Cytotoxic T-lymphocyte Precursor Frequencies Research Articles

After discontinuation of calcineurin inhibitors, tapering of mycophenolate mofetil further impairs donor‐directed cytotoxicity

Recently, we described a significant decrease in donor-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) after discontinuation of calcineurin inhibitors (CNI), while the proliferative capacity in mixed lymphocyte culture (MLC), and the number of interferon-gamma (IFN-gamma) producing cells (pc) in Elispot remained unchanged. We tested T-cell reactivity in CNI free patients with stable renal graft function, on mycophenolate mofetil (MMF) or azathioprine (AZA) plus prednisone, who were tapered to 50% of their MMF or AZA dose. Furthermore, tapering of the MMF or AZA dose resulted in a decrease of donor-reactive CTLpf in all patients with detectable CTLpf. Detectable numbers decreased from a median of 32 to 8 CTLp/10(6) peripheral blood mononuclear cell (PBMC). No effect on third-party reactive CTLpf was found, while the T-cell reactivity to donor and third-party cells as tested in MLC and in IFN-gamma Elispot was not affected either by tapering of immunosuppression. Third-party reactivity was significantly higher than donor-specific reactivity in all tests. A control group showed no changes in any of the in vitro assays. Both withdrawal of CNI and tapering of MMF or AZA dose decreases the donor-specific CTLpf. Our data suggest that reduction of immunosuppression results in a specific decrease of donor-directed cytotoxic capacity of immunocompetent cells, while their proliferation and cytokine production capacity remained unchanged. Immunosuppression hinders development of cytotoxic non-responsiveness.

Highly diverged MHC class I mismatches are acceptable for haematopoietic stem cell transplantation

A fully major histocompatilbility complex (MHC) matched donor is not available for the majority of patients in need of a haematopoietic stem cell transplantation (SCT), which illustrates the need for a tool to define acceptable MHC disparities. Previously, we noticed that a variety of single MHC class I mismatched allogeneic donor-recipient pairs did not elicit an allogeneic cytotoxic-lymphocyte (CTL) response in vitro if the MHC amino-acid sequences had five or more differences in the alpha-helices plus five or more differences in the beta-sheet (> or =5alpha5beta) (7). To address the clinical relevance of this observation, we analysed CTL precursor (CTLp) assay outcome and SCT outcome in 53 Dutch recipients of a single MHC class I mismatched graft from an unrelated donor. Overall patient survival was 44% after 4 years. In multivariate analysis, recipients of a > or =5alpha5beta mismatched graft with negative CTLp frequencies in vitro before transplantation demonstrated superior survival: survival at 4 years was 80% as compared to 47% in recipients of other mismatched grafts with negative CTLp frequencies (hazard ratio=0.131; 95% CI=(0.03-0.61); P=0.009). This option of acceptable mismatches may enlarge the pool of potentially acceptable stem cell donors.

Full-length EBNA1 mRNA-transduced dendritic cells stimulate cytotoxic T lymphocytes recognizing a novel HLA-Cw*0303- and -Cw*0304-restricted epitope on EBNA1-expressing cells

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is an attractive target for immunotherapy against EBV-associated malignancies because it is expressed in all EBV-positive cells. Although CD8+ cytotoxic T-lymphocyte (CTL) epitope presentation is largely prevented by its glycine-alanine-repeat domain (GAr), the use of mRNA-transduced dendritic cells (DCs) would offer the advantage of priming EBNA1-specific CTLs. After stimulation with GAr-containing EBNA1-transduced monocyte-derived DCs, two EBNA1-specific CTL clones, B5 and C6, were isolated successfully from a healthy donor. These CTLs recognize peptides in the context of HLA-B*3501 and HLA-Cw*0303, respectively. A novel epitope, FVYGGSKTSL, was then identified, presented by both HLA-Cw*0303 and -Cw*0304, which are expressed by >35% of Japanese, >20% of Northern Han Chinese and >25% of Caucasians. The mixed lymphocyte-peptide culture method revealed that FVYGGSKTSL-specific CTL-precursor frequencies in HLA-Cw*0303- or -Cw*0304-positive donors were between 1x10(-5) and 1x10(-4) CD8+ T cells. Moreover, both CTL clones inhibited growth of HLA-matched EBV-transformed B lymphocytes in vitro, and B5 CTLs produced a gamma interferon response to EBNA1-expressing gastric carcinoma cells in the context of HLA-Cw*0303. These data demonstrate that EBNA1 mRNA-transduced DCs may be useful tools for inducing EBNA1-specific CTLs that might be of clinical interest for CTL therapy of EBV-associated malignancies.

