Depletion of Alloreactive T Cells by a Specific Anti–Interleukin-2 Receptor p55 Chain Immunotoxin Does Not Impair In Vitro Antileukemia and Antiviral Activity

Preeclampsia is associated with increased cytotoxic T-cell capacity to paternal antigens

The predictive value of limiting dilution analyses (LDA) measuring cytotoxic and helper T-lymphocyte precursor (CTLp and HTLp) frequencies for outcome after stem cell transplantation (SCT) is still a matter of debate. One reason may be that CTLp and HTLp frequencies are determined in peripheral blood mononuclear cells (PBMC) and this responder cell population does not reflect the cell type composition of the graft. We assessed whether CTLp and HTLp LDA can predict complications after human leukocyte antigen (HLA)-identical SCT when CTLp and HTLp frequencies are analyzed in PBMC of the respective stem cell graft [bone marrow (BMMC) or granulocyte colony-stimulating factor (G-CSF)-mobilized PBMC] and compared to PBMC of PB. Host-specific CTLp frequencies measured in 25 patients and HTLp frequencies analyzed in 6 patients were low in all responder cell sources. CTLp and HTLp frequencies seen against HLA-mismatched unrelated third-party cells were high, but third-party-specific CTLp and HTLp frequencies were lower in G-CSF-PBMC than in PBMC ( p=0.047 for CTLp frequencies). Host-specific CTLp frequencies analyzed in all responder cell sources did not predict acute or chronic graft-versus-host disease (GVHD). Lower CTLp frequencies were detected in all responder cell sources from patients who relapsed after SCT than in patients without relapse, but the differences between both groups were statistically significant only in PBMC. In conclusion, a significant correlation was detected only between relapse and CTLp frequencies measured in PBMC. The lower frequency of third-party-specific cells in G-CSF-PBMC indicates that the mobilization procedure with G-CSF itself may influence results.

A model of mouse acute myeloid leukemia (mAML) was used to study the effector mechanism mediating the graft-versus-leukemia (GVL) effects in recipients of allogeneic bone marrow cells (BMC). mAML-bearing SJL/J (H-2s) mice were lethally irradiated and then transplanted with a mixture of BMC and spleen cells (SC) derived from normal syngeneic or allogeneic mice. To augment the GVL effect, recipients were injected intraperitoneally with recombinant human interleukin-2 (rIL-2) (1.2 x 10(5) IU) for 3 consecutive days, starting one day post BMC + SC transplantation. Spleen cells from treated recipients were adoptively transferred to untreated secondary SJL/J mice to test for the existence of residual tumor cells. All the secondary recipients of SC from mAML-bearing SJL/J mice rescued with syngeneic (SJL/J) or allogeneic (B10.S) BMC+SC (H-2s) differing at minor antigens of the histocompatibility complex (MiHC) developed leukemia and died. In sharp contrast, none of the secondary recipients of SC obtained from identical mAML-bearing mice rescued with B10.S BMC + SC but activated in vivo with IL-2 developed leukemia. Adoptive recipients of SC obtained from mAML-bearing recipients of major histocompatibility complex (MHC)-disparate (C57BL/6, H-2b) cells remained free of leukemia regardless of the use of rIL-2. In parallel with the in vivo findings, a 4-day in vitro exposure of splenocytes to 6 x 10(3) IU/ml rIL-2 resulted in a 5- to 20-fold increase in the frequency of alloreactive cytotoxic T-lymphocyte (CTL) precursors (CTLp) across MiHC and MHC barriers and a 2- to 6-fold increase in their cytotoxic activity. Our data suggest that augmentation of GVL effects by rIL-2 may be due to CTL activation by rIL-2, not excluding the potential beneficial role of rIL-2-activated allogeneic natural killer cells and MHC non-restricted killer cells. Cumulatively, our results suggest potentially beneficial effects of rIL-2, when used jointly with bone marrow transplantation or allogeneic cell therapy, on eradication of leukemia.

A better understanding of the immune response to live and formalin-inactivated respiratory syncytial virus (RSV) is important for developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c mice intranasally infected with live RSV or intramuscularly inoculated with formalin-inactivated RSV (FI-RSV). After RSV infection, both class I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV infection were less than class I-restricted CTLp frequencies through day 14 p.i., during which class I-restricted CTLp frequencies remained elevated, but then declined by 48 days p.i. The frequencies of class II-restricted CTLps in the BAL were 2- to 10-fold less than those of class I-restricted CTLps. For spleen cells, frequencies of both MHC class I- and II-restricted CTLps to live RSV were similar. In contrast, class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with FI-RSV immunization and help define possible mechanisms of enhanced disease after FI-RSV immunization. In addition, these studies provide a quantitative means to address potential vaccine candidates by examining both MHC class I- and II-restricted CTLp frequencies.

