langerhans cells and isolated tonsil DCs were noted, our results support the view that there is a unique hemopoietic lineage of DCs. The presence of DCs, which stimulate strong allogeneic T cell responses, in the portal triads is consistent with the fact that the histologic changes of graft-versus-host disease seen in bone marrow transplantation and the lymphocytic infiltrate in a rejecting liver allograft occur predominantly in the periportal region.

The effect of donor pretreatment on interstitial dendritic cell content and rat cardiac allograft survival.

Different pretreatment schedules were applied to rat heart donors and their effect on heart interstitial dendritic cell content was observed using monoclonal antibodies and immunofluorescence techniques. Interstitial dendritic cell (IDC) numbers were correlated with the histology and survival of hearts transplanted into untreated allogeneic recipients. Hearts from AS donors pretreated with cyclophosphamide and total-body irradiation showed prolonged survival in DA recipients only when more than 95% of the graft interstitial dendritic cells were depleted. Reconstitution studies established that prolonged graft survival following donor pretreatment depended on the removal of bone-marrow-derived cells and that these were IDCs. These results suggest that the IDC is the most significant “passenger leucocyte”, and that very small numbers of residual IDCs were still sufficient to cause rapid rejection. DA rat heart contained three times as many IDCs as the AS strain and DA hearts were much more difficult to deplete of IDCs. Pretreated DA hearts were rejected by AS recipients, although grafts with a lower IDC content resulted in an attenuated histological rejection response and a markedly decreased recipient lymphocytotoxin response. To be effective, donor pretreatment schedules had to be initiated 5 days prior to transplantation. Pretreatment 6 hr prior to transplantation failed to deplete IDC or prolong graft survival even in the weak (ASDA) F1 to DA model. Pretreatment protocols used clinically have probably not removed IDCs adequately and the development of methods that deplete IDCs effectively could improve graft survival at no risk to the recipient.

Interstitial Dendritic Cells

Interstitial dendritic cells (IDC) were first identified in the interstitium of non-lymphoid organs as leucocytes which stained intensely with anti-MHC class II antibodies. These cells have been identified in several species including man, and can be distinguished from tissue macrophages by their immunological phenotype and cytochemical and functional characteristics. IDC appear to be closely related to lymphoid dendritic cells (DC), and have the capacity to bind antigen and stimulate T lymphocyte responses. It seems probable that they represent a stage of nonlymphoid dendritic cell differentiation necessary for antigen surveillance, similar to the Langerhans cell of the skin. Exposure to antigen appears to induce migration of these cells into adjacent lymphatics and subsequent localization in the interfollicular areas of lymph node, where the DC present processed antigen to activate a primary T cell response. The IDC has been identified as the passenger leucocyte within organ allografts which contributes substantially to graft immunogenicity, so that eradication of donor organ IDC improves organ graft survival.

The soluble form of CD83 is present at elevated levels in a number of hematological malignancies

Recombinant forms of soluble CD83 (sCD83) inhibit anti-tumor responses. In this analysis of circulating sCD83 levels we report that although >95% of acute myeloid leukemia (AML) and multiple myeloma (MM) patients have normal or only weakly elevated sCD83 levels, 20% of chronic lymphocytic leukemia (CLL) and 5/7 mantle cell lymphoma (MCL) patients have significantly elevated levels (>1 ng/ml). Isolated CLL cells both weakly expressed membrane CD83 (mCD83), and released sCD83 during in vitro culture. We conclude that malignant cells are a potential source of sCD83 and that it may have functional and/or prognostic significance in hematological malignancies, particularly CLL and MCL.

Adhesion molecules on human tonsil dendritic cells.

Dendritic cells are specialist antigen-presenting cells that have a unique ability to stimulate a primary T cell response. Activation of T cells by DC depends on the formation of cell clusters creating DC-T cell membrane contact that probably involves adhesion molecules. Monoclonal antibodies were used to study adhesion molecules on DC, including members of the integrin and immunoglobulin supergene families. DC expressed LFA-1, ICAM-1, LFA-3, and the Hermes antigen, but no other integrin or immunoglobulin supergene family adhesion molecules were detected using a sensitive immunoperoxidase staining technique. Monoclonal antibodies to LFA-1 alpha and LFA-1 beta inhibited DC-stimulated allogeneic T cell (MLR) responses by 75 +/- 12% and 74 +/- 8%, respectively, as did the anti-LFA-3 (56 +/- 3% inhibition) and anti-LFA-2 (60 +/- 5% inhibition) antibodies. Three different anti-ICAM-1 antibodies inhibited only to a limited degree (mean range 8-24%). The inhibitory effect of the LFA-1 and LFA-3 antibodies was maximal if added early to the MLR. The inhibitory effect of the different antibodies was associated with variable decreases in DC-T cell cluster stability. The simultaneous addition of monoclonal antibodies to MLRs and preincubation washing experiments established that DC have at least 3 independent adhesion ligand interactions (LFA-1-ICAM-1, ICAM-1-LFA-1, and LFA-3-CD2) with T cells. It seems likely that the additional ligand for LFA-1, ICAM-2, is expressed on DC and contributes significantly to DC-T cell adherence and T cell activation. The membrane mobility of these molecules may also be important in the DC-T cell activation process.