Emergence of antitumor cytolytic T cells is associated with maintenance of hematologic remission in children with acute myeloid leukemia

AbstractAlthough the graft-versus-leukemia effect of allogeneic bone marrow transplantation (BMT) is of paramount importance in the maintenance of disease remission, the role played by the autologous T-cell response in antitumor immune surveillance is less defined. We evaluated the emergence of antileukemia cytotoxic T-lymphocyte precursors (CTLp's) and the correlation of this phenomenon with maintenance of hematologic remission in 16 children with acute myeloid leukemia (AML), treated with either chemotherapy alone (5 patients) or with autologous BMT (A-BMT, 11 patients). Antileukemia CTLp's were detectable in 8 patients in remission after induction chemotherapy; none of them subsequently had a relapse. Of the 8 patients who did not show detectable CTLp frequency while in remission after induction chemotherapy, 7 subsequently experienced leukemia relapse. In patients undergoing A-BMT, molecular fingerprinting of the TCR-Vβ repertoire, performed on antileukemia lines, demonstrated that selected antileukemia T-cell clonotypes, detectable in bone marrow before transplantation, survived ex vivo pharmacologic purging and were found in the recipient after A-BMT. These data provide evidence for an active role of autologous T cells in the maintenance of hematologic remission and also suggest that quantification of antileukemia CTLp frequency may be a useful tool to identify patients at high risk for relapse, thus potentially benefiting from an allogeneic antitumor effect.

Induction of Cytotoxic Granules in Human Memory CD8+ T Cell Subsets Requires Cell Cycle Progression

Memory CD8(+) T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8(+) T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8(+) T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA(-)CD8(+) T cells, or of CD8(+) T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA(+) and CD45RA(-) cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7(+) subpopulations, with poorest proliferation being observed in the CD45RA(-)CCR7(-) effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8(+) T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.

A cohort pilot study on therapeutic vaccination of advanced melanoma patients with dendritic cells loaded with multiple peptides or electroporated with mRNA

18011 Background: Therapeutic vaccination of melanoma patients with tumor specific antigenic peptides has been associated with increased frequency of anti-vaccine CTL precursor frequencies in peripheral blood and objective tumor regression in a low percentage of patients. Methods: We investigated the safety and activity of two autologous dendritic cell-based vaccines in 2 sequential cohorts of advanced melanoma patients. Patients first underwent a leucapheresis. Following enrichment in a semi-closed culture system using Cell Factories (Nunc), adherent cells were cultured in RPMI 1640 medium supplemented with 1% heat-inactivated autologous plasma, 500U/ml IL-4 and 1000 U/ml GM-CSF for 6 days and cryo-preserved. Upon thawing, DC were pulsed with 8 peptides or electroporated with synthetic mRNA encoding 7 antigens. Dendritic cell vaccines were administered by intradermal and subcutaneous biweekly injections for a total of 6 times (= cycle 1). Thereafter vaccination was repeated every 6w until clinical deterioration or patient refusal. Frequencies of blood anti-vaccine CTL were estimated by in vitro restimulation in limiting dilution conditions followed by detection with tetramer assay. Results: Fifteen patients were recruited (9M/6F; median age was 49 y, range 28–81). Seven HLA-A2 patients were treated with peptide pulsed DC (cohort A) and 7 patients (4 HLA-A2 and 3 HLA-A1) with mRNA electroporated DC (cohort B). One patient was sequentially treated with both vaccines. Five patients in cohort A and 3 patients in cohort B completed the first treatment cycle of 6 bi-weekly vaccinations (cycle 1 is ongoing in 3 patients of cohort B). Toxicity was limited to grade 1 or 2 local skin reactions at the vaccine administration sites in all patients. In cohort B one patient qualified for stable disease and 2 other patients with progressive disease displayed regression of individual metastases. A >10-fold increase in CTL precursor frequency was observed in 2 out of 3 patients of cohort A that have been analyzed at present. Conclusions: Vaccination of melanoma patients with peptide pulsed or mRNA electroporated autologous DC vaccines was found to be feasible and safe. Updated results will be presented at the meeting. [Table: see text]

Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors.

Human polyomavirus BK (BKV) has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag) to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals.Peptides selected from either p53-binding region (LTag351–450 and LTag533–626) by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-γ gene expression ex vivo and detected by cytotoxicity assays following in vitro culture.We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag579–587; LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations.These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection.

Large HIV‐specific CD8+ cytotoxic T‐lymphocyte (CTL) clones reduce their overall size but maintain high frequencies of memory CTL following highly active antiretroviral therapy

Cytotoxic T-lymphocytes (CTL) play an important role in the control of human immunodeficiency virus (HIV) and of human cytomegalovirus (HCMV) infection. Following highly active antiretroviral therapy (HAART), most studies have demonstrated a decline in the frequency of HIV-specific CTL. We analysed the effect of HAART on the size, phenotype and function of individual HIV- and HCMV-specific CTL clones, using clonotypic oligonucleotide probing specific for the T-cell receptor (TCR) beta-chain hypervariable sequence of defined immunodominant CTL clones specific for peptides of HIV or HCMV, and quantified the limiting dilution analysis frequencies of CTL precursors (CTLp) specific for the same viral peptides. We found that the clonal composition of CD8+ T cells specific for HIV gag and env epitopes was highly focused and did not change after HAART. Following HAART, there was progressive contraction of HIV-specific CD8+ clones, especially in the CD28- CD27- subpopulation--the remaining cells of contracting HIV-specific clones were predominantly CD28- CD27+ CD45RO(hi). We observed maintenance of strong functional HIV-specific CD8+ T-cell responses in limiting dilution analysis following HAART, indicating preferential loss of HIV-specific cells that have reduced cloning efficiency in vitro. Following HAART, we also observed selective expansion of HCMV-specific CD8+ clones. Most HCMV-specific CD8+ clones were predominantly CD28- CD27+/- CD45RA(hi) following HAART. In one subject, a Vbeta6.4+ clone specific for HCMV pp65 selectively expanded following HAART, without expansion of two other Vbeta6.4+ clones, indicating that individual clonotypes specific for the same peptide can show different kinetics and phenotypes in response to antiretroviral therapy.

Calcineurin Inhibitor Withdrawal in Stable Kidney Transplant Patients Decreases the Donor-Specific Cytotoxic T Lymphocyte Precursor Frequency

In a prospective study, calcineurin inhibitors (CNI) were withdrawn in patients two years after kidney transplantation. We questioned whether stopping CNI had an effect on the donor-specific reactivity, as CNI might hinder immune responses leading to graft acceptance. We measured the donor-specific cytotoxic T lymphocyte (CTL) precursor frequency (CTLpf) in 54 patients before and after withdrawal of CNI. In addition, the T-cell reactivity of PBMC to donor and third-party antigens was tested in MLR, and in IFNgamma-Elispot. Reactivity to tetanus toxoid (TET) was studied as well. Donor-specific CTLpf significantly decreased after CNI withdrawal (P=0.0001). In contrast, no difference was observed in third-party reactive CTLpf, donor and third-party reactive MLR and IFNgamma-Elispot. Proliferative responses and the number of IFNgamma-producing cells to TET also decreased after CNI withdrawal. The decrease in CTLpf correlated with the time between the two blood samples (before and after stopping CNI, P=0.05). This decrease was caused by stopping CNI, because there was no correlation between CTLpf and the duration of the CNI treatment after transplantation. Moreover, the percentage of regulatory T cells in the peripheral blood increased after CNI withdrawal. We report here that after withdrawal of CNI the donor-specific CTLpf decreases. We hypothesize that CNI suppress regulatory mechanisms that have the potential to down-regulate donor-specific CTL responses and reactivity to TET.

