ATIR101 administered after T-cell-depleted haploidentical HSCT reduces NRM and improves overall survival in acute leukemia.

Overcoming graft-versus-host disease (GvHD) without increasing relapse and severe infections is a major challenge after allogeneic hematopoietic stem-cell transplantation (HSCT). ATIR101 is a haploidentical, naïve cell-enriched T-cell product, depleted of recipient-alloreactive T cells to minimize the risk of GvHD and provide graft-versus-infection and -leukemia activity. Safety and efficacy of ATIR101 administered after T-cell-depleted haploidentical HSCT (TCD-haplo + ATIR101) without posttransplant immunosuppressors were evaluated in a Phase 2, multicenter study of 23 patients with acute leukemia and compared with an observational cohort undergoing TCD-haplo alone (n = 35), matched unrelated donor (MUD; n = 64), mismatched unrelated donor (MMUD; n = 37), and umbilical cord blood (UCB; n = 22) HSCT. The primary endpoint, 6-month non-relapse mortality (NRM), was 13% with TCD-haplo + ATIR101. One year post HSCT, TCD-haplo + ATIR101 resulted in lower NRM versus TCD-haplo alone (P = 0.008). GvHD-free, relapse-free survival (GRFS) was higher with TCD-haplo + ATIR101 versus MMUD and UCB (both P < 0.03; 1-year rates: 56.5%, 27.0%, and 22.7%, respectively) and was not statistically different from MUD (1 year: 40.6%). ATIR101 grafts with high third-party reactivity were associated with fewer clinically relevant viral infections. Results suggest that haploidentical, selective donor-cell depletion may eliminate requirements for posttransplant immunosuppressors without increasing GvHD risk, with similar GRFS to MUD. Following these results, a randomized Phase 3 trial versus posttransplant cyclophosphamide had been initiated.

Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers.

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes.

Simultaneous detection of circulating autoreactive CD8+ T-cells specific for different islet cell-associated epitopes using combinatorial MHC multimers.

OBJECTIVE: Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-cells in the pancreas. In this process, islet epitope-specific CD8(+) T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8(+) T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS: Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B(10-18), prepro-insulin (PPI)(15-24), islet antigen (IA)-2(797-805), GAD65(114-123), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(265-273), and prepro islet amyloid polypeptide (ppIAPP)(5-13)-specific CD8(+) T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS: Using this kit, islet autoreactive CD8(+) T-cells recognizing insulin B(10-18), IA-2(797-805), and IGRP(265-273) were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI(15-24) proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS: A kit was developed that allows simultaneous detection of CD8(+) T-cells reactive to multiple HLA-A2-restricted beta-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

Differences in baseline lymphocyte counts and autoreactivity are associated with differences in outcome of islet cell transplantation in type 1 diabetic patients.

OBJECTIVE: The metabolic outcome of islet cell transplants in type 1 diabetic patients is variable. This retrospective analysis examines whether differences in recipient characteristics at the time of transplantation are correlated with inadequate graft function. RESEARCH DESIGN AND METHODS: Thirty nonuremic C-peptide-negative type 1 diabetic patients had received an intraportal islet cell graft of comparable size under an ATG-tacrolimus-mycophenolate mofetil regimen. Baseline patient characteristics were compared with outcome parameters during the first 6 posttransplant months (i.e., plasma C-peptide, glycemic variability, and gain of insulin independence). Correlations in univariate analysis were further examined in a multivariate model. RESULTS: Patients that did not become insulin independent exhibited significantly higher counts of B-cells as well as a T-cell autoreactivity against insulinoma-associated protein 2 (IA2) and/or GAD. In one of them, a liver biopsy during posttransplant year 2 showed B-cell accumulations near insulin-positive beta-cell aggregates. Higher baseline total lymphocytes and T-cell autoreactivity were also correlated with lower plasma C-peptide levels and higher glycemic variability. CONCLUSIONS: Higher total and B-cell counts and presence of T-cell autoreactivity at baseline are independently associated with lower graft function in type 1 diabetic patients receiving intraportal islet cells under ATG-tacrolimus-mycophenolate mofetil therapy. Prospective studies are needed to assess whether control of these characteristics can help increase the function of islet cell grafts during the first year posttransplantation.

