IGRP and insulin vaccination induce CD8+ T cell-mediated autoimmune diabetes in the RIP-CD80GP mouse.

Autoimmune diabetes is characterized by autoantigen-specific T cell-mediated destruction of pancreatic islet beta cells, and CD8(+) T cells are key players during this process. We assessed whether the bitransgenic RIP-CD80 x RIP-LCMV-GP (RIP-CD80GP) mice may be a versatile antigen-specific model of inducible CD8(+) T cell-mediated autoimmune diabetes. Antigen-encoding DNA, peptide-loaded dendritic cells and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre-proinsulin 2 (100% of mice; median time after vaccination, 60 days) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (77%, 58 days) could induce diabetes. Vaccination with DNA encoding for zinc transporter 8, Ia-2, Ia-2ß, glutamic acid decarboxylase 67 (Gad67), chromogranin A, insulinoma amyloid polypeptide and homeobox protein Nkx-2.2 induced diabetes development in 25-33% of mice. Vaccination with DNA encoding for Gad65, secretogranin 5, pancreas/duodenum homeobox protein 1 (Pdx1), carboxyl ester lipase, glucagon and control hepatitis B surface antigen (HBsAg) induced diabetes in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin-antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8(+) T cell targets of IGRP were identified with a peptide library-based enzyme-linked immunospot assay, and diabetes could also be induced by vaccination with major histocompatibility complex (MHC) class I-restricted IGRP peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP-CD80GP mouse. We conclude that RIP-CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8(+) T cell-targeted prevention strategies.

Low dose streptozotocin-induced diabetes in rat insulin promoter-mCD80-transgenic mice is T cell autoantigen-specific and CD28 dependent.

Although transgenic mice expressing murine B7-1 (mCD80) on their pancreatic beta cells under the rat insulin-1 promoter (RIP-mCD80(+) mice) rarely develop spontaneous beta cell destruction and diabetes, we have previously reported the transgene-dependent induction of profound insulitis and lethal diabetes following multiple low dose injections of the beta cell toxin streptozotocin (MLDS) in RIP-mCD80(+) mice. Here, we have further characterized this MLDS-induced diabetes model using the RIP-mCD80(+) mice and now demonstrate that disease is critically dependent on T cell signaling via CD28. Thus, although naive RIP-mCD80(+) and nontransgenic littermates have comparable gross beta cell mass, and immediately following MLDS induction the mice display similar degrees of insulitis and decrements in the beta cell mass, only transgenic mice continued to destroy their beta cells and develop insulin-dependent diabetes mellitus. Strikingly, MLDS-induced diabetes was completely prevented in CD28-deficient mice (RIP-mCD80(+)CD28(-/-)) due to abrogation of leukocytes infiltrating their pancreatic islets. We further characterized MLDS-induced diabetes in the RIP-mCD80(+) mice by demonstrating that the MLDS-induced lymphocytic islet infiltrate contained a substantial frequency of autoantigen-specific, IFN-gamma-secreting, CD8(+) T cells. We conclude that MLDS-induced beta cell destruction and subsequent insulin-dependent diabetes mellitus in RIP-mCD80(+) mice is T cell-mediated as it involves both Ag-specific recognition of self-target molecules in the inflamed pancreatic islet (signal 1) and is CD28 costimulation dependent (signal 2).

Inflammatory cytokines IFN-gamma plus TNF-alpha induce regulated expression of CD80 (B7-1) but not CD86 (B7-2) on murine fibroblasts.

Optimal T cell activation requires signals delivered via both the TCR and the costimulatory receptors. Considerable experimental data now suggest that this costimulatory signal is generated predominantly by CD28 when engaged by its ligands CD80 (B7-1) and/or CD86 (B7-2). Whether T cell activation is controlled in part by regulated CD80 and/or CD86 expression has been incompletely explored. Here, we report that CD80 can be expressed constitutively by murine fibroblasts and up-regulated after treatment with IFN-gamma plus TNF-alpha. CD80 expression and function was confirmed by 1) Northern analysis, 2) specific immunoprecipitation of a approximately 69-kDa surface protein that comigrated with CD80 precipitated from CD80-transfected CHO cells, and 3) two independent assays for costimulation of Ag-specific T cell activation. Taken together, these observations suggest that CD28/CTLA-4 ligands are expressed on a wider variety of tissues than previously suspected and that their expression is dynamically regulated. Consequently, these results might explain previous observations that inflammatory cytokines can result in autoimmune responses.