CTLA4-Fas Ligand Gene Transfer Mediated by Adenovirus Induce Long-Time Survival of Murine Cardiac Allografts

Background Fas ligand gene transfer to induce peripheral allograft tolerance in animal models has shown controversial results. The immunosuppression effects mediated by engineered FasL depend on whether alloreactive T cells are selectively deleted. In the present study, we tested the feasibility of a strategy to induce long-time survival by fusing CTLA4-FasL gene transfer in vivo. Methods Cardiac allografts from DA(RT-1 a) rats were transplanted heterotopically into the abdomens of LEW(RT-1 1) rats. Plaque units (5×10 9) of either AdCTLA4-FasL, AdCTLA4Ig, or AdEGFP were administered via the portal vein immediately after cardiac transplantation. The frequencies of helper T lymphocyte precursors (HTLp) and cytotoxic T lymphocyte precursors (CTLp) were determined by a combined single limiting dilution assay on days 5 and 20 after transplantation. Results Cardiac allograft survival was significantly prolonged by either AdCTLA4-FasL or AdCTLA4Ig treatment(mean survival times [MST] of 71.0 ± 3.7 and 45.7 ± 2.4, respectively, n = 6) compared with untreated hosts or animals treated with AdEGFP(MST of 5.7 ± 0.5 and 5.2 ± 0.4, respectively, n = 6). In addition, treatment with AdCTLA4-FasL led to significantly prolonged allograft survival compared with AdCTLA4Ig treatment. Furthermore, the frequencies of HTLp and CTLp on day 20 among rats treated with AdCTLA4-FasL was lower than those on day 5, whereas frequencies of HTLp and CTLp on day 20 were similar with those on day 5 in the other groups. Conclusion These results suggest that administration of an adenovirus encoding fusion CTLA4-FasL gene to rat recipients effectively decreased the size of alloreactive T cells and induced long-term survival of cardiac allografts.

The Granzyme B and Interferon-?? Enzyme-Linked Immunospot Assay as Alternatives for Cytotoxic T-Lymphocyte Precursor Frequency after Renal Transplantation

The interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay has gained increased popularity as a surrogate marker of cytotoxic T-lymphocyte (CTL) activity. However, the functional activity of CTL might be a more relevant surrogate marker of CTL. Therefore, the authors wondered whether the granzyme B (GrB) ELISPOT assay is a better marker for determining the number of CTL than the IFN-gamma ELISPOT assay. METHOD.: Peripheral blood mononuclear cells (PBMC) from 19 kidney transplant patients were stimulated with donor cells or third-party cells. The authors determined the CTL precursor frequency (CTLpf) and simultaneously measured the number of IFN-gamma- and GrB-producing cells (pc) by ELISPOT assay. In all three different assays, the reactivity to donor cells was significant lower than the reactivity to third-party cells: CTLpf, median: 9 versus 60/10(6) PBMC (P=0.0002); number of IFN-gamma pc: 10 versus 90/10(6) PBMC (P=0.0001); number of GrB pc: 60 versus 205/10(6) PBMC (P=0.05). When the authors compared the CTLpf after third-party stimulation with the corresponding ELISPOT results, they found a positive correlation between the CTLpf and the number of IFN-gamma pc (r(s)=0.47, P=0.05). No correlation was found between the CTLpf and the number of GrB pc (r(s)=0.23, P=0.36). However, when they compared the donor-specific CTLpf with the corresponding ELISPOT results, no correlation with the ELISPOT for IFN-gamma (r(s)=0.10, P=0.69) or GrB (r(s)=-0.24, P=0.34) was found. The authors feel that the CTLpf, as a measure of the actual endpoint of cytolytic activity and independent of the pathway of killing, remains the "gold standard" for determining donor-specific cytolytic activity after clinical organ transplantation.

