Immunologic Unresponsiveness

Tolerance induction by megadose stem cell transplants: synergism between SCA-1⁺Lin⁻ cells and nonalloreactive T cells

Allogeneic HSCT with graft versus tumor (GVT) effect is an accepted therapeutic option in pediatric solid tumors. Due to donor availability and logistic reasons, unmanipulated family haploidentical transplants are becoming increasingly used in patients lacking an HLA identical family donor, or a well matched unrelated donor. Recently nonmyeloablative, haplo-identical T-cell replete bone marrow transplantation using high-dose cyclophosphamide post-HSCT (PTCy) to control GVHD and prevent graft rejection by inducing bi-directional tolerance was described. The objective of this report is an analysis of case of a child treated for relapsed neuroblastoma with haploidentical HSCT with PTCy. In this case following therapeutic modalities were combined: haploidentical HSCT in pediatric solid tumor relapsing after autologous HSCT, non-myeloablative haploidentical HSCT with unmanipulated T-repleted graft, and post-transplant use of high-dose cyclophosphamide as GVHD prophylaxis. This strategy was safe and efficient, as we observed low toxicity, relatively fast hematological engraftment, and hyperacute GVHD followed by mild GVHD. Patient stayed in remission for 12 months. Based on published data, it seems possible, that PTCy selectively depletes T cells that react against host allo-antigens, yet preserves tumor-specific and pathogen reactive T cells.

Successful allogeneic hematopoietic stem cell transplantation (HSCT) and solid organ transplantation require development of a degree of immune tolerance against allogeneic antigens. T lymphocytes play a critical role in allograft rejection, graft failure, and graft-versus-host disease (GVHD). T-cell tolerance occurs by two different mechanisms: (1) depletion of self-reactive T cells during their maturation in the thymus (central tolerance), and (2) suppression/elimination of self-reactive mature T cells in the periphery (peripheral tolerance). Induction of transplant tolerance improves transplantation outcomes. Adoptive immunotherapy with immune suppressor cells including regulatory T cells, natural killer (NK)-T cells, veto cells, and facilitating cells are promising therapies for modulation of immune tolerance. Achieving mixed chimerism with the combination of thymic irradiation and T-cell–depleting antibodies, costimulatory molecule blockade with/without inhibitory signal activation, and elimination of alloreactive T cells with varying methods including pre- or post-transplant cyclophosphamide administration appear to be effective in inducing transplant tolerance.

Induction of donor type chimerism in mildly prepared hosts without graft-versus-host disease (GvHD) is a most desirable goal in bone morrow transplantation. We have recently demonstrated in a mouse model that donor veto cytotoxic T lymphocytes (CTLs) can facilitate the induction of donor type chimerism in sublethally irradiated recipients without causing GvHD if they are effectively depleted of alloreactivity against host cells by means of stimulation against a third party. We extend this approach to human cells, by preparing CTLs in two major steps: primary culture in the absence of interleukin 2, leading to death by neglect of antihost clones, and addition of interleukin 2 and subsequent dilution of antihost clones as a consequence of the expansion of the anti-third-party clones. CTLs prepared in this way specifically suppress host cytotoxic T cells directed against antigens of the donor, but not against fourth-party antigens, as demonstrated in a standard ⁵¹Cr release assay. We conclude that human anti-third-party CTLs afford a new source of veto cells that are depleted of potential graft-versus-host-reactive clones. The cells generated by this approach could potentially be used to facilitate engraftment of allogeneic hematopoietic stem cells.

Previous studies suggest that cells within the CD34⁺ hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34⁺ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti–IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34⁺ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34⁺ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34⁺ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti–tumor necrosis factor-α (anti–TNF-α) and not by anti–transforming growth factor-β (anti–TGF-β), suggesting a potential role for TNF-α in the regulatory activity of CD34⁺ cells.

Type 1 diabetes is a systemic autoimmune disease that can be cured by transplantation of hematopoietic stem cells (HSCs) from disease-resistant donors. Nonobese diabetic (NOD) mice have a number of features that distinguish them as bone marrow transplant recipients that must be understood prior to the clinical application of chimerism to induce tolerance. In the present studies, we characterized NOD HSCs, comparing their engraftment characteristics to HSCs from disease-resistant strains. Strikingly, NOD HSCs are significantly enhanced in engraftment potential compared with HSCs from disease-resistant donors. Unlike HSCs from disease-resistant strains, they do not require graft-facilitating cells to engraft in allogeneic recipients. Additionally, they exhibit a competitive advantage when coadministered with increasing numbers of syngeneic HSCs, produce significantly more spleen colony-forming units (CFU-Ss) in vivo in allogeneic recipients, and more granulocyte macrophage–colony-forming units (CFU-GMs) in vitro compared with HSCs from disease-resistant controls. NOD HSCs also exhibit significantly enhanced chemotaxis to a stromal cell–derived factor 1 (SDF-1) gradient and adhere significantly better on primary stroma. This enhanced engraftment potential maps to the insulin-dependent diabetes locus 9 (Idd9) locus, and as such the tumor necrosis factor (TNF) receptor family as well as ski/sno genes may be involved in the mechanism underlying the autonomy of NOD HSCs. These findings may have important implications to understand the evolution of autoimmune disease and impact on potential strategies for cure.

Allogeneic reduced-intensity stem cell transplantation (RISCT) decreases regimen-associated morbidity and mortality, but it is unfortunately still constrained by the same immune T-cell reactions that limit myeloablative transplantation, including graft rejection, graft-versus-host disease (GVHD), and suboptimal graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effects. Graft rejection is mediated by host T cells, whereas GVHD and GVL/GVT effects are initiated by donor T cells, and to this extent, future advances in RISCT will likely benefit from an ability to modulate both donor and host T-cell immunity. As a step in this direction, we have developed a RISCT approach that first involves chemotherapy-induced host T-cell ablation, and second involves administration of allogeneic inocula enriched for donor CD4⁺ Th2 and CD8⁺ Tc2 T-cell subsets that in murine studies mediate reduced GVHD. In a pilot clinical trial, “immunoablative” RISCT with human leukocyte antigen (HLA)-matched related allografts resulted in rapid and complete donor chimerism and GVL effects early post-transplant, with GVHD being the primary toxicity. Using this immunoablative RISCT approach, we are now evaluating the feasibility and safety of augmenting allografts with additional donor CD4⁺ Th2 cells that are generated in vitro via CD3/CD28 costimulation in the presence of interleukin (IL)-4. We review the biology of host and donor T-cell immunity during allogeneic RISCT and discuss the strategies of host immunoablation and donor Th2 and Tc2 cell therapy as potential means to improve the clinical results in RISCT.