Monotherapy rapamycin allows an increase of CD4 CD25 FoxP3 T cells in renal recipients.

CD4(+) CD25(bright+) FoxP3(+) regulatory T cells (Tregs) may control donor-specific allogeneic responses in kidney transplant recipients. Recent evidence demonstrated that three phenotypical Treg-subsets, naive (CCR7(+)CD45RO(-)), central-memory (CCR7(+)CD45RO(+)) and effector-memory (CCR7(-)CD45RO(+)), are essential for the development and function of antigen-specific suppression in the lymphoid and peripheral tissues. Also, it has been appreciated that Tregs are affected by immunosuppressive agents. In clinical practice, however, the effect of a single drug remains to be determined. Therefore, we analyzed the effect of several immunosuppressive agents on the number, phenotype and function of peripheral Tregs from 46 stable kidney transplant recipients. These patients were converted to monotherapy with tacrolimus (n = 15), rapamycin (n = 17) or mycophenolate mofetil (n = 14). Blood was obtained at inclusion and 6 months thereafter. The number of Tregs increased significantly in patients on monotherapy with rapamycin (P < 0.001), which was caused by increased numbers of Tregs with a central-memory and an effector-memory phenotype (both P < 0.05). At 6 months after conversion, however, the suppressive function of Tregs did not significantly change in co-cultures stimulated with donor-Ag. Therefore, monotherapy with rapamycin allows the signals that are needed to increase the number of functional Tregs with a memory phenotype, thereby enhancing the potential capacity to regulate donor-specific responses in the lymphoid and the peripheral tissues.

Generation of donor-specific regulatory T-cell function in kidney transplant patients.

BACKGROUND: In the search for mechanisms that can induce and maintain transplant tolerance, donor-specific CD4CD25FoxP3 regulatory T cells have been frequently mentioned. However, it remains to be demonstrated, whether these cells are generated after clinical transplantation. METHODS: We prospectively analyzed the phenotype and function of peripheral regulatory CD4CD25 T cells of 79 patients before, 3, 6, and 12 months after kidney transplantation. The immune regulatory capacities of CD4CD25 T cells were assessed by their depletion from peripheral blood mononuclear cells and in co-culture with CD25 responder T-cells in the mixed lymphocyte reactions. RESULTS: In the first year after transplantation, the number and proportion of CD4CD25 T cells significantly decreased (P<0.05 and P<0.001, respectively). In the mixed lymphocyte reactions, we observed donor-specific hyporesponsiveness in the presence of significantly increased proliferation to third and fourth Party-Ag, (P<0.001 and P<0.05, respectively). Furthermore, functional analysis of CD25 cells showed that the effect of depletion of these cells from peripheral blood mononuclear cells, and their suppressive capacities in co-culture with donor-Ag stimulated CD25 responder T-cells (1:10 ratio) significantly improved (P<0.01 and P<0.001, respectively). Moreover, the difference between the stimulation with donor-Ag and third Party-Ag became apparent at 6 months after transplantation. CONCLUSIONS: These findings demonstrate that donor-specific CD4CD25 regulatory T-cell function is generated in fully immunosuppressed renal recipients in the first year after transplantation.

Intragraft FOXP3 mRNA expression reflects antidonor immune reactivity in cardiac allograft patients.