Reduction of Immunosuppressive Load in Renal Transplant Recipients With a Low Donor-Specific Cytotoxic T-Lymphocyte Precursor Frequency is Safe

Background Tapering of immunosuppressive medication is indicated to prevent long-term side effects. Recently, we have shown that renal transplant recipients can safely be converted from calcineurin inhibitors to MMF or AZA when their donor-specific cytotoxic T-lymphocyte precursor frequencies (CTLpf) are below 10/10 6 PBMC. We wondered whether a low CTLpf also had predictive value when immunosuppressive medication was reduced in patients only on MMF or AZA and steroid medication. Methods Renal transplant recipients with stable renal function at least 2 years after transplantation and with low (<10/10 6 PBMC) CTLpf were included. Their MMF or AZA dose was reduced to 75% and to 50% of the original dose at 4 months and 8 months after inclusion. Endpoint of the study was 12 months after inclusion or developing acute rejection. Results Forty-five patients have reached the 1-year follow up endpoint. Their median time after transplantation was 4.2 years (range 2.0–15.5 years). Acute rejection was seen in one patient only (who had discontinued all his medication). Conclusion In patients with low CTLpf long after kidney transplantation, a 50% reduction of immunosuppression is safe and further decreasing their immunosuppressive load is the obvious next step.

A Potent Immunogenic General Cancer Vaccine That Targets Survivin, an Inhibitor of Apoptosis Proteins

We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24(+) cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24(+) cancer patients.

CMV Specific Immunotherapy: How Many Patients Will Benefit?.

Cytomegalovirus (CMV) infection is associated with increased transplant related mortality and decreased overall survival after stem cell transplantation (SCT) despite major advances in early detection, prophylaxis and treatment. This effect is most marked in CMV seropositive patients who have a statistically significant decrement in overall or disease free survival of 20–46% when compared to low risk (−/−) transplants (Boeckh Blood 2004 103: 2003–8). Transfer of cellular immunity from a seropositive donor results in reconstitution of T cells to CMV and protection from CMV disease post-SCT, and this underlies the recent development of cellular immunotherapeutic manoeuvres. As a centre looking to develop such therapy, we have carried out a retrospective analysis of a patient cohort in order to estimate how many patients would require this intervention. We used twice weekly quantitative PCR surveillance to analyse the CMV reactivation profiles of 104 recipients of 106 allogeneic SCT, transplanted between April 2002 and April 2004. Hence follow up was 5–27 months. The cohort comprised 41 adults and 65 children. Transplants were from related (42), unrelated (52) and haploidentical donors (12). T cell depletion was performed either (i) in vivo using either CAMPATH-1H (44 transplants) or ATG (5), (ii) ex vivo by either CD34 (8) or CAMPATH-1H in vitro (1), or (iii) by combinations of (i) and (ii) (26). SCTs were for malignancies in 92 cases and non-neoplastic disorders in 14. Many patients were considered high risk, necessitating short search to transplant times. A hierarchy of donor selection factors was considered: HLA matching was the primary determinant, with other factors including stem cell dose, age, gender and CMV status. Seventy-one patients were at risk of CMV reactivation. There were 28 episodes of CMV reactivation in 27 of these patients, with the following Recipient/Donor serostatus combinations: 0 (of 16) −/+, 22 (of 38) +/− and 6 (of 17) +/+. Seven patients (Group A) resolved their infections without intervention. Nine patients (Group B) resolved infections after antiviral drug treatment, whilst the remaining 12 reactivators (Group C) did not clear their CMV DNA load despite antiviral treatment (of whom 3 died, 1 relapsed and the remainder have ongoing CMV PCR positivity). As a minimum a CMV immunotherapy programme should allow prophylaxis or early treatment of all patients in groups B and C. However, using our current selection criteria only 5/21 cases had a CMV seropositive donor. Logistical problems e.g. CTL precursor frequency or availability of an immunodominant tetramer might have rendered some donors inappropriate providers of anti-CMV CTL. Thus a maximum of 5 out of 106 transplanted patients in our unit could have benefited, although this figure could be slightly improved by deliberate selection of CMV positive donors for CMV positive patients. Such numbers should be borne in mind by any centre contemplating the development of antiviral immunotherapy programmes.