Mycobacteria-reactive gamma delta T cells in HIV-infected individuals: lack of V gamma 9 cell responsiveness is due to deficiency of antigen-specific CD4 T helper type 1 cells.

Human gamma delta T lymphocytes expressing the variable T cell receptor elements V gamma 9 paired with V delta 2 are activated by antigen derived from Mycobacterium tuberculosis (M. tb.) and presented by antigen-presenting cells (APC). The subsequent proliferation is strictly dependent on the presence of CD4+ TCR alpha beta+ T helper type 1 (Th1) cells producing interleukin-2 (IL-2). In this study, we report that the reactivity of V gamma 9 cells to M. tb. stimulation in vitro was drastically decreased or absent in the majority of the analyzed HIV-1-infected individuals (CDC stages III and IV). We show that the failure of V gamma 9 cells from HIV+ individuals to proliferate following M. tb. stimulation was not due to an intrinsic qualitative or quantitative defect of gamma delta T cells but rather to a deficiency of M. tb.-reactive CD4 Th1 cells. Thus, V gamma 9 responsiveness could be restored if cultures of M. tb.-stimulated T cells from HIV+ donors were reconstituted with one of the following: (i) exogenous IL-2 (ii) purified CD4 T cells from allogeneic donors; or (iii) T cell-depleted APC from allogeneic donors. In the majority of HIV+ patients, the defective Th1 activity of M. tb.-stimulated CD4 T cells could be decreased neither by cytokines known to favor Th1 development (IL-12, interferon-gamma) nor by neutralization of the Th1-suppressing Th2 cytokine IL-10. We suggest that measurement of V gamma 9 cell expansion within M. tb.-stimulated peripheral blood mononuclear cells provides a sensitive assay for the functional capacity of antigen (M. tb.)-specific CD4 Th1 cells in HIV-infected individuals.

Apoptosis of mature T lymphocytes: putative role in the regulation of cellular immune responses and in the pathogenesis of HIV infection.

In this chapter, some aspects of programmed cell death, or apoptosis, of T lymphocytes are discussed. It has been recognized that transformed T cells and immature T lymphocytes can be triggered to undergo apoptosis. As in other cell systems, apoptosis is characterized by cell shrinkage, nuclear condensation, and DNA fragmentation that displays the characteristic "ladder" pattern of approximately 180-200 bp fragments. More recently, however, it has become clear that apoptosis is not restricted to immature thymocytes or transformed T lymphocytes, but can also occur in mature peripheral T cells. This raises the question of whether apoptosis plays a role as a mechanism in regulating cellular immune responses, which will be discussed in the following sections. We will also address the issue of the potential role of T cell apoptosis in pathophysiology. Here, we will concentrate on the infection with human immunodeficiency virus (HIV), where apoptosis is thought to contribute to the continuous decline in CD4+ T cells.

Antigen-induced death of alloreactive human T-lymphocytes occurs in the absence of low molecular weight DNA fragmentation.

