Key Aspects of the Immunobiology of Haploidentical Hematopoietic Cell Transplantation

Abstract

Hematopoietic stem cell transplantation from a haploidentical donor is increasingly used and has become a standard donor option for patients lacking an appropriately matched sibling or unrelated donor. Historically, prohibitive immunological barriers resulting from the high degree of HLA-mismatch included graft-vs.-host disease (GVHD) and graft failure. These were overcome with increasingly sophisticated strategies to manipulate the sensitive balance between donor and recipient immune cells. Three different approaches are currently in clinical use: (a) ex vivo T-cell depletion resulting in grafts with defined immune cell content (b) extensive immunosuppression with a T-cell replete graft consisting of G-CSF primed bone marrow and PBSC (GIAC) (c) T-cell replete grafts with post-transplant cyclophosphamide (PTCy). Intriguing studies have recently elucidated the immunologic mechanisms by which PTCy prevents GVHD. Each approach uniquely affects post-transplant immune reconstitution which is critical for the control of post-transplant infections and relapse. NK-cells play a key role in haplo-HCT since they do not mediate GVHD but can successfully mediate a graft-vs.-leukemia effect. This effect is in part regulated by KIR receptors that inhibit NK cell cytotoxic function when binding to the appropriate HLA-class I ligands. In the context of an HLA-class I mismatch in haplo-HCT, lack of inhibition can contribute to NK-cell alloreactivity leading to enhanced anti-leukemic effect. Emerging work reveals immune evasion phenomena such as copy-neutral loss of heterozygosity of the incompatible HLA alleles as one of the major mechanisms of relapse. Relapse and infectious complications remain the leading causes impacting overall survival and are central to scientific advances seeking to improve haplo-HCT. Given that haploidentical donors can typically be readily approached to collect additional stem- or immune cells for the recipient, haplo-HCT represents a unique platform for cell- and immune-based therapies aimed at further reducing relapse and infections. The rapid advancements in our understanding of the immunobiology of haplo-HCT are therefore poised to lead to iterative innovations resulting in further improvement of outcomes with this compelling transplant modality.

Keywords: NK-cells; graft-vs.-leukemia; haploidentical; immunobiology; stem cell transplantation.

Figure 1

HLA-matching in Haplo-HCT. (A) Distribution of HLA alleles on chromosome 6. All HLA alleles exist on the short arm of the chromosome, specifically 6p21.3. The classical HLA classification system is used clinically for matching donors and recipients in the transplant setting. HLA class I alleles -A, -B, and -C are expressed on all nucleated cells and display antigen to CD8⁺ T-cells, while HLA Class II alleles -DR, -DQ, -DP are expressed on antigen-presenting cells and initiate a response by CD4⁺ T-cells. Not shown are the non-classical HLA Class I alleles -E, -F, -G, -H, -J that are also present on the same chromosome arm. (B) A representative inheritance pattern of HLA alleles is demonstrated. For a patient with HLA allele distribution b and d as shown in the middle, each sibling has a 25% chance of being a full match based on inheritance of the same maternal (b) and paternal (d) alleles as the patient. Each sibling has a 50% chance of being a haploidentical match by virtue of having inherited one identical allele (b) from the parents. The likelihood of having inherited neither of the parental alleles that were inherited by the patient is 25% (complete HLA-mismatch).

Figure 2

Immunological balance determines outcomes after haplo-HCT. The graft contains CD34⁺ and CD34⁻ hematopoietic cells. CD34⁺ progenitor and stem cells are required for engraftment and reconstitution of the bone marrow after transplantation into the host. T cells in the graft facilitate neutrophil engraftment, immune reconstitution, post-transplant infectious immunity and exert GVL effect (Right). However, without an ex vivo (T cell depletion or CD34 positive selection) or in vivo (ATG or Campath) T cell depletion strategy, they mediate prohibitively severe GVHD (Right). In contrast, extensive T cell depletion from the graft results in an immunologic imbalance between residual host and donor T cells favoring graft rejection (Left). Extensive T cell depletion of the graft also results in slow immune constitution, infections and poor GVL control. To achieve an optimal immunologic balance, novel graft manipulation approaches selectively deplete T cells involved in GVHD (CD45RA⁺ T cell and αβ- T cell depletion strategies), while maintaining beneficial immune cells such as NK cells and γδT cells in the graft.

