Dieter Kabelitz

A rapid colorimetric assay for the determination of IL-2-producing helper T cell frequencies

Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.

Apoptosis, graft rejection, and transplantation tolerance.

Transplantation tolerance can be achieved through several mechanisms, including the action of suppressor cells, the induction of anergy, or the deletion of graft-reactive donor T cells. In this article, the possible involvement of programmed cell death (apoptosis) in allograft rejection and transplantation tolerance is discussed. The recent characterization of members of the tumor necrosis factor-alpha gene family has identified ligands (Fas ligand and TRAIL) and corresponding death receptors (DR). In rejected organ transplants, apoptotic cells are frequently encountered. Conversely, high-level expression of Fas ligand on the allograft correlates with graft acceptance in some models of organ transplantation. Furthermore, some of the immunosuppressive drugs currently in clinical use might exert their activity at least in part through effects on apoptotic pathways. From the available data, it can be inferred that apoptosis contributes to the outcome after organ transplantation, being involved both in graft rejection and in transplantation tolerance.

Selective reduction of donor-specific cytotoxic T lymphocyte precursors in patients with a well-functioning kidney allograft.

We have recently developed a sensitive limiting dilution (LD) culture system to measure human alloreactive cytotoxic T lymphocyte precursors (CTL-p) in a given lymphoid cell population. We have now used this system to determine frequencies of donor HLA antigen-inducible CTL-p in the peripheral blood of human allograft recipients at various stages after transplantation. All patients (1 pancreas recipient and 9 kidney recipients) were on continuous cyclosporine treatment throughout the study. We report that, in patients with a well-functioning kidney graft (6/9), the number of donor-reactive CTL-p among peripheral blood lymphocytes decreased within 3–8 months after transplantation—in some cases (2/6) more than 10-fold. In contrast, frequencies of CTL-p with specificity for third-part HLA antigens remained largely unaltered in these patients. Furthermore, no decrease of donor-reactive CTL-p frequencies was seen in 3 of 4 patients showing clinical symptoms of graft rejection. These results indicate that functional clonal deletion of antigraft-reactive CTL-p may contribute to the state of graft tolerance in certain patients with a well-functioning kidney allograft.

Human cytotoxic T lymphocytes. I. Limiting-dilution analysis of alloreactive cytotoxic T-lymphocyte precursor frequencies.

A limiting‐dilution system was established to measure the frequency of alloreactive cytotoxic T‐lymphocyte precursors (CTL‐p) in human peripheral blood T cells. Culture medium supple mented with recombinant interleukin‐2 enabled clonal expansion of all CTL‐p stimulated by allogeneic peripheral blood or spleen cells. The range of CTL‐p frequencies in fully HLA‐mismatched responder‐stimulator combinations was 1:240 to 1:1230. Split‐well analysis of individual microwells showed that the cytotoxic T‐cell clones generated under limiting‐dilution conditions showed exquisite specificity for the stimulating alloantigens. Alloreactive CTL‐p were enriched in the OKT4 T‐cell subset. This limiting‐dilution system was highly reproduci ble and can thus be applied to investigate human cytotoxic T‐eell precursor frequencies in various clinically relevant situations.

Growth inhibition of Epstein-Barr virus-transformed B cells by anti-HLA-DR antibody L243: Possible relationship to L243-induced down-regulation of CD23 antigen expression

Among five anti-HLA class II monoclonal antibodies (mabs) tested, the anti-HLA-DR mab L243 selectively inhibited the in vitro proliferation of Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL). Saturating amounts of L243 mab induced 50% suppression of LCL growth as revealed by measuring [3H]thymidine incorporation or counting cell numbers. Preincubation for 20 hr at 37 degrees C in the presence of L243 mab dramatically reduced the expression of certain B cell-specific antigens on LCL, notably CD23 (BLAST-2, low affinity Fc epsilon receptor). In view of the known function of the CD23 antigen as a B cell growth factor receptor, we discuss the possibility that the suppressive effect of L243 mab on LCL proliferation is due to down-regulation of CD23 antigen expression.

Recombinant interleukin 2 rapidly augments human natural killer cell activity

Recombinant human interleukin 2 (r-IL-2) rapidly stimulated human natural killer cell activity in vitro. Augmentation of NK activity occurred within 1 hr of preincubation with r-IL-2. Responsive killer cells were typical NK cells as shown by cell fractionation procedures. These included Percoll density gradient separation and depletion of OKT3+ T cells by an indirect rosetting method. Analysis with a panel of polyclonal and monoclonal antibodies against alpha and gamma interferon revealed that this early enhancement of NK activity by r-IL-2 was independent of the production of both types of interferon.

Quantitative assessment of interleukin-2-producing alloreactive human T cells by limiting dilution analysis

A limiting dilution (LD) culture system was developed to estimate the frequency of IL-2 producing alloantigen-reactive human helper T lymphocytes (HTL). E rosette-purified T cells or cell sorter-separated CD4+ and CD8+ subsets were co-cultured under LD conditions with irradiated allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) in the absence of additional growth factors. IL-2 in microculture supernatants was detected in a bioassay combined with rapid colorimetric visualization (cleavage of MTT). Under these conditions, one out of 250-800 E+, 180-280 CD4+, and 440-1000 CD8+ T cells produced IL-2 after a culture period of 3 days. This quantitative analysis of alloantigen-specific human HTL is complete within 4 days and thus provides a rapid method with potential applications in various clinical situations, e.g., transplantation medicine.