Alteration in lymphocyte recognition repertoire during aging: II. Changes in the expressed T-cell receptor repertoire in aged mice and the persistence of that change after transplantation to a new differentiative environment

The frequencies of HLA class II-specific cytotoxic T lymphocyte precursors (CTLp) were studied in number of unrelated individuals using a limiting dilution analysis system optimized for the detection of CD4+ CTLp. Peripheral blood mononuclear cells (PBMC) were enriched for CD4+ T cells by immunomagnetic depletion of CD8+ T cells. In some allogeneic combinations high CTLp frequencies were obtained with no significant difference between PBMC and CD4-enriched PBMC populations. In other combinations CTLp frequencies in CD4-enriched PBMC were found to be at least twentyfold lower than in the starting, unfractionated PBMC, suggesting a predominance in these pairs of CD8+ CTLp. In addition there was variation in CTLp frequencies against the same set of HLA class II gene products between individuals, and variation in CTLp frequencies against different HLA class II gene products within individuals. The HLA class II specificity of the assay system was demonstrated unequivocally with detection of CTLp against HLA-DR1 expressed on a murine L cell transfectant.

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8(+) MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon gamma (IFNgamma), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (-/-) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNgamma + VV-IL-2, VV-IFNgamma + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNgamma had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (-/-) mice demonstrated that mice that are IL-2 -/- and IL-7 receptor -/- produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 -/-, IL-10 -/- and IFNgamma -/- mice were marginally improved and responses to M-FP in IL-4 -/- and tumour necrosis factor receptor 2 -/- mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1(+) P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone.

Major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to human persistent virus infections. Measurements of the frequency and specificity of human immunodeficiency virus type 1 (HIV-1)-specific CTL and their variation with time may indicate their relative importance in modulating the progression of HIV-1 infection. We have used limiting dilution analysis (LDA) to derive quantitative estimates of the frequency of HIV-1-specific CTL precursors in a cross-sectional study of 23 patients at different clinical stages of HIV-1 infection and to compare these with the frequency of CTL precursors specific for another persistent virus (Epstein-Barr virus [EBV]) in the same patients. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with autologous HIV-1-infected lymphoblasts and assayed for cytotoxicity in 51Cr release assays against autologous and MHC-mismatched lymphoblastoid B cells infected with recombinant vaccinia viruses expressing the three HIV-1 structural gene products. The frequency of MHC-restricted precursors was high in asymptomatic HIV-1-infected patients (env-specific CTL precursors up to 73/10(6) PBMC; gag-specific CTL precursors up to 488/10(6) PBMC), although the relative frequency against the different structural gene products varied from patient to patient. The HIV-1-specific CTL precursor frequency was reduced in patients who had more severe (< 400/microliters) CD4+ lymphocyte depletion, while in the majority of such patients the frequency of CTL precursors against EBV was maintained at levels observed in healthy controls. Direct CTL activity in unstimulated PBMC was observed in three of nine patients but no correlation was found between the presence of an activated CTL response and the magnitude of the CTL response detected after stimulation in LDA. Thus, CTL precursors were detected against all three HIV-1 structural gene products in patients with CD4+ lymphocyte counts > 400/microliters, at frequencies that are high compared with those reported for other persistent viruses. A CTL response directed against multiple protein antigens of HIV-1 may protect the patient against epitope variation. The fact that the EBV- specific CTL precursor frequencies were maintained in advanced HIV-1 infection suggests that there may be selective impairment of the HIV-1- specific CTL response associated with disease progression.

Tolerance to allografted hearts in human recipients has been observed both in clinical situations and in in vitro experiments. To elucidate whether a quantitative change of alloreactive CTL is one of the mechanisms accounting for this graft tolerance, CTL precursor (CTLp) frequencies in the peripheral blood of 10 heart recipients were measured against spleen cells from donors and HLA nonidentical third-party persons. In this longitudinal follow-up study, we showed that the rejection reaction(s) in the grafted heart correlated with CTLp frequencies in samples taken before transplantation against the donor spleen cells, but not with the CTLp frequencies against the spleen cells from the third-party persons. The CTLp frequencies against the spleen cells from donors decreased 4-6 months after transplantation, and remained at a low level afterward. However, the CTLp frequencies against spleen cells from third-party persons in blood samples obtained 1 year after transplantation were not significantly different from those before transplantation. Therefore, we conclude that donor-reactive CTLs are important in rejecting allografted heart. The decrease in donor-specific CTLp after transplantation could explain the donor-specific tolerance. The decrease may be due to homing of the specific CTLp to the graft, or by clonal deletion of the donor-reactive CTL caused by chronic alloantigen stimulation in the presence of immunosuppressive therapies.

