T regulatory cells in xenotransplantation.

The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long-term graft survival. In this regard, the induction of donor-specific tolerance through isolation and expansion of xenoantigen-specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4(+)CD25(+)Foxp3(+) T cells, CD8(+)CD28(-) Treg, double negative CD4(-)CD8(-) T cells, and natural killer Treg. Although only studied in vitro so far, human CD4(+)CD25(+)Foxp3(+) Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8(+)CD28(-) Treg and double negative CD4(-)CD8(-) Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4(+)Valpha14(+) Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4(+)CD25(+)Foxp3(+) Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.

Differential requirement for CD4 help in the development of an antigen-specific CD8+ T cell response depending on the route of immunization.

Previous studies in our laboratory have shown that DBA/2 mice injected i.p. with syngeneic P815 tumor cells transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8+CD62L- T cells expressing exclusively the Vbeta10 segment. We have used this model to study the regulatory mechanisms involved in the development of the CW3-specific CD8+ response, with respect to different routes of immunization. Whereas both intradermal (i.d.) and i.p. immunization of DBA/2 mice with P815-CW3 cells led to a strong expansion of CD8+CD62L-Vbeta10+ cells, only the i.d. route allowed this expansion after immunization with P815 cells transfected with a minigene coding for the antigenic epitope CW3 170-179 (P815 miniCW3). Furthermore, depletion of CD4+ T cells in vivo completely abolished the specific response of CD8+CD62L-Vbeta10+ cells and prevented the rejection of P815-CW3 tumor cells injected i.p., whereas it did not affect CD8S+CD62L-Vbeta10+ cell expansion after i.d. immunization with either P815-CW3 or P815 miniCW3. Finally, the CW3-specific CD8+ memory response was identical whether or not CD4+ T cells were depleted during the primary response. Collectively, these results suggest that the CD8+ T cell response to P815-CW3 tumor cells injected i.p. is strictly dependent upon recognition of a helper epitope by CD4+ T cells, whereas no such requirement is observed for i.d. injection.

Tacrolimus and cyclosporine have differential effects on the risk of development of bronchiolitis obliterans syndrome: results of a prospective, randomized international trial in lung transplantation.

BACKGROUND: Chronic lung allograft dysfunction, which manifests as bronchiolitis obliterans syndrome (BOS), is recognized as the primary cause of morbidity and mortality after lung transplantation. In this study we assessed the efficacy and safety of two de novo immunosuppression protocols to prevent BOS. METHODS: Our study approach was a multicenter, prospective, randomized (1:1) open-label superiority investigation of de novo tacrolimus vs cyclosporine, with both study arms given mycophenolate mofetil and prednisolone after lung transplantation. Cytolytic induction therapy was not employed. Patients were stratified at entry for cystic fibrosis. Primary outcome was incidence of BOS 3 years after transplant (intention-to-treat analysis). Secondary outcomes were survival and incidence of acute rejection, infection and other adverse events. RESULTS: Group demographic data were well matched: 110 of 124 tacrolimus vs 74 of 125 cyclosporine patients were treated per protocol (p < 0.01 by chi-square test). Cumulative incidence of BOS Grade ≥1 at 3 years was 11.6% (tacrolimus) vs 21.3% (cyclosporine) (cumulative incidence curves, p = 0.037 by Gray's test, pooled over strata). Univariate proportional sub-distribution hazards regression confirmed cyclosporine as a risk for BOS (HR 1.97, 95% CI 1.04 to 3.77, p = 0.039). Three-year cumulative incidence of acute rejection was 67.4% (tacrolimus) vs 74.9% (cyclosporine) (p = 0.118 by Gray's test). One- and 3-year survival rates were 84.6% and 78.7% (tacrolimus) vs 88.6% and 82.8% (cyclosporine) (p = 0.382 by log-rank test). Cumulative infection rates were similar (p = 0.91), but there was a trend toward new-onset renal failure with tacrolimus (p = 0.09). CONCLUSIONS: Compared with cyclosporine, de novo tacrolimus use was found to be associated with a significantly reduced risk for BOS Grade ≥1 at 3 years despite a similar rate of acute rejection. However, no survival advantage was detected.

Aganirsen Antisense Oligonucleotide Eye Drops Inhibit Keratitis-Induced Corneal Neovascularization and Reduce Need for Transplantation: The I-CAN Study.