Stimulation via the CD3/TCR molecular complex induces proliferation of resting T cells, but triggers programmed cell death (apoptosis) in immature thymocytes and preactivated mature T cells. Activation-induced cell death (AICD) triggered by anti-CD3/TCR mAb or by staphylococcus enterotoxin superantigen is associated with fragmentation of genomic DNA into oligonucleosomal fragments of 200 bp length, thus displaying the characteristic features of apoptosis. Here, we show that a fraction (20-50%) of cells in alloreactive CD8 human short-term T cell lines, generated by repeated restimulation with EBV-transformed B cell lines, undergo AICD when restimulated with the appropriate (but not with third party) stimulator cells. AICD of responder T cells is inhibited when stimulator cells are preincubated with anti-HLA class I mAb but not with anti-HLA class II mAb, indicating that T cell death is dependent on alloantigen (HLA class I) recognition by responding CD8 T cells. Importantly, alloantigen-induced T cell death occurs in the absence of detectable DNA fragmentation. Thus, several independent assay systems all failed to reveal low molecular weight DNA fragmentation, even though DNA fragmentation was readily detected in T cell lines exposed to PHA or gamma-irradiation. Alloantigen-induced T cell death was prevented by aurintricarboxylic acid, which has previously been shown to inhibit apoptosis in experimental systems where no DNA fragmentation occurs. Taken together, these results demonstrate that alloantigen can trigger AICD in mature responding T cells in the absence of low molecular weight DNA fragmentation.

Very-low-dose streptozotocin induces diabetes in insulin promoter-mB7-1 transgenic mice.

Transgenic mice that express mouse B7-1 (mB7-1, recently designated CD80) on their pancreatic beta-cells maintain normal islet architecture, have normal pancreatic insulin content, and only rarely spontaneously develop insulitis and diabetes. Nevertheless, these mice display an extreme sensitivity to streptozotocin (STZ)-induced diabetes. Female mice were administered two STZ doses intraperitoneally, 20 and 40 mg/kg body wt, each for five consecutive days. Nontransgenic but otherwise syngeneic mice responded to the STZ with a moderate diminution in pancreatic insulin content but not with persistent glycosuria. In striking contrast, STZ administered to transgenic mice resulted in a severe diminution of pancreatic insulin content and in diabetes. Notably, the lower STZ dose resulted in diabetes only after a prolonged (26- to 100-day) latency. STZ-induced diabetes appears to be T-cell dependent, since treatment with T-cell-depleting (and in particular CD8+ subset-depleting) antibodies ameliorated the response. Anti-mB7-1 monoclonal antibody administration also prevented STZ-induced diabetes. Thus, unmasked mB7-1 is a required component in the pathway resulting in beta-cell killing. Immunohistological analysis revealed that early after STZ administration, both mB7-1 transgenic and nontransgenic mice developed insulitis. While this insulitis resolved in the nontransgenic mice, the islet-infiltrating CD4+ and CD8+ T-cells in the transgenic mice were associated with complete beta-cell destruction. These data suggest that STZ-induced diabetes in mB7-1 transgenic mice is an immune-mediated process with distinct potential advantages over existing insulin-dependent diabetes models.

Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry.

Determination of the number of viable cells or quantification of lymphocyte subsets in heterogeneous cell populations is critically important for cytotoxicity assays, apoptosis assays, or the analysis of differential activation of T-cell subsets by distinct stimuli. In this report, we describe a rapid flow cytometry method termed Standard Cell Dilution Analysis (SCDA) specifically to quantify any subset of phenotypically definable, viable cells in heterogeneous populations using a FACScan flow cytometer. This method combines: (1) specific detection of lymphocyte subsets by phycoerythrin-conjugated monoclonal antibodies, (2) electronic exclusion of dead cells or cell debris by propidium-iodide staining and gating on forward vs. sidescatter, respectively, and (3) admixture of a known amount of fixed, fluorescein isothiocyanate stained cells immediately before analysis as a constant parameter to allow for calculation of cell quantity. We have used SCDA to analyze the in vitro growth characteristics of various human T-lymphocyte subpopulations in response to different activation stimuli.

Primary activation of V gamma 9-expressing gamma delta T cells by Mycobacterium tuberculosis. Requirement for Th1-type CD4 T cell help and inhibition by IL-10.

