Thomas E. Mandel

The maintenance and regulation of the humoral immune response: persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells.

The evidence indicating that antigen can persist for months or years in the spleens and draining lymph nodes of immunized animals is persuasive. This retained antigen Is almost exclusively, if not exclusively, associated with processes of dendritic cells in the follicles of these lymphoid organs (reviewed in this volume by Mandel et al. 1980), However, the role persisting antigen plays, once an immune response has been initiated, is open to question. It has been suggested that persisting antigen and specific antibody are the major components in an antibody feedback system which maintains and regulates antibody levels in vivo (Richter et al. 1965, Britton & MoUer 1968, Bystryn et at, 1971, Weigle 1975). It is well known that no additional antigen is required to maintain circulating antibody levels in hyperimmunized animals. Even when large volumes of blood are removed, antibody levels soon return to their previous levels. TTiis phenomenon might be explained by assuming that persisting antigen is unable to stimulate antibody synthesis

Administration of Silica Particles or Anti-Lyt2 Antibody Prevents β-Cell Destruction in NOD Mice Given Cyclophosphamide

The cellular pathway of β-cell destruction in type I (insulin-dependent) diabetes is still undefined. L3T4+ T-lymphocytes have a role in both the initiation of insulitis and in recurrent disease in transplanted allogeneic islets in nonobese diabetic (NOD) mice. The roles of macrophages and Lyt2+ T-lymphocytes in β-cell destruction were studied in cyclophosphamide-induced diabetic NOD mice with silica particles and a rat anti-Lyt2 monoclonal antibody. After administration of cyclophosphamide, 10 of 26 untreated mice and 1 of 21 anti-Lyt2-treated mice became diabetic. Insulitis was significantly reduced in anti-Lyt2-treated mice, and immunocytochemical staining showed a lack of Lyt2+ cells. Only 1 of 19 silica-treated mice became diabetic, compared to 8 of 19 control mice. This study demonstrates that both Lyt2+ T-lymphocytes and macrophages are necessary, but not sufficient, for β-cell destruction in NOD mice. Therefore, we propose that macrophages present β-cell antigen to L3T4+ cells, which induce cytotoxic Lyt2+ cells to specifically destroy β-cells.

Cyclophosphamide-Induced Diabetes in NOD/WEHI Mice: Evidence for Suppression in Spontaneous Autoimmune Diabetes Mellitus

Nonobese diabetic (NOD) mice spontaneously develop a lymphocytic infiltration of pancreatic islets (insulitis) that may progress to overt diabetes. Virtually all NOD/WEHI mice develop insulitis, but very few progress to diabetes. However, cyclophosphamide (CY) can promote the onset of diabetes in NOD mice, including the NOD/WEHI strain. The means by which CY produces diabetes was investigated in NOD/WEHI mice, in which it was hypothesized that active suppression mechanisms prevented the progression from insulitis to diabetes. A study of the time course of insulitis in the islets after CY was given showed that insulitis was initially reduced but rapidly increased over 16 days, and T-lymphocytes were predominant in the lesion. This suggested a compression of the normal time course of the disease seen in NOD mice. CY did not produce diabetes in any of 11 non-NOD strains studied. Fetal isografts in NOD mice given CY several days before were subjected to lymphocytic infiltration and β-cell destruction. These findings suggested that CY was not directly (β-cell toxic and that altered β-cells were not essential for β-cell destruction. This was further demonstrated with subdiabetogenic doses of streptozocin, which significantly damaged β-cells but did not increase the severity of insulitis or induce diabetes as did CY. Most important, the transfer of mononuclear cells from nondiabetic NOD mice to mice given CY prevented diabetes, which indicated that the likely effect of CY was via immunomodulation, possibly by allowing poised effector cells to act on (β-cells. The NOD/WEHI mice appear to have suppressor mechanisms acting to halt the progression of the early insulitis lesion and so preventing diabetes occurring in most mice. We propose that CY removes these suppressors and thereby induces a rapid progression to diabetes by compressing the normal immune destruction process into a 2-wk period. This model affords the opportunity to study the process in ways not practicable in the usual time course of events.