Evidence that functional deletion of donor-reactive T lymphocytes in kidney allograft recipients can occur at the level of cytotoxic T cells, IL-2-producing T cells, or both. A limiting dilution study.

The frequencies of circulating donor-reactive cytotoxic lymphocyte precursors (CLP) and Il-2-producing helper lymphocyte precursors (HLP) were determined by limiting dilution analysis in 19 kidney allograft recipients before and at various intervals (up to 2 years) after transplantation. A significant, selective, and stable reduction of the frequencies of donor-reactive (but not of third party—reactive) CLP and/or HLP was observed in some patients beginning 3 to 6 months after transplantation. One patient developed reduced frequency of CLP only, 3 patients reduced frequencies of HLP only, and 2 patients reduced frequencies of both CLP and HLP. The selective reduction of donor-reactive CLP and/or HLP frequencies ranged from 5–25-fold when compared with the pretransplantation level and was associated with stable graft function. These data indicate that functional deletion of circulating donor-reactive T cells can occur at the level of cytotoxic T lymphocytes, Il-2-producing helper T lymphocytes, or both. Implications of these findings for the individualization of immunosuppressive regimens will be discussed.

Frequency analysis of donor-reactive cytotoxic T lymphocyte precursors in in allograft recipients. Lack of correlation with clinical outcome.

The frequency of donor-reactive and of third party-reactive cytotoxic T lymphocyte precursors (CTLp) was determined by limiting dilution analysis in the peripheral blood of 13 liver allograft recipients before (pre-tpx) and at various intervals (4 to 22 months) after (post-tpx) transplantation. In 6 of 13 patients with stable graft function, no change of donor-reactive CTLp occurred within 9 to 17 months post-tpx, and high levels of donor-reactive CTLp (frequencies ranging from 1/1024 to 1/13,295) were maintained. In contrast, a 4-11-fold reduction of donor-reactive CTLp frequencies was observed in 5 patients within 4 to 22 months post-tpx; in 3 of these patients the measured frequency of donor-specific CTLp dropped to biologically irrelevant levels (1/43,800 to 1/150,000). However, a 5-7-fold increase of the post-tpx frequency of donor-reactive CTLp was observed in two additional patients with stable graft function. Taken together, these results demonstrate that the frequency analysis of donor-reactive CTLp in the peripheral blood does not closely correlate with the clinical outcome. Thus, stable liver allograft function after > 1 year of transplantation can be associated with a specific deletion, unaltered levels, or even an increase in the frequency of circulating donor-reactive CTLp.The frequency of donor-reactive and of third partyreactive cytotoxic T lymphocyte precursors (CTLp) was determined by limiting dilution analysis in the peripheral blood of 13 liver allograft recipients before (pre-tpx) and at various intervals (4 to 22 months) after (post-tpx) transplantation. In 6 of 13 patients with stable graft function, no change of donor-reactive CTLp occurred within 9 to 17 months post-tpx, and high levels of donor-reactive CTLp (frequencies ranging from 1/1024 to 1/13,295) were maintained. In contrast, a 4–11-fold reduction of donorreactive CTLp frequencies was observed in 5 patients within 4 to 22 months post-tpx; in 3 of these patients the measured frequency of donor-specific CTLp dropped to biologically irrelevant levels (1/43,800 to 1/150,000). However, a 5–7-fold increase of the post-tpx frequency of donor-reactive CTLp was observed in two additional patients with stable graft function. Taken together, these results demonstrate that the frequency analysis of donorreactive CTLp in the peripheral blood does not closely correlate with the clinical outcome. Thus, stable liver allograft function after >1 year of transplantation can be associated with a specific deletion, unaltered levels, or even an increase in the frequency of circulating donor-reactive CTLp.

A previously unrecognized large fraction of cytotoxic lymphocyte precursors is present in CD4+ human peripheral blood T cells.

We describe a limiting dilution (LD) culture system in which cell sorter-purified CD4+ (and CD8+) peripheral blood T cells are cocultured with irradiated, anti-CD3 mab-producing OKT3 hybridoma cells. Under these conditions, one out of 2-3 CD4+ (and CD8+) T cells is induced to clonal proliferation. In striking contrast to previously described LD culture systems, every growing CD4+ cell clone displayed cytotoxic activity when tested in a lectin-facilitated 51Cr release assay against P815 target cells. This contrasts with the development of cytotoxic CD4+ T cells in alloantigen-stimulated LD cultures, where only one out of 15-20 proliferating CD4+ cells killed P815 in the presence of PHA, and one out of 300-500 proliferating CD4+ cells displayed alloantigen-specific cytotoxic activity. Furthermore, we have established antigen-specific proliferating CD4+ T cell clones which do not exert antigen-specific cytotoxicity but can be cytotoxic when crosslinked to target cells via lectin or monoclonal antibody (anti-CD3, anti-TCR). Our results show that a previously unrecognized large fraction (at least 30-50%) of all peripheral blood CD4+ T cells can give rise to cytotoxic effector cells. The mode of CD4+ T cell activation (OKT3 hybridoma versus alloantigen) thus determines whether the intrinsic cytotoxic capacity of CD4+ T cells is functionally activated or not.