Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation.

Because of the expression of inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I allotypes, a person's natural killer (NK) cells will not recognize and will, therefore, kill cells from individuals lacking his/her KIR epitopes. This study investigated the role of NK cell alloreactivity in human HLA haplotype-mismatched hematopoietic stem cell transplantation and, specifically, the role of the three major NK specificities, ie, those for HLA-C group 1, HLA-C group 2, and HLA-Bw4 alleles. In 20 of 60 donor-recipient pairs, KIR epitope incompatibility and functional analyses of donor NK cell clones predicted donor NK cells could cause graft-versus-host (GVH)/graft-versus-leukemia (GVL) reactions. NK cell clones of donor origin were obtained from transplanted recipients and tested for lysis of recipient's cryopreserved pretransplant lymphocytes. Despite the absence of GVH disease, we detected high frequencies of NK clones which killed recipient's target cells. Lysis followed the rules of NK cell alloreactivity, being blocked only by the MHC class I KIR epitope which was missing in the recipient. The alloreactive NK clones also killed the allogeneic leukemia. Transplants from these KIR epitope incompatible donors had higher engraftment rates. Therefore, a GVL effector and engraftment facilitating mechanism, which is independent of T-cell-mediated GVH reactions, may be operational in HLA mismatched hematopoietic cell transplants.     

T-cell alloreactivity dominates natural killer cell alloreactivity in minimally T-cell-depleted HLA-non-identical paediatric bone marrow transplantation

Natural killer (NK) cell alloreactivity resulting from killer immunoglobulin-like receptor (KIR) ligand incompatibility improves outcomes in patients receiving extensively T-cell-depleted bone marrow (BM) grafts. Patients with KIR ligand incompatibility are at risk for donor T-cell alloreactivity. We investigated the relative significance of NK-cell and T-cell alloreactivity in 105 paediatric patients who received a minimally T-cell-depleted human leucocyte antigen-non-identical BM transplantation. Donor NK-cell incompatibility did not improve patient outcome [engraftment, graft-versus-host disease (GVHD), relapse or overall survival]. In contrast, donor T-cell incompatibility was a risk factor for acute GVHD, chronic GVHD and death. Thus, T-cell alloreactivity dominated that of NK cells in minimally T-cell-depleted grafts.     

Role of HLA in hematopoietic SCT

HLA disparity between hematopoietic stem cell (HSC) donor and recipient triggers T-cell and NK-cell allorecognition, and induces the GVHD, GVL effect and/or may cause an engraftment failure. This review will cover the scope of human genomic variation, the methods of HLA typing and interpretation of high-resolution HLA results. We describe the main subsets of related and unrelated HSC donors and outline the main aspects of HLA disparity and their effect on the outcome of the patients after allogeneic HSC transplantation (HSCT). The HLA match between HSCT donor and recipient is crucial, but for many patients a perfectly matched donor is not available. The HSCT from the alternative mismatched donor with one allele/antigen mismatch (9/10) can be as beneficial as a HSCT from a fully matched donor, especially in younger patients. For the remaining patients, the donors with permissive mismatches may be the option. The permissiveness depends not only on the potential adverse effect of the HLA mismatches, but also on the urgency of the transplantation, the desirable GVL effect and the potential efficacy of the alternative therapy available for the patient.     

Alloreactive natural killer cells in mismatched hematopoietic stem cell transplantation

Natural killer (NK) cell-mediated, donor-vs.-recipient alloresponses occur following transplantation of human leukocyte antigen (HLA) haplotype-mismatched hematopoietic stem cells (HSCs). NK cell alloreactivity reduced the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-vs.-host disease (GvHD). NK cells are primed to kill by several activating receptors. NK killing of autologous cells is prevented because NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) MHC class I alleles. As KIRs are clonally distributed, the NK population in any individual is constituted of a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and in some cases, functional assessment of donor NK clones will identify haploidentical donors who are able to mount donor-vs.-recipient NK alloreactions.     

Beta2 integrins separate graft-versus-host disease and graft-versus-leukemia effects

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. beta2 integrins are critically important for leukocyte extravasation through vascular endothelia and for T-cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing the graft-versus-leukemia (GVL) effect. In murine allogeneic bone marrow transplantation models, we found that recipients of CD18-/- donor T cells had significantly less GVHD morbidity and mortality compared with recipients of wild-type (WT) donor T cells. Analysis of alloreactivity showed that CD18-/- and WT T cells had comparable activation, expansion, and cytokine production in vivo. Reduced GVHD was associated with a significant decrease in donor T-cell infiltration of recipient intestine and with an overall decrease in pathologic scores in intestine and liver. Finally, we found that the in vivo GVL effect of CD18-/- donor T cells was largely preserved, because mortality of the recipients who received transplants of CD18-/- T cells plus tumor cells was greatly delayed or prevented. Our data suggest that strategies to target beta2 integrin have clinical potential to alleviate or prevent GVHD while sparing GVL activity.     