CD4+ T cell mediated destruction of xenografts within cell-impermeable membranes in the absence of CD8+ T cells and B cells

Xenogeneic cells encapsulated in cell-impermeable diffusion chambers die within 3 weeks when implanted into immunocompetent animals but not when implanted into immunodeficient animals. To determine which cells are necessary for this observation, we depleted normal mice in vivo of either CD4+ or CD8+ T cells using monoclonal antibodies. We also reconstituted the immune system of athymic CBA mice (T-lymphocyte deficient) and C.B17 SCID mice (T- and B-lymphocyte deficient) with different cell subsets from normal CBA and BALB/C mice, respectively. Depleted or reconstituted mice were implanted with a diffusion chamber containing COS (monkey kidney) cells. Membrane enclosed xenografts survived in CD4+ T cell depleted mice but not in CD8+ T cell depleted or nondepleted control mice. Encapsulated xenografts survived when implanted into either athymic or SCID mice but were destroyed in reconstituted athymic and SCID mice. Furthermore, encapsulated xenogeneic cells were destroyed in athymic or SCID mice reconstituted with CD4+ cell preparations depleted of CD8+ cells and/or B cells. In contrast, encapsulated xenogeneic cells were not destroyed in athymic or SCID mice reconstituted with CD8+ cell preparations depleted of CD4+ cells. These studies highlight the critical role of CD4+ T cells, in the absence of CD8+ cells and B cells, in the processes leading to the ultimate destruction of encapsulated xenografts. Because of the use of cell-impermeable membranes in these studies, the most likely involvement of CD4+ T cells is in the indirect antigen recognition by these cells and subsequent stimulation of inflammatory cells.

Evidence that macrophages are required for T-cell infiltration and rejection of fetal pig pancreas xenografts in nonobese diabetic mice.

BACKGROUND Host macrophages are abundant within fetal pig pancreas xenografts undergoing rejection, but their role is unknown. Therefore, we examined the effect of host macrophage depletion on xenograft rejection. METHODS Nonobese diabetic (NOD) mice were given clodronate-loaded liposomes intravenously to deplete macrophages. Controls received phosphate-buffered saline (PBS) or PBS-liposomes. General immune status was assessed after 2, 3, and 7 days by (1) fluorescence-activated cell sorter analysis of peripheral blood, spleen, and lymph node cells, (2) immunohistochemistry on spleens, and (3) mixed lymphocyte reaction. Organ-cultured fetal pig pancreas was transplanted under the kidney capsule of NOD mice 3 days after clodronate or PBS injection. Grafts were assessed histologically at 4, 5, 6, and 8 days after transplantation. RESULTS Splenic macrophages and peripheral blood monocytes were depleted 2 days after clodronate treatment but had recovered within 11 days. T cell, B cell, and dendritic cell numbers were normal in spleen, peripheral blood, and lymph nodes of clodronate-treated mice, and T cells and antigen-presenting cells from these mice functioned normally in mixed lymphocyte reaction. Clodronate treatment markedly reduced graft infiltration by macrophages, T cells, and eosinophils at 4, 5, and 6 days after transplantation, and was associated with maintenance of endocrine cell viability and insulin expression. However, all grafts were rejected 8 days after transplantation, concordant with reappearance of splenic macrophages. CONCLUSIONS Short-term, specific depletion of macrophages markedly delayed cellular infiltration and rejection of xenografts. The results provide the first evidence that macrophages promote T-cell infiltration and rejection of fetal pig pancreas xenografts in NOD mice.

Progression From Insulitis to β-Cell Destruction in NOD Mouse Requires L3T4+ T-Lymphocytes

The identity of the cells responsible for beta-cell destruction in type I (insulin-dependent) diabetes is still uncertain. L3T4+ T-lymphocytes have a role in the initiation of insulitis and in damaging transplanted allogeneic islets in nonobese diabetic (NOD) mice. The role of L3T4+ T-lymphocytes in destruction of beta-cells of the NOD mouse was studied in cyclophosphamide (CY)-induced diabetic NOD mice with a rat anti-L3T4 monoclonal antibody (MoAb). After administration of CY, most untreated animals became diabetic, whereas all antibody-treated animals remained normoglycemic. Insulitis was still present in MoAb-treated animals, but immunocytochemical staining showed rat antibody blocking the L3T4 antigen on T-lymphocytes. This study provides further evidence that L3T4+ T-lymphocytes are critical to the process of beta-cell destruction in NOD mice. The means by which L3T4+ cells exert their effect remains to be clarified.