Correlation between tumor regression and T cell responses in melanoma patients vaccinated with a MAGE antigen.

The cancer-germline gene MAGE-3 codes for tumor-specific antigens recognized on many tumors by T lymphocytes. A MAGE-3 antigen presented by HLA-A1 has been used in several vaccination trials on metastatic melanoma patients. Only a small minority of patients have shown evidence of tumor regression. Attempts to correlate the tumor rejections with the cytotoxic T lymphocyte (CTL) response against the vaccine have been hampered by the low level of these responses. In noncancerous individuals, the frequency of the T cell precursors against antigen MAGE-3.A1 is approximately 4 x 10(-7) CD8 T cells. The diversity of the T cell receptor repertoire of these anti-MAGE-3.A1 precursors was analyzed in one individual. The results indicate that it is very likely that the repertoire comprises >100 clonotypes. On this basis, it is possible to use not only the frequency of CTL precursors in the blood but also the presence of dominant clonotypes to ascertain in patients the existence of anti-MAGE-3.A1 responses as low as 10(-6) of CD8. With this approach, we observed a correlation between tumor regression and anti-MAGE-3.A1 CTL responses in patients vaccinated with a recombinant virus encoding the antigen and also in patients vaccinated with peptide-pulsed dendritic cells. In contrast, for patients showing tumor regression after vaccination with peptide alone, CTL responses were almost never observed. It is possible that even those CTL responses that are below our present detection level can trigger a sequence of events that leads to tumor regression.

Partial activation of neonatal CD11c+ dendritic cells and induction of adult-like CD8+ cytotoxic T cell responses by synthetic microspheres.

Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8(+) cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8(+) T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-gamma-secreting CD8(+) splenocytes. Neonatal CD8(+) T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c(+) dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c(+) DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4(+) T cell help. The effective induction of neonatal CD8(+) T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c(+) DC may mature sufficiently to stimulate naive CD8(+) neonatal T cells, even in the absence of strong maturation signals.

HLAMatchmaker algorithm is not a suitable tool to predict the alloreactive cytotoxic T-lymphocyte response in vitro.

Both donor-specific anti-human leukocyte antigen (HLA) antibodies and cytotoxic T lymphocytes are important mediators of graft rejection. HLAMatchmaker determines the amino acid triplets on antibody-accessible sites of the HLA molecule that are not shared between patient and donor. A previous study showed a strong positive correlation between the number of triplet mismatches and the percentage of individuals producing HLA antibodies. In the present study, we tested whether the number of triplet mismatches is predictive for the cytotoxic T-lymphocyte precursor (CTLp) frequency in vitro. The analysis was performed on 108 HLA-DRB1 and DQB1 identical patient-donor combinations registered by the Europdonor foundation, with a single HLA class I mismatch and in healthy responder-stimulator combinations mismatched for at least one HLA class I antigen. The results show that there is no strong correlation between the number of triplet mismatches and the CTLp frequency. Even in the case of zero triplet mismatches, a high CTLp frequency can be found. This lack of correlation is probably caused by the fact that HLAMatchmaker considers only triplets on antibody-accessible positions, whereas CTLs also recognize other epitopes on the HLA molecule, including the bound peptides.