BACKGROUND: Regulatory FOXP3+ T cells control immune responses of effector T cells. However, whether these cells regulate antidonor responses in the graft of cardiac allograft patients is unknown. Therefore, we analyzed the gene expression profiles of regulatory and effector T-cell markers during immunological quiescence and acute rejection. METHODS: Quantitative real-time polymerase chain reaction was used to analyze mRNA expression levels in time-zero specimens (n=24) and endomyocardial biopsies (EMB; n=72) of cardiac allograft patients who remained free from rejection (nonrejectors; n=12) and patients with at least one histologically proven acute rejection episode (rejectors; International Society for Heart and Lung Transplantation [ISHLT] rejection grade>2; n=12). RESULTS: For all analyzed regulatory and effector T-cell markers, mRNA expression levels were increased in biopsies taken after heart transplantation compared with those in time-zero specimens. Posttransplantation, the FOXP3 mRNA levels were higher in EMB assigned to a higher ISHLT rejection grade than the biopsies with grade 0: the highest mRNA levels were detected in the rejection biopsies (rejection grade>2; P=0.003). In addition, the mRNA levels of CD25, glucocorticoid-induced TNF receptor family-related gene, cytotoxic T lymphocyte-associated antigen 4, interleukin-2, and granzyme B were also significantly higher in rejecting EMB than in nonrejecting EMB (rejection grade

An improved method to study NK-independent mechanisms of MTLn3 breast cancer lung metastasis.

To study the tumor cell autonomous processes of metastasis, an in vivo tumor metastasis model is required that excludes the involvement of the innate immune system. For this purpose we used the established syngeneic MTLn3 cell - Fischer 344 tumor model. MTLn3 cells are efficiently eradicated by NK cells in vivo. Using isolated cell systems, we provide evidence for apoptosis-induction by IL-2 activated NK cells, but not T-cells, despite the expression of MHC class I. This is largely mediated by the perforin/granzyme B pathway in MTLn3 cells in a caspase-dependent manner. Temporal in vivo depletion of NK cells by an antibody-based method, dramatically improved colonization of the lungs by MTLn3 cells, from 5 metastases in the untreated animals to 130 metastases in the NK-depleted animals. Thus, we improved the syngeneic MTLn3-Fischer 344 tumor model by temporal depletion of NK cells of which the advantages over the use of immunodeficient animals are evident.

Functional CD25(bright+) alloresponsive T cells in fully immunosuppressed renal allograft recipients.

BACKGROUND: Evidence from animal studies indicate a crucial role for CD25(bright+) regulatory T cells in transplantation tolerance. METHODS: To assess whether peripheral CD25(bright+) T cells control immune responses in immunosuppressed kidney transplant patients, we analyzed the suppressive capacities of these cells using mixed lymphocytes reactions. RESULTS: Allogeneic stimulation of patients peripheral blood mononuclear cells was associated with IL-2 production and T-cell proliferation. Depletion of CD25(bright+) T cells resulted in a 35% (median) higher IL-2 production and a 38% higher proliferative response against third party cells, showing that functional regulatory CD25(bright+) T cells were present (p = 0.03 and 0.02 respectively). In eight out of 11 patients, we also demonstrated regulation activity against donor-activated T cells (p = 0.03). These data were confirmed in coculture experiments with isolated CD25(-/dim) T cells plus CD25(bright+) T cells. At a 1:2 ratio, the CD25(bright+) T cells suppressed the proliferation of CD25(-/dim) donor- and third party-stimulated responder T cells. CONCLUSIONS: CD25(bright+) T cells with immune regulatory activities against anti-donor-responsive T cells are readily detectable in renal allograft recipients during treatment with full dosage immunosuppression. Whether CD25(bright+) T cells indeed play a role in graft acceptance after organ transplantation in patients remains to be elucidated.

Donor-specific cytotoxic hyporesponsiveness associated with high interleukin-10 messenger RNA expression in cardiac allograft patients.