Prevention of cyclophosphamide-induced diabetes by anti-Vβ8 T-lymphocyte-receptor monoclonal antibody therapy in NOD/Wehi mice

Walter & Eliza Hall Institute nonobese diabetic (NOD/Wehi) mice exhibit a low incidence of spontaneous diabetes mellitus, but one large dose of cyclophosphamide (CY) can lead to a rapid progression to overt diabetes. Macrophages and Lyt-2+ and L3T4+ cells have been demonstrated to be involved in β-cell destruction in this model. The role of a specific subset of T-lymphocytes expressing a particular T-lymphocyte–receptor segment was examined in CY-induced diabetic NOD mice with a mouse anti-Vβ8 T-lymphocyte–receptor monoclonal antibody (F23.1). After administration of CY, only 4 of 51 treated mice became hyperglycemie compared to 23 of 47 untreated mice, 13 of 26 mice treated with an isotype-matched control ascites, and 4 of 6 mice given antibody-negative ascites. Insulitis was significantly reduced in the F23.1-treated group, and immunocytochemistry revealed the absence of Vβ8 expression on cells in the lymphoid organs and insulitis of these mice. This investigation revealed that Vβ8+ cells were implicated in CY-induced diabetes in NOD/ Wehi mice.

Comparison of High— and Low—Diabetes-Incidence NOD Mouse Strains

The nonobese diabetic (NOD) mouse is a model of insulin-dependent diabetes mellitus. These mice develop insulinopenia and hyperglycemia secondary to α-cell destruction, which is associated with insulitis and autoantibody production. We have two strains of NOD mice: a low-incidence strain (NOD/Wehi), in which <10% females and <1% males develop diabetes by 150 days despite intense insulitis, and a highincidence strain (NOD/Lt), in which most females and many males develop diabetes by 150 days. This phenotypic difference has been maintained for 24 mo despite identical housing in our specific pathogenfree unit. Reciprocal skin grafting and allozyme electrophoresis have not identified a difference between the strains. Mixed-lymphocyte cultures were performed with splenic T-lymphocytes cultured with equal numbers of irradiated stimulator splenocytes for 3–6 days. NOD/Wehi mice demonstrated a heightened syngeneic mixed-lymphocyte response (SMLR), averaging 19% of the allogeneic response to CBA/CaHWehi cells. The response to NOD/Lt stimulator cells was not significantly different from the syngeneic response. In contrast, NOD/Lt mice had an SMLR similar to that of BALB/cAnBradleyWehi control mice, averaging 5% of the allogeneic response. NOD/Lt cells also responded similarly to NOD/Wehi stimulator cells and briskly to allogeneic cells. The heightened SMLR in NOD/Wehi mice may reflect active generation of suppressor function, and this may account for the low incidence of diabetes.

Retention of intact HSA for prolonged periods in the popliteal lymph nodes of specifically immunized mice

Abstract The rate of clearance of radiolabel was studied from the popliteal lymph nodes of either nonimmune or specifically immunized mice following footpad injection of 125 I-labeled HSA. The 125 I was more rapidly and more completely eliminated from the nodes of nonimmune mice than from the nodes of specifically immunized animals. Autoradiographs showed that, whereas the radiolabel was strictly confined for long periods to the follicles of the nodes of immune mice, no such localizaton was present in nonimmune animals. Solubilization of the whole nodes with 5 M guanidine-HCl released the labeled material which could then be specifically precipitated with anti-HSA but not with anti-EA. Such specifically precipitable material could be extracted from the draining popliteal lymph nodes of the immune mice even 12 weeks after challenge. Analysis of the solubilized radioiodinated lymph node extract on Sephacryl S-200 gels showed that the majority of the material was indistinguishable from the 125 I-labeled HSA used for challenge. The radiolabel therefore remains attached to intact HSA for long periods in vivo in immune animals which have a highly developed mechanism for clearance of simple protein antigens. These data suggest that intact antigenic determinants can persist for prolonged periods and may play a role in the long-term maintenance and regulation of immune responses.

High and Low Diabetes Incidence Nonobese Diabetic (NOD) Mice: Origins and Characterisation

The Nonobese Diabetic mouse (NOD mouse) is an established model of autoimmune diabetes mellitus. While all colonies of NOD mice are derived from a single diabetic female detected during the breeding of a cataract-prone strain of mice, some of the dispersed colonies have been separated for many generations and express varying levels of diabetes. It is unclear to what extent this is due to environmental factors such as diet factor or a result of the varied origins of the colonies. Here we compare the incidence of diabetes and severity of insulitis in two divergent lines of NOD mice that differ in incidence of disease, but are maintained in the same environment. F1 crosses were performed and the progeny found to express the disease incidence of the low incidence line. This finding is consistent with either a dominant resistance gene(s) being responsible for reduced penetrance of disease or a transmissible environmental agent reducing the severity of the autoimmune process.