The frequency of murine CTL precursors (CTLp) that recognize the human histocompatibility Ag HLA-A2 and HLA-B7 was measured and found to be approximately two orders of magnitude lower than the frequency of CTLp that recognize murine H-2 alloantigens. The possible contribution of other cell surface molecules to this difference in response was addressed by expression of the H-2Ld molecule on a human cell and the HLA-B7 molecule on a murine cell. It was found that both human and murine H-2Ld expressing cells elicited comparable levels of H-2Ld specific CTL. Although murine HLA-B7 positive cells stimulated a higher frequency of HLA-B7-specific CTLp than did human cells, this appeared to be largely due to stimulation of CTLp that recognized HLA-B7 in the context of H-2 molecules; consequently, it was concluded that the difference in the frequency of murine CTLp elicited by human and murine class I Ag is due to species specific structural differences in these molecules. The regions of the class I molecule that were responsible for this difference were mapped using chimeric class I molecules constructed to replace domains of the human molecule with their murine counterparts. It was found that the frequency of CTLp is controlled by structures within the alpha 1 and alpha 2 domains of the molecule. These results are discussed in the light of models for T cell recognition of class I Ag and the diversification of the T cell receptor repertoire.

Determination of equid herpesvirus 1-specific, CD8 +, cytotoxic T lymphocyte precursor frequencies in ponies

A reply to the letter from Affaticati et al. There is no doubt that the frequency analysis of helper T lymphocyte precursors (HTLp) and cytotoxic T lymphocyte precursors (CTLp) has great impact on the detection of alloreactivity. However, the debate on the clinical values of these functional assays for the prediction of graft-versus-host disease (GVHD) occurrence after bone marrow transplantation (BMT) has carried on for the last decade. One can easily find several reports in the literature either for (1–4) or against (5–9) the usefulness of these functional assays for predicting GVHD in HLA-matched sibling BMT. Although our observation (9) showed no significant correlation between the HTLp/CTLp frequencies and the incidence of acute GVHD after HLA-matched sibling BMT, there are other reports showing the contrary (5,7). Due to the lack of standard protocols for these functional assays, there are considerable variations in the methodology used by different investigators, which makes it difficult, sometimes impossible, to compare or correlate data from different research groups. For a rigorous examination on the association between the frequencies of HTLp/CTLp and the incidence of GVHD, it is crucial to establish standard protocols and to study a sufficient number of transplant recipients with homogeneity of disease and uniformity of conditioning and GVHD prophylaxis. The effectiveness of the immunosuppressive regimen after transplantation and the precision of the HLA matching also need to be taken into consideration. The discordance in one or more of these factors could be attributable to the controversy regarding the usefulness of HTLp/CTLp frequency estimation for GVHD prediction. During the last few years, the sensitivity of these assays has been improved by eliminating antigen-presenting cells from the responder population and/or T cells from the stimulator population (10,11). However, the complexity of the assay procedure makes these protocols impracticable for routine clinical use, and researchers are still searching for a better method. The combined limiting dilution assay (LDA) used in our study was established by Bouma (12) et al. and has proven to be as sensitive as single LDA to detect alloreactive HTLp and CTLp frequencies in HLA-mismatched settings. Our observation (9) has demonstrated that this combined LDA may not be ideal in HLA-identical sibling settings. The possible reasons have been discussed in detail in our previous articles (8,9). Although Affaticati et al. (13), using a single LDA, showed a positive correlation of CTLp frequency with clinical acute GVHD grades II–IV, the statistical significance remained marginal (P =0.04), with a third of patients who did not develop severe GVHD demonstrating high frequencies. It may well be that the positive CTLp frequencies detected in both publications by Affaticati et al. and ourselves were against minor histocompatibility antigens. Some of these responses detected in cohorts of patients developing no or low-grade GVHD may, however, not be relevant or strong enough in the in vivo clinical setting (in the presence of prophylaxis regimens for example) to give rise to GVHD. However, in our view, CTLp frequency estimation remains an inconclusive predictive indicator of GVHD occurrence and severity in the HLA-matched sibling setting, mainly due to overlapping results of high frequency within cohorts of patients demonstrating either no or low-grade GVHD (grades 0–1). Anne Dickinson Xiao-Nong Wang

Tolerance to allografted hearts in human recipients has been observed both in clinical situations and in in vitro experiments. To elucidate whether a quantitative change of alloreactive CTL is one of the mechanisms accounting for this graft tolerance, CTL precursor (CTLp) frequencies in the peripheral blood of 10 heart recipients were measured against spleen cells from donors and HLA nonidentical third-party persons. In this longitudinal follow-up study, we snowed that the rejection reaction(s) in the grafted heart correlated with CTLp frequencies in samples taken before transplantation against the donor spleen cells, but not with the CTLp frequencies against the spleen cells from the third-party persons. The CTLp frequencies against the spleen cells from donors decreased 4–6 months after transplantation, and remained at a low level afterward. However, the CTLp frequencies against spleen cells from third-party persons in blood samples obtained 1 year after transplantation were not significantly different from those before transplantation. Therefore, we conclude that donor-reactive CTLs are important in rejecting allografted heart. The decrease in donor-specific CTLp after transplantation could explain the donor-specific tolerance. The decrease may be due to homing of the specific CTLp to the graft, or by clonal deletion of the donor-reactive CTL caused by chronic alloantigen stimulation in the presence of immunosuppressive therapies.

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.

Relapse of Leukemia with Loss of Mismatched HLA Due to Uniparentaldisomy Follwing Haploidentical Hematopoietic Transplantation.