Figure 3

Frequently used haplo-HCT regimens. (A) Non-myeloablative (NMA) conditioning with administration of post-transplant cyclophosphamide (PTCy) as part of the Hopkins protocol for haplo-related donor HCT uses cyclophosphamide 50 mg/kg/day on days +3 and +4 and additional GVHD prophylaxis with oral MMF and tacrolimus (Tacro) starting on day +5. (B) Myeloablative conditioning (MAC) protocol with administration of post-transplant cyclophosphamide 50 mg/kg/day given on days +3 and +4 and additional GVHD prophylaxis with oral MMF and tacrolimus starting on day +5. (C) GIAC haplo-HCT protocol using a combination of G-CSF primed bone marrow (BM) and peripheral blood stem cells (PBSC) administered after a conditioning regimen including ATG on days −5 to −2. GVHD prophylaxis includes short-course Methotrexate in addition to MMF and cyclosporine (CSA).

Figure 4

Comparison of the three major haplo-HCT platforms. (A) Four different ex vivo T-cell depletion protocols are shown, with the resulting cell composition in the graft. CD34-positive selection preferentially isolates the hematopoietic stem and progenitor fraction required for engraftment with minimal immune cell content (top panel). Depletion of CD3⁺ T-cells results in a graft composed predominantly of CD34⁺ and NK cells (2nd panel). Depletion of αβ-T cells depletes T cells involved in mediating GVHD but retains beneficial immune cells such as NK cells and γδ- T cells in the graft. CD45RA-Depletion removes naïve T cells including cells responsible for alloreactivity and GVHD, while retaining memory T cells including cells vital for immunity against infections (3rd panel). Additional immunosuppression (IS) and/or infusion of T-cell subsets may be employed post-transplant to optimize engraftment (3rd panel). (B) Representation of the GIAC protocol indicating a G-CSF primed bone marrow (BM) and peripheral blood stem cell (PBSC) graft, with ATG targeting T-cells derived from both the donor and recipient. GVHD prophylaxis with methotrexate, a calcineurin inhibitor (CNI), and MMF targets residual T cells (3rd panel). (C) Post-transplant cyclophosphamide (PTCy) functionally impairs actively proliferating recipient and graft derived T cells while favoring Treg recovery (color of cells corresponds to Figure 2; Tregs are depicted in bright yellow).

Figure 5

NK-cell receptor repertoire. (A) NK-cell activity is mediated by a balance of activating and inhibitory signaling. Key activating receptors and their corresponding ligands are listed in the green table, while inhibitory receptors are displayed in the red table. (B) KIR genes are highly polymorphic and organized in centromeric and telomeric motifs with structural variation that creates multiple gene content haplotypes. Group A haplotype motifs are characterized by fewer genes and predominantly those encoding for inhibitory KIRs. In contrast, Group B haplotype motifs are enriched for activating KIRs. (C) Centromeric and telomeric motifs are paired together to generate either a KIR A haplotype (composed of centromeric and telomeric A motifs) or a KIR B haplotype (containing at least one centromeric or telomeric B motif). Representative KIR haplotypes by recombination are shown here. Prominent linkage disequilibrium has been noted within the centromeric and telomeric motifs but not between them, suggesting that pairing occurs by recombination between the centromeric and telomeric regions.