Natural killer cells as a therapeutic tool in mismatched transplantation

Natural-killer-cell-mediated, donor-vs-recipient alloresponses occur following transplantation of human-leukocyte-antigen (HLA)-haplotype-mismatched haematopoietic stem cells. Natural killer (NK) cell alloreactivity reduces the risk of relapse in acute myeloid leukaemia patients, while improving engraftment and protecting against graft-vs-host disease. NK cells are primed to kill by several activating receptors. NK cell killing of autologous cells is prevented as NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) major histocompatibility complex class I alleles. As KIRs are distributed clonally, the NK cell population in any individual constitutes a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High-resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes and, in some cases, functional assessment of donor NK clones will identify haplo-identical donors who are able to mount donor-vs-recipient NK alloreactions.     

Haploidentical hematopoietic cell transplantation

Haploidentical hematopoietic stem cell transplantation (HSCT) provides an opportunity for nearly all patients to benefit from HCT when a HLA genotypically matched sibling is not available. Initial results with the use of mismatched allografts led to limited enthusiasm due to GVHD and infectious complications resulting in unacceptable treatment-related morbidity and mortality. Recent advances with effective T-cell depletion, the use of 'megadoses' of stem cells, better antimicrobial therapy and reduced intensity conditioning has significantly decreased the early transplant-related mortality and GVHD. These modifications also enabled robust and prompt engraftment and led to enhancing the therapeutic benefits of haploidentical transplantation. However, the cardinal problems related to delayed immune reconstitution causing post-transplant infectious complications and relapse remain, limiting the efficacy of haploidentical transplant. Preliminary data have demonstrated the great potential in the use of adoptive cellular immunity and selective allodepletion in rapidly reconstituting immunity without GVHD. The encouraging reports from haploidentical transplant using noninherited maternal antigen (NIMA)-mismatched donors or natural killer (NK) alloreactive donors may greatly increase the donor availability and open a way to more appropriate donor selection in HLA-haploidentical HSCT. Future challenges remain in determining the safest approach for haploidentical transplant with minimal risk of GVHD, while preserving effective GVL activity and promoting prompt immune reconstitution.     

Immunoregulatory Cells for Transplantation Tolerance and Graft-versus-Leukemia Effect

Various immunoregulatory cells that inhibit graft-versus-host disease (GVHD) and induce the graft-versus-leukemia (GVL) effect are found after allogeneic hematopoietic stem cell transplantation. These cells comprise CD4+CD25+ regulatory T-cells, regulatory dendritic cells (rDCs), gamma(delta) T-cells, natural killer (NK) T-cells, and NK cells and T-cells with inhibitory NK receptors. Although the first 4 types of cells effectively inhibit GVHD in animal models, with rDCs showing an inhibitory effect on GVHD in humans as well, the GVL effect was observed only in rDCs. Additional analyses are required to determine whether these cells can inhibit GVHD and exert the GVL effect in humans. In contrast, NK cells and T-cells with inhibitory NK receptors have been shown in humans to possess a suppressive activity against GVHD while preserving the GVL effect. These results indicate that immunoregulatory cells may be used to modulate GVHD and the GVL effect in clinical settings.     

Gene transfer applied to the modulation of alloreactivity

Allogeneic hematopoietic stem cell transplantation is associated with a severe complication induced by the T-cells present in the graft: graft-vs-host disease (GVHD). While effectively preventing GVHD, ex vivo T-lymphocyte depletion of the graft unfortunately increases graft rejection and reduces the graft-vs-leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T-cells before their infusion with the hematopoietic stem cells should allow for selective in vivo depletion of these T-cells with ganciclovir (GCV) if subsequent GVHD was to occur. In patients not experiencing GVHD, and therefore at a higher risk of relapse, one could preserve the beneficial effects of the donor T-cells on tumor control. Lastly, the early presence of donor T-cells in all patients should contribute to successful engraftment. We have demonstrated that retro-viral-mediated transfer of HS-tk and Neomycine resistance genes in T-lymphocytes, followed by G418 selection, results in T-cells specifically inhibited by GCV with no bystander effect. In a phase I study, escalating amounts of HS-tk expressing T-cells will be infused in conjunction with a T-cell depleted marrow graft to allogeneic HLA identical recipients. Toxicity, survival, alloreactivity and GCV-sensitivity of the gene-modified cells will be monitored. If successful, such an approach could significantly contribute to expanding the use of alloreactivity as a treatment modality.     