OBJECTIVE: Eye drops of aganirsen, an antisense oligonucleotide preventing insulin receptor substrate-1 expression, inhibited corneal neovascularization in a previous dose-finding phase II study. We aimed to confirm these results in a phase III study and investigated a potential clinical benefit on visual acuity (VA), quality of life (QoL), and need for transplantation. DESIGN: Multicenter, double-masked, randomized, placebo-controlled phase III study. PARTICIPANTS: Analysis of 69 patients with keratitis-related progressive corneal neovascularization randomized to aganirsen (34 patients) or placebo (35 patients). Patients applied aganirsen eye drops (86 μg/day/eye) or placebo twice daily for 90 days and were followed up to day 180. MAIN OUTCOME MEASURES: The primary end point was VA. Secondary end points included area of pathologic corneal neovascularization, need for transplantation, risk of graft rejection, and QoL. RESULTS: Although no significant differences in VA scores between groups were observed, aganirsen significantly reduced the relative corneal neovascularization area after 90 days by 26.20% (P = 0.014). This improvement persisted after 180 days (26.67%, P = 0.012). Aganirsen tended to lower the transplantation need in the intent-to-treat (ITT) population at day 180 (P = 0.087). In patients with viral keratitis and central neovascularization, a significant reduction in transplantation need was achieved (P = 0.048). No significant differences between groups were observed in the risk of graft rejection. However, aganirsen tended to decrease this risk in patients with traumatic/viral keratitis (P = 0.162) at day 90. The QoL analyses revealed a significant improvement with aganirsen in composite and near activity subscores (P = 0.039 and 0.026, respectively) at day 90 in the per protocol population. Ocular and treatment-related treatment-emergent adverse events (TEAEs) were reported in a lower percentage with aganirsen compared with placebo. Only 3 serious TEAEs (2 with aganirsen and 1 with placebo) were considered treatment-related. CONCLUSIONS: This first phase III study on a topical inhibitor of corneal angiogenesis showed that aganirsen eye drops significantly inhibited corneal neovascularization in patients with keratitis. The need for transplantation was significantly reduced in patients with viral keratitis and central neovascularization. Topical application of aganirsen was safe and well tolerated.

Beyond the usual suspects: positive attitudes towards positive symptoms is associated with medication noncompliance in psychosis.

Antipsychotic medication represents the treatment of choice in psychosis according to clinical guidelines. Nevertheless, studies show that half to almost three-quarter of all patients discontinue medication with antipsychotics after some time, a fact which is traditionally ascribed to side-effects, mistrust against the clinician and poor illness insight. The present study investigated whether positive attitudes toward psychotic symptoms (ie, gain from illness) represent a further factor for medication noncompliance. An anonymous online survey was set up in order to prevent conservative response biases that likely emerge in a clinical setting. Following an iterative selection process, data from a total of 113 patients with a likely diagnosis of schizophrenia and a history of antipsychotic treatment were retained for the final analyses (80%). While side-effect profile and mistrust emerged as the most frequent reasons for drug discontinuation, 28% of the sample reported gain from illness (eg, missing voices, feeling of power) as a motive for noncompliance. At least every fourth patient reported the following reasons: stigma (31%), mistrust against the physician/therapist (31%), and rejection of medication in general (28%). Approximately every fifth patient had discontinued antipsychotic treatment because of forgetfulness. On average, patients provided 4 different explanations for noncompliance. Ambivalence toward symptoms and treatment should thoroughly be considered when planning treatment in psychosis. While antipsychotic medication represents the evidence-based cornerstone of the current treatment in schizophrenia, further research is needed on nonpharmacological interventions for noncompliant patients who are willing to undergo intervention but refuse pharmacotherapy.

Melan-A-specific cytotoxic T cells are associated with tumor regression and autoimmunity following treatment with anti-CTLA-4.

PURPOSE: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. EXPERIMENTAL DESIGN: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. RESULTS: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A-specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A-specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A-expressing tumor cells. CONCLUSIONS: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

Strength and limitations of regulatory T Cells for immunotherapy in transplantation.

In many experimental models, CD4+CD25+Foxp3+ regulatory T cells (nTreg) have been identifi ed as key players in promoting peripheral transplantation (Tx) tolerance. We have been focusing on therapies based on antigen-specifi c nTreg that can control effector T cells (Teff) and prevent allograft rejection. The use of nTreg in immunotherapeutic protocols for solid organ Tx is however limited by their overall low numbers as well as the low precursor frequency of alloantigen cross-reactive nTreg expected to be found in a normal individual. Moreover, although we previously described robust protocols to generate and expand antigen-specifi c nTreg in vitro, the process requires careful selection of highly pure nTreg and cumbersome ex-vivo manipulations, rendering this strategy not easily applicable in clinical solid organ Tx. In this study, we aimed to expand Treg directly in vivo and determine their suppressive function, effi cacy and stability in promoting donor-specifi c tolerance in a stringent murine Tx model. Our data suggest that IL-2-based therapies lead to a signifi cant increase of Treg in vivo. The expanded Treg suppressed Teff proliferation (albeit slightly less effi ciently than nTreg isolated from control mice) and allowed prolonged graft survival of major MHC-mismatched skin grafts in wild-type non-lymphopenic recipients. The expanded Treg alone were however not suffi cient to induce tolerance in stringent experimental conditions. Rapamycin reduced the frequency of Teff but did not impede expansion of Treg. Pro-infl ammatory stimuli hindered the expansion of Treg and resulted in an increase in the frequency of CD4+IFN-γ+ and CD4+IL17+ T cells. We propose that IL-2-based treatments would be an effi cient method for expanding functional Treg in vivo without affecting other immune cell populations, thereby favorably shifting the pool of alloreactive T cells towards regulation in response to an allograft. However, we also highlight some potential limitations of Treg expansion such as concomitant infl ammatory events.

Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.