Circulating levels and clinical significance of soluble CD40 in patients with hematologic malignancies

CD40 plays a critical role in immunoregulation, and CD40 ligation is being investigated as a therapy for hematologic malignancies. Although soluble CD40 (sCD40) is a potential modulator of both antitumor responses and CD40‐based therapies, the levels and significance of sCD40 in patients with hematologic malignancies are unknown.

Renal transplant recipients have elevated frequencies of circulating myeloid-derived suppressor cells

BACKGROUND Cancer, particularly cutaneous squamous cell carcinoma (SCC), is a major cause of mortality in renal transplant recipients (RTRs). Myeloid-derived suppressor cells (MDSC) play a central role in suppressing cancer immunosurveillance but their potential mobilisation in RTRs and levels relative to those of other immunoregulatory dendritic cell (DC) populations have not been analysed. METHODS The circulating frequencies of MDSC and DC were analysed by multicolour flow cytometry in immunocompetent patients without (n = 13) or with (ICI-SCC(Pos), n = 14) current SCC, normal donors (NDs, n = 34), chronic kidney disease patients (CKD patients, n = 22) and RTRs (n = 31). RESULTS Compared to NDs, RTRs had significantly elevated levels of both CD14(Neg) and CD14(Pos) MDSC subsets (P < 0.001), while CKD patients and ICI-SCC(Pos) had significantly elevated levels of only the CD14(Neg)-MDSC subset. DC frequencies were significantly decreased in RTRs and CKD patients but were at normal levels in ICI-SCC(Pos). The MDSC/DC ratio was significantly elevated (P < 0.05) in RTRs (median = 5.7), CKD patients (median = 3.2) and ICI-SCC(Pos) (median = 3.5) relative to NDs (median = 0.7). The use of immunosuppressive drugs in CKD patients and past/current occurrence of SCC in RTRs was associated with significantly increased CD14(Neg)-MDSC frequencies. MDSC enriched from RTRs, when co-cultured with activated NDs T cells significantly suppressed extracellular IL-10 levels and can, when activated with formyl-methionyl-leucyl-phenylalanine, inhibit T-cell proliferation. CONCLUSIONS RTRs, CKD patients and ICI-SCC(Pos) have increased MDSC frequencies and MDSC/DC ratios. These changes may impact on cancer immunosurveillance. Therefore, MDSC represent both a potential therapeutic target and prognostic marker in these patients, with respect to the development of SCC and other malignancies.

Effect of activated human polymorphonuclear leucocytes on T lymphocyte proliferation and viability

Human polymorphonuclear leucocytes (PMN) are thought to be immunosuppressive. The suppressive mechanism(s) used by PMN are, however, not well defined and in this study they were analysed using T‐cell responses to CD3+ CD28 monoclonal antibodies (mAb) as a readout. We demonstrate that in vitro activated PMN (PMNact) can, without any T‐cell interaction, induce apparent T‐cell suppression by inhibiting the stimulatory capacity of the CD3 mAb. However, a cell‐directed suppression of T‐cell proliferation was observed when PMNact were added to pre‐activated T cells that are already committed to polyclonal proliferation. This suppression was partially reversed by catalase addition (P < 0·01) and largely reversed by addition of exogenous interleukin‐2 (P < 0·001) but was not significantly reduced by nitric oxide synthase inhibition, myeloperoxidase inhibition or addition of excess arginine. Following removal of PMNact, suppressed T cells could respond normally to further stimulation. In addition to suppressing proliferation, co‐culture with PMNact also induced a significant decrease in T‐cell viability that was reversed by catalase addition (P < 0·05). The addition of the arginase inhibitor N‐hydroxy‐nor‐l‐arginine induced both a further significant, catalase‐sensitive, loss in T‐cell viability and increased nitrite release (P < 0·001). These data demonstrate that PMN, when activated, can both induce T‐cell death and reversibly inhibit proliferation of activated T cells. The mechanisms underlying these distinct processes and the effects of arginase inhibitors on PMN induced cytotoxicity merit further investigation.