Purified peripheral blood gamma delta T cells proliferated vigorously in response to killed Mycobacterium tuberculosis (M. tb.) in the presence of PBMC but not in the presence of T cell-depleted (E-) feeder cells. Addition of graded numbers of autologous CD4 T cells to E- feeder cells reconstituted in a dose-dependent fashion the response of V gamma 9-expressing gamma delta T cells to M. tb. IL-2 was identified as the major CD4 T cell-derived helper factor required for gamma delta T cell proliferation after stimulation with M. tb. In addition, neutralizing anti-IFN-gamma but not anti-IFN-alpha Ab inhibited the responsiveness of V gamma 9 T cells, suggesting that endogenously produced IFN-gamma was involved in the activation of gamma delta T cells by M. tb. Although gamma delta T cells could not proliferate on their own in the absence of CD4 T cells (or exogenous IL-2), the appearance of IL-2 receptors (CD25) was triggered in the absence of CD4 T cells. Furthermore, IL-10 strongly inhibited the activation of V gamma 9 T cells among unfractionated PBMC responder cells. Similarly, the responsiveness of purified gamma delta T cells to M. tb. occurring in the presence of CD4 T cells was strongly inhibited by IL-10, whereas the activation occurring in the presence of exogenous IL-2 was not impaired. These results show that interactions with Th1-type CD4 T cells are required for efficient activation of peripheral blood gamma delta T cells by M. tb. In addition, our results have practical implications for creating experimental conditions aimed at identifying V gamma 9-selective (myco)bacterial ligands.

V gamma gene usage in peripheral blood gamma delta T cells.

The majority (50-90%) of gamma delta T cells in the peripheral blood of adult individuals expresses a T-cell receptor (TCR) which uses V gamma 9 and V delta 2 as variable elements. Little is known about the distribution of other V gamma gene elements in the remaining 10-50% of gamma delta T cells. Here we have studied the V gamma gene expression in peripheral blood gamma delta T cells by 3-color flow cytometry analysis applying established monoclonal antibodies (mAb) directed against V gamma 9 and V gamma 4, as well as a novel mAb directed against V gamma 2, V gamma 3 and V gamma 4. On average, 79.9% of gamma delta T cells expressed V gamma 9, 11.9% V gamma 2/V gamma 3, 4.4% V gamma 4, and 7.5% one of the remaining V gamma 5, V gamma 8, V gamma 10 or V gamma 11 elements. There were remarkable variations in the gamma delta subset composition between individual donors. The majority (69.8%) of V gamma 2/V gamma 3/V gamma 4-bearing cells co-expressed V delta 1, while on average only 17.8% of V gamma 2/V gamma 3/V gamma 4-bearing cells co-expressed V delta 2. This is in contrast to V gamma 9-bearing gamma delta T cells, of which 83.1% used V delta 2 and only 12.7% V delta 1. Taken together, this data identifies V gamma 2/V gamma 3 as the second most frequently used set of V gamma elements in human peripheral blood gamma delta T cells.

Activation-induced cell death (apoptosis) of mature peripheral T lymphocytes.

Programmed cell death (apoptosis) is triggered in immature thymocytes and T-cell hybridomas by signalling via the CD3-T-cell receptor pathway. In this paper, Dieter Kabelitz and colleagues catalogue the recently accumulating evidence that apoptosis can also be initiated in mature peripheral T cells; this may constitute an important aspect of cellular immune regulation.

Human peripheral blood gamma delta T cells are uniformly sensitive to destruction by the lysosomotropic agents leucine methyl ester and leucyl leucine methyl ester.

Treatment of peripheral blood mononuclear cells (PBMC) with the lysosomotropic agent leucine methyl ester (Leu-OMe) eliminates monocytes/macrophages and cytotoxic lymphocytes including CD3- CD16+ natural killer (NK) cells and a fraction of T cell receptor (TcR) alpha beta + CD8+ T cells. We report that freshly isolated peripheral blood gamma delta T cells are highly sensitive to Leu-OMe treatment as well. After incubation of PBMC with 5 mM Leu-OMe or incubation of purified T cells with 50 microM leucyl leucine methyl ester (Leu-Leu-OMe) and subsequent overnight culture, CD3-CD16+ NK cells and gamma delta T cells were no longer detectable by immunofluorescence analysis. The two major gamma delta T cell subsets V gamma 9+V delta 2+ and V gamma 9-V delta 1+ were equally susceptible to Leu-OMe and Leu-Leu-OMe treatment. The elimination of V gamma 9+ T cells by Leu-OMe treatment was confirmed in functional assays. Stimulation of peripheral blood T cells with killed mycobacteria resulted in selective expansion of V gamma 9+ T cells. In contrast, no activation of gamma delta T cells was elicited in Leu-OMe-treated responder T cells stimulated with killed mycobacteria.