Tapering immunosuppression in recipients of living donor kidney transplants.

We have previously suggested that the in vitro donor-specific cytotoxic T-lymphocyte precursor (CTLp) assay can guide us to identify patients in which the immunosuppressive load can be tapered. In a clinical trial we had observed that a low (<10/10(6) PBMC) frequency of these CTLp was predictive for an uneventful rejection-free clinical course in patients that were converted from calcineurin inhibitors to mycophenolate mofetil or azathiopine. In the present prospective study in 81 stable kidney transplant recipients, already converted from calcineurin inhibitors, we measured CTLp frequencies and reduced the immunosuppressive load on a routine basis when CTLp were <10/10(6) PBMC. Donor-specific cytotoxicity could not be measured in 50/81 patients, while their reactivity against third-party lymphocytes was not impaired. These 50 patients were tapered in their immunosuppression. Only in one patient, who had stopped all his medication, was a rejection episode diagnosed. We conclude that in patients with a low donor-specific CTLp frequency it is safe to reduce the immunosuppression.

IMMUNOMONITORING IN ALLOGRAFT RECIPIENTS TREATED WITH A TOLERANCE-ENHANCING IMMUNOSUPPRESSIVE REGIMEN

O147* Aims: Recipients of whole organ allografts were treated in accordance with two therapeutic principles of (1) recipient pretreatment with Thymoglobulin (THY, 3-6 mg/kg) or Campath (CAM, 30 mg) before graft reperfusion and (2) minimum posttransplant immunosuppression consistent with graft survival and function using tacrolimus monotherapy (target trough level of 10 ng/ml). Other agents (e.g. steroid, rapamycin) were added only to treat biopsy-proven rejection. This report described changes in immunological and hematological parameters with a minimum followup of >12 months. Methods: Blood samples were obtained at 1, 3, 6, and 12 months after transplantation for four-color flow cytometry of leukocyte lineages, in vitro cell proliferation assays, and ELISPOT using cadaveric donor splenocytes or live donor PBMC as alloantigens. Results: Pretreatment with THY or CAM resulted in significant posttransplant lymphocyte depletion, which slowly recovered over the next 6-12M. CAM had stronger and prolonged impacts on lymphocyte depletion. Recovery of CD4 population was slower than CD8, resulting in remarkably low CD4/CD8 ratios (<0.8) at 6-12M with a higher incidence in the CAM group. Likewise, DC1 and DC2 populations considerably decreased after transplantation. DC1 gradually recovered and became nearly normal by 12M (49% vs. 57% in normal volunteers); however, recovery of DC2 was remarkably slow, particularly in the CAM group, and was 5.5 ± 5.0% at 12M compared to 15.0 ± 9.0% in the normal. CAM, but not THY, also effectively depleted B cells, and significantly low B cell counts persisted for nearly 12M. At 1 year, 48 of 155 (31%) functioning kidney recipients were on daily immunosuppression with single or multiple drugs. The remaining 107 (69%) were on tacrolimus, cyclosporine, or rapamycin monotherapy, with doses spaced from one every other day to once weekly. Of 95 liver recipients, 26 (27%) are on daily therapy with single or multiple agents, while 69 (73%) are on spaced monotherapy. Detailed in vitro studies (MLR, CML, limiting dilution, ELISPOT) were performed in 20 kidney and 16 liver recipients with >1 year follow-up. Kidney recipients on continuous multiple drugs showed global suppression to mitogens and alloantigens. In contrast, two-thirds of recipients on spaced weaning showed donor-specific hyporeactivity with vigorous responses to mitogens and 3rd party alloantigens. The remaining recipients on spaced monotherapy had intact anti-donor reactivity by MLR; but, CML responses were low with CTLp frequencies of less than 1:90,000-300,000. Patients (n=2) with modest killing (10-16%) with CTLp of 1:60,000-150,000 showed a low frequency of γ-IFN positive cells in ELISPOT. On the contrary, although the majority of liver recipients were in more advanced stages of weaning, correlation between immunosuppression and in vitro results was less clear probably due to the complicated immunological status in this population (e.g. hepatitis). Approximately 50% showed donor-specific hyporeactivity or total suppression in MLR and CML. The remaining half showed reactivity to mitogens and alloantigens (donor and 3rd party) in MLR with a significantly higher background. Donor killing in CML was 8-43% with low CTLp frequencies of 1:<200,000. Conclusions: Donor-specific immune modulation was observed in a subset of allograft recipients pretreated with THY or CAM and given minimal posttransplant immunosuppression. In contrast, patients on continuous multiple drugs showed overall suppression.