Figure 6

NK cell alloreactivity in haplo-HCT is demonstrated via the different models of receptor-ligand mismatch. (A) The donor-derived NK cell is licensed when its KIR2DL1 receptor had been engaged by expression of its cognate C2 ligand in the donor environment. Upon infusion of the licensed NK cell into the recipient, a leukemia cell expressing the C2 ligand will not activate the NK cell due to a receptor-ligand match. (B) A receptor-ligand mismatch occurs when the donor-derived NK cell is licensed, but the recipient does not express the C2 ligand (missing ligand). Provided that it is further driven by stimulation through activating receptors, this results in activation of the licensed donor NK cell upon infusion into the recipient, leading to a graft-vs.-leukemia effect. (C) If the donor does not express the appropriate class I ligand for its KIR receptor (HLA and KIR segregate independently), the donor NK cell is unlicensed. In this case, donor NK cells are accustomed to a missing ligand. They may be activated when encountering strong activating signals (like activating cytokines) or be further inhibited when encountering the inhibitory ligand in the recipient. (D) Licensing of the NK cell for the C1 ligand occurs in the donor. Upon transplant into the host, the missing C1 ligand coupled with binding of the activating ligand with the activating receptor on the NK cell results in alloreactivity. Binding of the activating KIR receptor KIR2DS1 to the C2 ligand on the target leukemia cell enhances NK cell alloreactivity.

Figure 7

Immune reconstitution with different haploidentical transplant platforms. Only subsets which have been characterized by published primary data for each platform are included in this figure (e.g., data on dendritic cell reconstitution has only been described for the GIAC approach). Cells are depicted at the approximate time-point of reaching the lower range of normal. T-cell depleted (TCD) haploidentical transplant is associated with early recovery of neutrophils, monocytes, immature NK cells and rapid NK cell maturation which takes 6–8 weeks (top panel). Additionally, αβ-T cell/CD19 depletion is associated with early detection of γδT cells and mature NK cells that are infused with the graft infusion (2nd panel from the top). B cell recovery is delayed with CD19 depletion relative to CD34⁺ selection. T-cell replete (TCR) haploidentical transplant performed with post-transplant cyclophosphamide (PTCy) and GIAC protocols is associated with early reconstitution of immature NK cells (3rd panel from the top). It is also associated with earlier reconstitution of CD8⁺ T-cells than TCD protocols. The GIAC protocol is associated with delayed dendritic cell recovery (bottom panel). This figure was created using BioRender.com.

Figure 8

Mechanisms of relapse post haploidentical HCT. Late relapse after haploidentical allogeneic transplantation can be driven by a number of immunologic mechanisms as shown. Under the immune pressure of graft-vs.-leukemia (GVL) via HLA mismatch in a haploidentical environment, loss of heterozygosity for the mismatched HLA allele is a mechanism of escape from immune surveillance and relapse (1). Another mechanism involves transcriptional silencing of HLA class II molecule, thereby reducing T-cell mediated GVL. This effect can be partially reversed in the presence of immunomodulatory molecules such as IFN-y or the epigenetic regulator 5-azacitidine (5-aza) (2). Modification of the tumor microenvironment via suppression of release of mediators that promote GVL is another mechanism used by relapsing leukemia cells, which may be partially reversed via administration of IL-15 agonists and NK cell infusions that promote the secretion of proinflammatory cytokines (3). An additional common mechanism of relapse involves the emergence of T-cell exhaustion with associated upregulation of PD-L1 and other inhibitory receptors. The latter may be reversed through administration of checkpoint inhibitors (4). Blue arrows indicate possible therapeutic strategies to overcome the different mechanisms of immune evasion. MRD, minimal residual disease; LOH, loss of heterozygosity; Chr, chromosome; DLI, donor lymphocyte infusion. This Figure was created using BioRender.com.

Figure 9

Haplo-HCT offers a platform for post-transplant immune therapies to prevent and treat relapse. In the context of a haploidentical transplant, there are several options to administer cellular therapies in order to address relapse, infection and GVHD either pre-emptively or therapeutically. In the event of a relapse, enhancing GVL effect using cellular therapy that either relies on the haploidentical mismatch between donor and recipient or gene-modified donor immune effector cells T cells are potential options. Donors haploidentical to the recipient may also readily serve as a source of cells for the production of CAR-T or CAR-NK. In the event of significant viral infection post relapse, administration of antiviral cytotoxic T-cells may promote viral clearance without increasing the risk of GVHD. Finally, Treg infusions may be utilized to treat GVHD. DLI, Donor lymphocyte infusion; CIML NK cells, Cytokine-induced memory-like NK cells; TCR, T-cell receptor; CAR, Chimeric antigen receptor; CTLs, Cytotoxic T-lymphocytes.