Gene transfer applied to the modulation of alloreactivity

Allogeneic hematopoietic stem cell transplantation is associated with a severe complication induced by the T-cells present in the graft: graft-vs-host disease (GVHD). While effectively preventing GVHD, ex vivo T-lymphocyte depletion of the graft unfortunately increases graft rejection and reduces the graft-vs-leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T-cells before their infusion with the hematopoietic stem cells should allow for selective in vivo depletion of these T-cells with ganciclovir (GCV) if subsequent GVHD was to occur. In patients not experiencing GVHD, and therefore at a higher risk of relapse, one could preserve the beneficial effects of the donor T-cells on tumor control. Lastly, the early presence of donor T-cells in all patients should contribute to successful engraftment. We have demonstrated that retro-viral-mediated transfer of HS-tk and Neomycine resistance genes in T-lymphocytes, followed by G418 selection, results in T-cells specifically inhibited by GCV with no bystander effect. In a phase I study, escalating amounts of HS-tk expressing T-cells will be infused in conjunction with a T-cell depleted marrow graft to allogeneic HLA identical recipients. Toxicity, survival, alloreactivity and GCV-sensitivity of the gene-modified cells will be monitored. If successful, such an approach could significantly contribute to expanding the use of alloreactivity as a treatment modality.     

NK cell alloreactivity and allogeneic hematopoietic stem cell transplantation

As only 60% of leukaemia patients find a matched donor, the Perugia Bone Marrow Transplant Centre developed transplantation from HLA haplotype-mismatched family donors to provide a cure for more patients [F. Aversa, A. Tabilio, A. Terenzi, et al., Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum, Blood 84 (1994) 3948-3955] [F. Aversa, A. Tabilio, A. Velardi, et al., Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype, N. Engl. J. Med. 339 (1998) 1186-1193] [F. Aversa, A. Terenzi, A. Tabilio, et al., Full haplotype-mismatched hematopoietic stem-cell transplantation: a phase II study in patients with acute leukemia at high risk of relapse, J. Clin. Oncol. 23 (2005) 3447-3454]. HLA-mismatches trigger donor vs. recipient NK cell alloreactivity which improves engraftment, protects from GvHD and reduces relapse in AML patients [L. Ruggeri, M. Capanni, E. Urbani, et al., Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants, Science 295 (2002) 2097-2100], [L. Ruggeri, A. Mancusi, M. Capanni, E. Urbani, A. Carotti, T. Aloisi, M. Stern, D. Pende, K. Perruccio, E. Burchielli, F. Topini, E. Bianchi, F. Aversa, M.F. Martelli, A. Velardi, Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value, Blood, in press]. We are using murine transplant models to determine whether NK cell alloreactivity can be exploited to reduce transplant-related mortality (TRM) which remains a major issue. Data from these on-going studies show pre-transplant infusion of alloreactive NK cells: (1) ablates AML cells, (2) kills recipient T cells, permitting a reduced toxicity conditioning regimen, and (3) ablates the recipient dendritic cells (DCs) which trigger GvHD, thus protecting from GvHD while permitting a higher T cell content in the graft. We are designing a clinical haploidentical transplant trial using alloreactive NK cells in the conditioning regimen, with the aim of reducing TRM and improving outcomes and overall survival.     

Minor histocompatibility antigens in human stem cell transplantation

Minor histocompatibility antigens (mHags) play a major role in graft rejection, the induction of detrimental graft-vs-host disease (GVHD), and the development of the beneficial graft-vs-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT). mHags can be defined as amino acid polymorphisms in cellular proteins that can lead to differential presentation of antigenic peptides in HLA molecules and therefore to differential recognition by T cells. The tissue distribution of the mHags and the HLA molecules by which they can be presented play a significant role in the clinical outcome of T-cell responses against these antigens. In part, differential recognition by T cells of mHags specifically expressed in hematopoietic cells, including the malignant cells from the recipient may result in GVL reactivity without concurrent GVHD. Furthermore, T-cell responses against proteins solely expressed in hematopoietic cell lineages from which the malignancy is derived may be appropriate mediators of GVL reactivity without GVHD induction. Characterization of clinical immune responses in patients treated for relapsed leukemia after allogeneic SCT with donor lymphocyte infusion in the absence of GVHD may lead to the characterization of new mHags that can be exploited to generate tumor-specific immune responses. By in vitro generation of T-cell responses against defined mHags, the efficacy and specificity of cellular immunotherapy against hematologic malignancies in the context of allogeneic transplantation may be improved.     

