Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation and Adoptive Immunotherapy

The immune system is itself adversely affected by graft-versus-host disease. The varying period of prolonged immunodeficiency after allogeneic hematopoietic stem cell transplantation (HSCT) results in significant morbidity and mortality from bacterial, viral, and fungal infection, EBV-induced lymphoproliferative disease and associated disorders. Delays in immune reconstitution can also contribute to the relapse of malignant disease when allo HSCT is used as a therapy for cancer.

Consequently, improved strategies to enhance post-transplant immune reconstitution could decrease the incidence of fatal infectious complications and significantly improve the overall survival after allo HSCT.

Our laboratory uses murine models for MHC-matched and mismatched allo HSCT with young (3-month-old) and middle-aged (9-month-old) recipients to study immune reconstitution after HSCT. Our results show that T cell reconstitution, in contrast to myeloid, erythroid, and B cell recovery, was not complete at 12 weeks post allo HSCT, even in young recipients without graft-versus-host disease (GVHD), thus suggesting a focus on T cell reconstitution.

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Post-transplant T-cell reconstitution involves thymus-dependent and independent T cell generation and the expansion of mature T cells in the periphery. The prolonged T cell deficiency after transplant continues even after quantitative regeneration has taken place, suggesting that a T cell dysfunction occurs in the post-transplant period, which is at present still poorly understood.

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Thymic output is dependent on MHC differences, the occurrence of GVHD and the age of recipients. We observed that most donor T cells during reconstitution had a memory T cell phenotype. However, we have demonstrated, by in vivo BrdU labeling, that a subpopulation of these cells in time will revert to naïve T cell phenotype.

As an immune reconstitution strategy, we have found that administration of Interleukin-7 (IL-7) to allo HSCT recipients enhances thymopoiesis and peripheral B and T cell numbers independent of MHC differences or age of the recipient, as measured by BrdU pulse-chase, cell cycle, and Annexin-V analysis.

IL-7 is a potent lymphopoietic growth factor in allo HSCT recipients and affects thymopoiesis and proliferation, expression of the anti-apoptotic Bcl-2 protein, and apoptosis of peripheral T cells without aggravating GVHD.

Peripheral CD8+ T cells in allo HSCT recipients contain significantly higher levels of apoptotic cells than in non-transplanted control mice. Peripheral CD4+ T cells in middle-aged (but not young) allo HSCT recipients also have increased numbers of apoptotic cells. We found that apoptosis in peripheral CD4+ T in allo HSCT recipients was associated with a decrease in intracellular expression and intensity of Bcl-2. IL-7 administration could enhance Bcl-2 levels in CD4+ T cells in middle-aged allo HSCT recipients and decrease the percentage of apoptotic cells.

In contrast, IL-7 had no effect on the percentage of apoptotic CD8+ T cells. Upon cell cycle analysis, we found an increase of peripheral T cells in S phase after IL-7 administration to allo HSCT recipients. Finally, we observed no effect of IL-7 administration on the development of GVHD or graft-versus-tumor (GVT) activity. Further analysis by CFSE labeling demonstrated that alloreactive T cells downregulate their IL-7 receptors upon activation, which suggest that they become insensitive to IL-7 stimulation.

Apart from general immune reconstitution, we are also now studying immune reconstitution in the gut. As the gut is exposed on a regular basis to a variety of foreign antigens, including pathogens, immune surveillance of the gut is important. We are establishing the baseline kinetics of gut immune reconstitution after allogeneic MHC-matched and MHC-mismatched HSCT with and without GVHD, and characterizing the phenotypes of associated hematopoietic populations. We are also analyzing the effects of immunostimulatory cytokine administration (IL-7 and IL-15) on the kinetics of gut immune reconstitution.

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Finally, we are also analyzing the effects of combination immune reconstitution therapies, using IGF-1, IL-7, IL-15, and KGF.

In related studies, we are analyzing the effects of sex steroid inhibition (e.g., via GnRH agonists) and peripheral T cell apoptosis in allo HSCT recipients on immune reconstitution.

Yet another approach to immune reconstitution involves the infusion of cultured thymic precursors into HSCT recipients.

Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a novel OP9-DL1 co-culture system, consisting of OP9 bone marrow stromal cells expressing Delta-like 1 Notch ligand (which binds to the Notch 1 receptor) and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand).

We have determined the effects of ex vivo-generated T cell precursors on T cell reconstitution after allo HSCT. By selecting for Lin-, Sca-1hi, c-kithi HSC from donor bone marrow and culturing these on a monolayer of OP9-DL1 cells in the presence of growth factors, these HSC can be expanded 850-5000-fold and consist of >95 percent CD4-CD8-double negative (DN) T cell precursors after 16 to 28 days of culture.

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Zuniga-Pflucker JC. T cell development made simple. Nat Rev Immunol. 2004;4:67-72.

Cells in this in vitro culture system can progress from the DN1 stage through the DN4 stage as evidenced by CD44/CD25 staining.

Transfer of these T cell precursors, along with the allograft, to lethally irradiated allogeneic hematopoietic stem cell transplantation (HSCT) recipients resulted in increased thymic cellularity and chimerism, as well as enhanced peripheral T and NK cell reconstitution. Combination of T cell precursor administration and treatment with keratinocyte growth factor (KGF) had additive effects on thymic reconstitution. In thymectomized recipients, adoptively transferred T cell precursors enhanced extrathymic T cell development. OP9-DL1 derived T cell precursors gave rise to host-tolerant CD4 and CD8 populations with normal T cell receptor repertoires, cytokine secretion, and proliferative responses to antigen. Administration of OP9-DL1 derived T cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD). Adoptive transfer of in vitro generated T cell precursors therefore represents a promising novel strategy to enhance T cell reconstitution and GVT activity after T cell-depleted allogeneic HSCT.

We also evaluated if allogeneic T cell precursors can be safely used for adoptive transfer across MHC barriers in the absence of allogeneic HSCs to overcome radiation injury, enhance T cell function and improve anti-tumor activity in immunosuppressed recipients. We found that that adoptively transferred allogeneic T cell precursors develop into host-MHC restricted T cells characterized by host tolerance and selection of a functional TCR repertoire even in a fully mismatched thymic epithelial MHC environment. This strategy overcomes important limitations of conventional adoptive T cell therapies: rejection, alloreactivity and impaired antigen recognition due to restriction to MHC disparate from the one expressed on antigen-presenting cells (APCs).

Furthermore, we demonstrated the feasibility of genetic engineering of antigen-specific T cell precursors, by transducing them to express a chimeric antigen receptor (CAR). Adoptive transfer of CAR-expressing T cell precursors resulted in the in vivo generation of high numbers of appropriately selected T cells expressing the CAR, which were capable of significantly enhanced anti-tumor activity (compared with CAR-negative T cell precursors) against a CAR-sensitive tumor, without any undesirable auto/alloreactivity.

Adoptive transfer of allogeneic and genetically enhanced T cell precursors therefore represents a promising novel strategy for targeted 'off-the-shelf' immunotherapy in immunosuppressed patients.

As with most of our other studies, we employ bioluminescence imaging and other techniques to monitor the effects of therapeutic strategies on post-transplant immune reconstitution. We also continue to evaluate the success of immune reconstitution strategies by challenging allo HSCT recipients with tumor or microbial agents and assessing the anti-microbial and anti-tumor activity after our therapies, as seen above.