Leukaemic T cells from patients with chronic lymphocytic leukaemia of T-cell origin respond to Staphylococcus aureus enterotoxin superantigens.

We investigated the in vitro responsiveness of peripheral blood lymphocytes from two patients with T-cell chronic lymphocytic leukaemia (T-CLL) to Staphylococcus aureus enterotoxin (SE) superantigens. T-cell receptor (TcR) alpha beta (V beta 7.1)-expressing CD4+ leukaemic T cells from patient HE (white blood cell count 480,000/microliters) proliferated in response to SEA and, only at 1000-fold higher concentrations, to SEB, SED, and SEE. CD4+CD8+ TcR alpha beta (V beta 12.1)-expressing leukaemic T cells from patient KO (white blood cell count 120,000/microliters) were activated by SEB but not by the other tested SEs. In both instances, the activation of leukaemic T cells by SE was dependent on the presence of HLA-DR+ cells. Southern blot analysis of TcR beta gene rearrangement confirmed that the proliferating cells were derived from the leukaemic T-cell clone and not from contaminating normal T cells. These data indicate that leukaemic T cells from patients with T-CLL exert a clonally variable responsiveness to SE superantigens. We conclude that recognition of specific antigen and subsequent signal transduction can be initiated via the TcR of leukaemic T-CLL cells.

A rapid staining procedure for two-color analysis of lymphocyte antigen expression.

Two color immunofluorescence analysis of lymphocyte cell surface antigen expression using an unconjugated plus a biotinylated monoclonal antibody (mAb) requires four incubation steps: (1) unconjugated mAb; (2) fluorochrome-labelled goat anti-mouse Ig; (3) biotinylated mAb; (4) fluorochrome-labelled avidin or streptavidin. We describe a time-saving modification of this procedure which requires only two incubation steps: (1) simultaneous unconjugated and biotinylated mAbs; (2) fluorochrome-labelled avidin/streptavidin followed by fluorochrome-labelled goat anti-mouse Ig. The slightly delayed (5 min) addition of the goat anti-mouse Ig prevents it from binding to the mAb which has already interacted with avidin/streptavidin. Both procedures yield identical results with a variety of different mAbs.

Perspectives in transplantation immunology 1991.

The clinical success of organ transplantation depends to a large degree on the immunological acceptance of the grafted organ. This paper summarizes from an immunological point of view the recent progress that has been made to improve graft acceptance, and discusses some future aspects in the field. Over the last few years, major emphasis has been put on the development of new immunosuppressive drugs, including FK 506, rapamycin, and Deoxyspergualin. Together with monoclonal antibodies against defined T-cell surface antigens, there are now new and effective means available to prevent or treat rejection episodes. Progress has also been made in the field of HLA typing, where the introduction of molecular biology-based methods significantly increased the accuracy of HLA class II typing. The ultimate goal of transplantation immunology is the induction of (donor-) specific tolerance. While some protocols are effective in inducing peripheral tolerance in experimental animals, these regimens are at present not yet applicable in the clinical situation. To overcome the shortage of donor organs, alternative strategies are currently being considered. Among these, xenotransplantation may eventually prove successful, despite the massive immunological problems such as, e.g., the presence of preformed xenoreactive antibodies.

The primary response of human gamma/delta + T cells to Mycobacterium tuberculosis is restricted to V gamma 9-bearing cells.