Human leukocyte antigen haploidentical hematopoietic stem cell transplantation: indications and tentative outcomes in Japan

The stem cell banking system in Japan by the Japan Marrow Donor Program (JMDP) and Japan Cord Blood Bank Network (JCBBN) has provided increased opportunities for patients who might benefit from stem cell transplant from allogeneic sources but who lack human leukocyte antigen (HLA)-matched related donors. Nevertheless, most patients probably do not undergo transplantation because of the absence of suitable stem cell sources. To fulfill this potential need, the outcomes of transplants from HLA-mismatched relatives with or without T-cell depletion were retrospectively analyzed: the rates of engraftment and survival were insufficient in transplants with T-cell depletion, and the actual increase in transplantable donor numbers was small because only a single locus mismatched donor was the realistic choice in those without T-cell depletion. Since prophylaxis with tacrolimus reduced the incidence of grade I-IV acute graft-versus-host disease (GVHD) in HLA class I allele mismatched unrelated donors, we studied transplantation from HLA one haploidentical family donors who showed microchimerism of noninherited maternal antigens, without T-cell depletion but with tacrolimus prophylaxis. The rates of engraftment and survival in this circumstance were similar to those obtained with transplantation from HLA-matched sibling donors.     

Natural Killer Cell Alloreactivity in Haploidentical Hematopoietic Stem Cell Transplantation

Natural killer (NK) cells are primed to kill by several activating receptors. NK cell killing of autologous cells is prevented because NK cells coexpress inhibitory receptors (killer cell immunoglobulin-like receptors [KIR]) that recognize groups of (self) major histocompatibility complex class I alleles. Because KIRs are clonally distributed, the NK cell population in any individual are constituted of a repertoire with a variety of class I specificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express the class I alleles that block them. After haploidentical hematopoietic transplantation, NK cell-mediated donor-versus-recipient alloresponses reduce the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-versus-host disease. High-resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and, in some cases, functional assessment of donor NK clones identify haploidentical donors who are able to mount donor-versus-recipient NK alloreactions.     

Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor

One of the functions of HLA class I alleles is interaction with natural killer (NK) cells. Receptors termed killer immunoglobulin-like receptors (KIRs) on NK cells recognize groups of HLA class I alleles, and interaction between receptor and class I allele inhibits reactivity of the NK cell. Failure to recognize the appropriate KIR ligand on a mismatched cell can trigger NK cell elimination of that target cell. Recent analysis of haploidentical hematopoietic transplantations has shown a reduction of graft failure, graft-versus-host disease, and relapse in those with KIR ligand incompatibility in the graft-versus-host direction. In this study we analyzed the effect of KIR ligand incompatibility on outcomes of unrelated donor bone marrow transplantations. The data show no advantage for KIR ligand incompatibility in this clinical setting as assessed by HLA-Bw4 and HLA-C alleles. It is possible that there will be a benefit of NK cell alloreactivity if strategies of haploidentical transplantation are used: high stem cell doses, extensive T-cell depletion, and no postgrafting immune suppression.     

Does haploidentical transplantation in children with primary immunodeficiencies have the potential to exploit donor NK cell alloreactivity?

Donor potential to exert NK cell alloreactivity has been shown to confer survival advantage in haploidentical hematopoietic cell transplantation for hematological malignancies. We investigated killer immunoglobulin receptor (KIR) ligand incompatibility in 40 children receiving haploidentical transplantation for primary immunodeficiencies. The conditioning regimen consisted of busulfan and cyclophosphamide. T-cell depletion of the graft used complement-dependent lysis or CD34+ selection. Two patients died in the first month. The remaining 38 patients were divided into those with (n=13) and those without (n=25) donor potential to exert NK cell alloreactivity. Engraftment was similar in the two groups (61.5 and 64%, respectively). The incidence of grade II-IV acute graft-versus-host disease (GVHD) tended to be lower in the group with donor potential to exert NK cell alloreactivity, but the difference was not significant. In conclusion, in this series of patients with primary immunodeficiencies, donor potential to exert NK cell alloreactivity was not associated with significant advantages in engraftment and prevention of acute GVHD.     

