Induction of antigen-specific tolerance through hematopoietic stem cell-mediated gene therapy: the future for therapy of autoimmune disease?

Based on the principle that immune ablation followed by HSC-mediated recovery purges disease-causing leukocytes to interrupt autoimmune disease progression, hematopoietic stem cell transplantation (HSCT) has been increasingly used as a treatment for severe autoimmune diseases. Despite clinically-relevant outcomes, HSCT is associated with serious iatrogenic risks and is suitable only for the most serious and intractable diseases. A further limitation of autologous HSCT is that relapse rates can be high, suggesting disease-causing leukocytes are incompletely purged or the environmental and genetic determinants that drive disease remain active. Incorporation of antigen-specific tolerance approaches that synergise with autologous HSCT could reduce or prevent relapse. Further, by reducing the requirement for highly toxic immune-ablation and instead relying on antigen-specific tolerance, the clinical utility of HSCT could be significantly diversified. Substantial progress has been made exploring HSCT-mediated induction of antigen-specific tolerance in animal models but studies have focussed on primarily on prevention of autoimmune diseases. However, as diagnosis of autoimmune disease is often not made until autoimmune disease is well developed and populations of autoantigen-specific pathogenic effector and memory T cells have become well established, immunotherapies must be developed to address effector and memory T-cell responses which have traditionally been considered the key impediment to immunotherapy. Here, focusing on T-cell mediated autoimmune diseases we review progress made in antigen-specific immunotherapy using HSCT-mediated approaches, induction of tolerance in effector and memory T cells and the challenges for progression and clinical application of antigen-specific ‘tolerogenic’ HSCT therapy

Facilitating Engraftment After MHC-Matched, Allogeneic BMT by IL-2 / Anti IL-2 Complex Treatment Requires Targeting CD25 On, and Activation in Situ of, Residual CD4 Tregs

Abstract Abstract 66 We recently demonstrated that administration of IL-2 complexed to the anti IL-2 antibody JES6-A12 (IAC) induced stable chimerism and engraftment of donor HSCT (BBMT 15:785, 2009). Based on suppression of anti-donor MiHA-specific host T cells, it was concluded that IAC administration enhanced chimerism by suppression of HVG. We proposed that in situ manipulation of host Tregs was crucial to facilitating engraftment and establishing tolerance in this model and hypothesized that the enhanced chimerism induced by this strategy was a direct result of host Treg activation, expansion and function following engagement of the IL-2 receptor CD25. To directly test this hypothesis, B6 CD4−/− (H-2b, Ly9.1−) mice were infused with highly enriched CD4 cells from B6-WT or B6-IL-2Rβ−/− mice deficient in CD25 expression and 4 days later conditioned with 5.5 Gy TBI. One day later, these mice were transplanted with MHC-matched, MiHA-mismatched 4 × 106 BALB.B (H-2b, Ly9.1+) TCD-BM. At days +3 and +5, all recipients were administered IAC and subsequently assessed for peripheral donor chimerism. By 2 weeks post-HCT, untreated control mice had increased circulating levels of CD8TETRAMER+ T cells (representing specific host anti-donor H60 MiHA reactive T cells) vs. IAC-treated recipients. Three months post-HCT, CD4−/− recipients of WT but not IL-2Rβ−/− CD4 cells were chimeric as evidenced by high levels of circulating donor cells (60% vs. <1%). These findings demonstrate that IAC effects require host CD25+ Treg cells and we propose that facilitation of engraftment by this strategy was a direct result of Treg cell activation and expansion following engagement of IAC with CD25. To assess Treg activation, we examined these cells in our standard BALB.B à B6 HSCT model. Tregs isolated 7 days post-HCT from IAC-treated but not untreated recipients expressed readily demonstrable levels of pStat-5a expression (∼2X increase in IAC-treated vs. PBS controls). Moreover, culture of the former Tregs in the presence of rmIL-2 illustrated their heightened sensitivity to activation by this cytokine as virtually all Tregs from IAC-treated animals exhibited high levels of pStat-5a expression (3.2 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.6) compared to control mice (0.4 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.5). Following this activation, increased numbers of host CD4+ FoxP3+ Tregs were readily identified in the PB and spleen of IAC vs. PBS-treated recipients. We next directly examined functional capacity of residual host Tregs exposed to IAC post-5.5Gy TBI and-transplant. Eight days post-HSCT, host Tregs (0 – 25 × 103/well) were isolated and highly enriched populations assessed for suppression of TCONV in anti-CD3 activation assays. Tregs from IAC-treated recipients efficiently mediated suppression at least equivalent to that by normal, untreated Tregs in these assays. Additionally, these IAC treated residual B6 Tregs also effectively inhibited allogeneic MLR responses by B6 CD4+ CD25− responder cells. Therefore, the capacity of host Tregs to respond to IAC activation signals and suppress T cell activation remained intact following conditioning and HSCT. We conclude that host Treg cell activation/expansion is central to the suppression of host effector cell responses to donor hematopoietic antigens leading to the inhibition of HVG following IAC administration resulting in enhanced engraftment. Disclosures: No relevant conflicts of interest to declare

Targeting Treg Cells In Situ: Emerging Expansion Strategies for (CD4 +CD25 +) Regulatory T Cells

Recognition of the ability of CD4 +FoxP3 + T cells (Treg) to influence the generation of peripheral immune responses has engendered enthusiasm for the development of strategies utilizing these cells to regulate immune responses in clinically important settings including transplantation, autoimmunity and cancer. A number of studies have reported effective regulation utilizing ex-vivo expansion approaches and subsequent transfer of Treg populations in experimental models. This commentary discusses recently emerging strategies to activate and expand Treg cells in situ which include antibodies, antigen presenting cells and the use of IL2 / anti-IL2 antibody complex. The development of reagents which can stimulate and / or remove Treg cells in situ would represent an important advance towards facilitating new opportunities to harness this compartment for the augmentation of ‘wanted’ or suppression of ‘unwanted’ immune responses. Simultaneous targeting of multiple molecules on Treg cells may ultimately enable more effective control of this regulatory sector

Identification of a subpopulation of macrophages in mammary tumor-bearing mice that are neither M1 nor M2 and are less differentiated

Systemic and local immune deficiency is associated with cancer, and the role of M2 tumor-associated macrophages in this phenomenon is well recognized. However, the immune status of macrophages from peripheral compartments in tumor hosts is unclear. Peritoneal macrophages (PEM) are derived from circulating monocytes and recruited to the peritoneal cavity where they differentiate into macrophages. We have previously shown that PEMs from mice bearing D1-DMBA-3 mammary tumors (T-PEM) are deficient in inflammatory functions and that this impairment is associated with diminished expression of transcription factors nuclear factor kappaB and CAAT/enhancer-binding protein. We now provide evidence that T-PEMs display neither M1 nor M2 phenotypes, yet exhibit deficiencies in the expression of several inflammatory cytokines and various proinflammatory signaling pathways. Moreover, due to nuclear factor kappaB down-regulation, increased apoptosis was observed in T-PEMs. We report for the first time that macrophage depletion is associated with increased macrophage progenitors in bone marrow. Furthermore, T-PEMs have a lower expression of macrophage differentiation markers F4/80, CD68, CD115, and CD11b, whereas Gr-1 is up-regulated. Our results suggest that T-PEMs are less differentiated and represent a newly derived population from blood monocytes. Lastly, we show that transforming growth factor-beta and prostaglandin E(2), two immunosuppressive tumor-derived factors, may be involved in this phenomenon