BACKGROUND: After transplantation, CD4+CD25+FOXP3+ and interleukin (IL) 10-producing regulatory cells might regulate immune responses toward donor antigens. In this study, we determined whether cardiac allograft recipients show donor-specific hyporesponsiveness and studied the underlying mechanisms. METHODS: We analyzed the donor-specific T-cell responses by mixed lymphocyte reactions and limiting dilution assays to define whether cardiac allograft recipients (n = 21) show proliferative and cytotoxic hyporesponsiveness to donor antigens long after transplantation (range, 1.5-7 years). The mechanisms controlling immune responses, that is, FOXP3+/GITR+ T cells, and IL-10-producing cells, were studied by quantitative real-time polymerase chain reaction. RESULTS: In the presence of a proliferative response to donor antigens, no cytotoxic responsiveness could be measured in a number of patients in the absence (73%) and presence of exogenous IL-2 (29%), IL-15 (31%), and IL-15 plus IL2Ralpha blockade (88%). Overall, the cytotoxic response to donor cells was significantly lower than the reactivity to third-party cells after the addition of IL-2 (p = 0.004) and IL-15 plus IL2Ralpha blockade (p < 0.001). After donor stimulation, the peripheral blood mononuclear cells expressed higher messenger RNA (mRNA) levels of IL-10, but not of FOXP3 or GITR, than after third-party stimulation (p = 0.003). Moreover, the IL-10 mRNA expression was inversely correlated with the donor-specific cytotoxic responsiveness (p = 0.01). CONCLUSIONS: A significant proportion of patients showed donor-specific cytotoxic hyporesponsiveness long after heart transplantation, which was associated with high mRNA levels of IL-10 but not of FOXP3 or GITR. This observation suggests that IL-10-producing cells participate in the donor-specific cytotoxic hyporeactivity.

Impaired activation of caspases and prevention of mitochondrial dysfunction in the metastatic colon carcinoma CC531s-m2 cell line.

In a previous paper we described the properties of a rapidly metastasizing cell line CC531s-m2 derived from the poorly metastasizing CC531s cell. The m2-cell line was relatively resistant to killing by NK cells. Both CD95L and TRAIL mediated apoptosis was decreased in the m2-cell line. Now, by flow cytometrical analysis of intra- and extra-cellular expressed receptors, we show that the localization of the receptors for CD95L and TRAIL was not altered in the CC531s-m2 cells as compared to the parental cell line. Subsequently caspase-activation and mitochondrial function were studied by enzymatic cleavage of fluorescent caspase-substrates and retention of the mitochondrial dye rhodamine-123, respectively. The activation of caspases as well as the loss of the mitochondrial membrane potential (MMP) was less in the CC531s-m2 cell line upon CD95L- and TRAIL-signalling. Furthermore, the sensitivity of the CC531-m2 towards cisplatin-induced apoptosis was strongly decreased. This was consistent with less mitochondrial damage, delayed caspase cleavage and decreased caspase activity. Altogether, we conclude that an Natural Killer-cell insensitive cell is less sensitive to CD95L- and TRAIL-induced apoptosis as well as anti-cancer drug induced apoptosis by prevention of mitochondrial damage and activation of caspases.

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells.

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-alpha pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-alpha pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.

Rat colon carcinoma cells that survived systemic immune surveillance are less sensitive to NK-cell mediated apoptosis.

In order to form distant metastases, cells from the primary tumor have to detach, enter the blood- or lymph-compartment and escape immune surveillance. Here, we describe the selection of rat colon carcinoma cell lines (CC531s-m1 and CC531s-m2) that escaped from systemic immune surveillance; CC531s cells were injected into the v. jugularis of Wag/Rij rats, after three weeks the lung tumors were isolated, the tumor cells were cultured, characterized and injected again. The m1- and m2-cell lines were less susceptible for killing by syngeneic NK cells. Further characterization of this cell line showed a decreased sensitivity towards TRAIL- and CD95L-, but not to granzyme B-mediated apoptosis. In the m1- and m2-cells log-phase growth started earlier as compared to the parental cell line, whereas no changes were found in anchorage-dependent or anchorage-independent growth. After subcapsular injection of the m2-cell line into the liver of rats much more lung metastases were formed in comparison to injection of the parental cell line. In conclusion, the results suggest that the resistance of the m1- and m2-cells to NK cell-mediated apoptosis was associated with their capability to survive systemic immune surveillance and form metastases in vivo.

Distinct intracellular signaling in tumor necrosis factor-related apoptosis-inducing ligand- and CD95 ligand-mediated apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.