We have previously reported that peripheral blood gamma/delta + T cells proliferate in high frequency (1 in 2-20) in response to heat-killed Mycobacterium tuberculosis (M.tb.). In the present study, the T cell receptor phenotype of mycobacteria-responsive human gamma/delta + T cells was analyzed in primary cultures with a set of monoclonal antibodies (mAbs) directed against V gamma 9, V delta 1, and V delta 2. When unseparated T cells were stimulated with M.tb., all proliferating gamma/delta + T cells expressed V gamma 9 (and V delta 2) after culture. Selective depletion of V gamma 9-bearing cells before culture completely abolished the proliferative response of all gamma/delta + cells (but did not inhibit reactivity of alpha/beta + T cells). In addition, when CD4- CD8- thymocytes were stimulated with M.tb., there was again selective outgrowth of V gamma 9+ cells. In this case, the starting responder population contained few (0.5-1.8%) V gamma 9+ and many (11.5-31.5%) V delta 1+ cells that did not coexpress V gamma 9. These V delta 1+ cells were not activated by M.tb. but could be readily stimulated by anti-V delta 1 mAb A13. Finally, a V gamma 9-specific mAb selectively suppressed the proliferative response of gamma/delta + T cells to M.tb. Taken together, our results demonstrate that, within gamma/delta + T cells, reactivity towards M.tb. is an exclusive property of V gamma 9+/V delta (2+)-bearing cells.

Activation and activation-driven death of human gamma/delta T cells.

We have concentrated on three functional aspects of human gamma/delta+ T cells. (1) Peripheral blood gamma/delta+ T cells proliferate at a high frequency in response to heat-killed mycobacteria (M.tb.). We present evidence that the primary response of human gamma/delta+ T cells is restricted to those cells bearing a V gamma 9/V delta 2 receptor. (2) T cells expressing the alpha/beta TCR can be activated through the CD2 antigen via an "alternative" pathway. Activation requires the combined signaling of 2 mAb directed against two distinct epitopes of CD2. In striking contrast, cloned gamma/delta+ T cells are activated by a single immobilized anti-CD2 mAb directed against the sheep E-binding epitope of CD2. Stimulation of cloned gamma/delta+ T cells by a single anti-CD2 mAb results in IL-2 production, proliferation, and triggering of cytolytic effector function. (3) Cloned gamma/delta+ T cells are susceptible to programmed cell death (apoptosis) when stimulated simultaneously by anti-CD3/TCR mAb plus exogenous IL-2. The very same clones are activated, however, by the same stimuli in the absence of exogenous IL-2 (but in the presence of LCL feeder cells). This provides the basis for a model where extensive gamma/delta+ T cell proliferation is negatively regulated by antigen plus IL-2.

Selective activation of gamma/delta + T cell clones by single anti-CD2 antibodies.

The CD2 antigen is the target for an "alternative" T cell activation pathway. Numerous studies have demonstrated that pairs of monoclonal antibodies (mAbs) directed toward two different epitopes are required for activation of T cell receptor (TCR)-alpha/beta + T cells via CD2. We have now explored the activation of human TCR-gamma/delta + T cell clones by a panel of anti-CD2 mAbs directed against the sheep erythrocyte-binding (T11.1) epitope of CD2. Seven of seven gamma/delta + clones expressing different molecular forms of the TCR-gamma/delta responded to stimulation by a single anti-CD2 mAb (OKT11, 9E8, BW0110, M-T910) with IL-2 secretion and/or proliferation. Immobilization of anti-CD2 mAbs in microculture plates was essential for activation of gamma/delta + clones, which occurred in the absence of feeder cells. In addition to interleukin 2 (IL-2) production and proliferation, anti-CD2 mAbs also triggered cytotoxic effector activity in gamma/delta + clones as measured against FcR+ P815 target cells. In contrast to gamma/delta + clones (but in line with established data), none of five CD4+ or CD8+ TCR-alpha/beta + clones were activated by any of the tested individual anti-CD2 mAbs. Taken together, our results reveal a striking difference between cloned gamma/delta + and alpha/beta + T cells in that gamma/delta + T cells are selectively activated by a single anti-CD2 (T11.1) mAb, without need for the simultaneous signal of a second anti-CD2 mAb directed against another (T11.2 or T11.3) CD2 epitope.