ALLORESPONSES IN TOLERANT RECIPIENTS OF COMBINED KIDNEY/BONE MARROW TRANSPLANTATION WITH NON-MYELOABLATIVE CONDITIONING

P931 Introduction: We analyzed in vitro responses in 6 patients who received combined non-myeloablative HLA-matched sibling bone marrow (BM) and kidney transplantation (Tx) for the treatment of multiple myeloma (MM) with renal failure. Patients 3 - 6 were participants in an Immune Tolerance Network study. Methods: Mixed lymphocyte reaction (MLR), cell-mediated lymphocytotoxicity (CML), and limiting dilution assays (LDA) were performed. Donor chimerism was monitored by microsatellite analysis. T cell recovery was followed by flow cytometry. Results: In Patient 1, initial multilineage mixed chimerism declined to undetectable levels after Day 105. CyA was discontinued on Day 73 and the transplanted kidney has shown no evidence of rejection > 5.5 years. Early CD8 T cell recovery and strong anti-donor cytotoxic T lymphocyte (CTL) responses were seen in CML and LDA, without anti-donor MLR responses. The myeloma is in complete remission (CR) > 5.5 years. In Patient 2, initial mixed chimerism became undetectable after Day 123. CyA was discontinued on Day 77 and the kidney has shown no rejection > 3 years. Early recovery of anti-3rd party responses but not anti-donor responses were seen in MLR and CTL and helper T lymphocyte (HTL) LDA. Urinary light chain was reduced by 90% after Tx, but the myeloma later progressed. In Patient 3, multilineage mixed chimerism converted to donor full chimerism by Day 62. MMF was added to control mild chronic GvHD and CyA was discontinued on Day 379. The patient showed transient anti-donor responses in MLR and CTL-LDA prior to conversion to donor full chimerism, and high anti-host CTLp frequencies following conversion to donor full chimerism. The kidney has shown no rejection. The patient is in CR > 30 months. In Patient 4, initial mixed chimerism declined to undetectable levels by Day 71. CyA was discontinued on Day 60. The kidney has shown no rejection > 21 months. Early recovery of anti-3rd party responses but not anti-donor responses were seen in MLR and CTL and HTL-LDA. Urinary light chain persisted post-Tx. In Patient 5, multilineage mixed chimerism converted to donor full chimerism by Day 90. CyA was discontinued on Day 35. The patient showed transient anti-donor responses in HTL and CTL-LDA prior to conversion to donor full chimerism, and high anti-host CTLp frequencies following conversion to donor full chimerism. The kidney has shown no rejection. In Patient 6, initial mixed chimerism was observed, but became undetectable by Day 98. CyA was discontinued on Day 60. The kidney has shown no rejection > 3 months. No anti-donor responses were seen in MLR and CTL and HTL-LDA, but a high anti-host HTLp frequency was seen on POD70. Conclusions: Combined kidney/BMTx with non-myeloablative conditioning for MM patients is a promising strategy for successful induction of renal allograft tolerance and also provides potent anti-myeloma effects. Donor marrow rejection and anti-donor alloresponses can appear in patients despite maintenance of tolerance to donor kidneys.