Graft engineering using ex vivo methods to limit GVHD: fludarabine treatment generates superior GVL effects in allogeneic BMT

OBJECTIVE: To compare the abilities of different ex vivo methods of treating donor lymphocytes to inhibit graft-vs-host disease (GVHD) while preserving graft-vs-leukemia (GVL) activity in murine models of allogeneic bone marrow transplantation. METHODS: Donor/recipient pairs included MHC fully mismatched, MHC haplomismatched, and MiHA mismatched strain combinations. T cell-depleted BM (TCD-BM) was transplanted in combination with untreated, fludarabine-treated, 7.5-Gy gamma-irradiated, or psoralen/UVA (PUVA)-treated splenocytes. GVL activity was studied by adding a lethal number of recipient-type lymphoma cells. Posttransplant survival was determined, and GVHD and GVL activity were assessed by clinical and pathological scoring. Hematopoietic chimerism and donor T-cell expansion were analyzed by flow cytometry of peripheral blood samples at 30 and 60 days posttransplant. RESULTS: Allogeneic splenocytes treated with fludarabine, 7.5 Gy gamma-irradiation, or PUVA had significantly diminished GVHD activity, and all treated donor T cells facilitated engraftment by low-dose TCD-BM. Allogeneic splenocytes treated with fludarabine and, to a lesser extent, PUVA retained GVL activity and contributed more to donor T-cell chimerism compared to gamma-irradiated donor splenocytes. CONCLUSION: Among ex vivo methods of treating donor T cells to limit their proliferative capacity, fludarabine exposure had the greatest differential ability to inhibit the GVHD activity of allogeneic lymphocytes, while preserving their GVL activity and ability to engraft recipients. Thus, ex vivo treatment with fludarabine was superior to gamma-irradiation or PUVA in separating GVL from GVHD activity in murine models of allogeneic bone marrow transplantation.     

Memory T cells: A helpful guard for allogeneic hematopoietic stem cell transplantation without causing graft-versus-host disease

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (AHSCT) and the major cause of nonrelapse morbidity and mortality of AHSCT. In AHSCT, donor T cells facilitate hematopoietic stem cell (HSC) engraftment, contribute to anti-infection immunity, and mediate graft-versus-leukemia (GVL) responses. However, activated alloreactive T cells also attack recipient cells in vital organs, leading to GVHD. Different T-cell subsets, including naïve T (T_N) cells, memory T (T_M) cells, and regulatory T (T_reg) cells mediate different forms of GVHD and GVL; T_N cells mediate severe GVHD, whereas T_M cells do not cause GVHD, but preserve T-cell function including GVL. In addition, metabolic reprogramming controls T-cell differentiation and activation in these disease states. This minireview focuses on the role and the related mechanisms of T_M cells in AHSCT, and the potential manipulation of T cells in AHSCT.     

Haploidentical stem cell transplantation in leukemia.

In acute leukemia patients, infusing a megadose of extensively T-cell-depleted hematopoietic stem cells after an immuno-myeloablative conditioning regimen ensures sustained engraftment of full-haplotype mismatched transplants without graft-vs-host disease. Besides the conditioning regimen and the megadose of stem cells donor natural killer cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in 112 very high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.     

Association of natural killer cell immune recovery with a graft-versus-leukemia effect independent of graft-versus-host disease following allogeneic bone marrow transplantation

 There is good evidence that T lymphocytes play an important role in the graft-versus-leukemia (GVL) effect following allogeneic bone marrow transplantation (BMT) for hematologic malignancies. However, the role of natural killer (NK) cells in GVL is less clear. To further investigate a possible association of NK cells with GVL we studied 15 patients undergoing BMT for chronic myeloid leukemia (CML), correlating T-cell (CD4+ and CD8+) and NK-cell (CD16+56+) recovery with relapse and graft-versus-host disease (GVHD). Patients were studied on three occasions up to 9 months after BMT, for lymphocyte surface phenotype and for spontaneous and IL-2-stimulated (LAK cell) cytotoxic function. Circulating CD8+ and NK but not CD4+ cell numbers were significantly lower in five patients who relapsed compared with those remaining in remission after BMT (mean 0.03 vs 0.32×10⁹/l, p=0.002 for CD8+ cells; mean 0.03 vs 0.11×10⁹/l, p=0.002 for NK cells). There was no correlation of CD4+, CD8+, or NK cell numbers and development of grade-II or more acute GVHD. Spontaneous NK cytotoxic function rose to within the normal range in the first month after BMT. LAK function remained low during the study period. These results link NK cell recovery more closely with a GVL than with a GVH effect. Received: 1 May 1996/Accepted: 19 September 1996