Donor natural killer cells trigger production of ß-2-microglobulin to enhance post-bone marrow transplant immunity.

Allogeneic hematopoietic transplantation is a powerful treatment for hematologic malignancies. Posttransplant immune incompetence exposes patients to disease relapse and infections. We previously demonstrated that donor alloreactive natural killer (NK) cells ablate recipient hematopoietic targets, including leukemia. Here, in murine models, we show that infusion of donor alloreactive NK cells triggers recipient dendritic cells (DCs) to synthesize ß-2-microglobulin (B2M) that elicits the release of c-KIT ligand and interleukin-7 that greatly accelerate posttransplant immune reconstitution. An identical chain of events was reproduced by infusing supernatants of alloreactive NK/DC cocultures. Similarly, human alloreactive NK cells triggered human DCs to synthesize B2M that induced interleukin-7 production by thymic epithelial cells and thereby supported thymocyte cellularity in vitro. Chromatography fractionation of murine and human alloreactive NK/DC coculture supernatants identified a protein with molecular weight and isoelectric point of B2M, and mass spectrometry identified amino acid sequences specific of B2M. Anti-B2M antibody depletion of NK/DC coculture supernatants abrogated their immune-rebuilding effect. B2M knock-out mice were unable to undergo accelerated immune reconstitution, but infusion of (wild-type) NK/DC coculture supernatants restored their ability to undergo accelerated immune reconstitution. Similarly, silencing the B2M gene in human DCs, before coculture with alloreactive NK cells, prevented the increase in thymocyte cellularity in vitro. Finally, human recombinant B2M increased thymocyte cellularity in a thymic epithelial cells/thymocyte culture system. Our studies uncover a novel therapeutic principle for treating posttransplant immune incompetence and suggest that, upon its translation to the clinic, patients may benefit from adoptive transfer of large numbers of cytokine-activated, ex vivo-expanded donor alloreactive NK cells.

Blinatumomab Redirects Donor Lymphocytes against CD19+ Acute Lymphoblastic Leukemia without Relevant Bystander Alloreactivity after Haploidentical Hematopoietic Stem Cell Transplantation.

Blinatumomab alone or with donor leukocyte infusions (DLI) has been used after allogeneic hematopoietic stem cell transplantation (HSCT) as a salvage therapy in relapsing patients with CD19+ hematological malignancies. It was effective in a fraction of them, with low incidence of Graft-versus-Host Disease (GvHD). Immunosuppressive drugs used as GvHD prophylaxis hinder T cell function and reduce the efficacy of the treatment. Because T cell-depleted haploidentical HSCT with donor regulatory and conventional T cells (Treg/Tcon haploidentical HSCT) does not require post-transplant immunosuppression, it is an ideal platform for the concomitant use of blinatumomab and DLI. However, the risk of GvHD is high because the donor is haploidentical. We treated two patients with CD19+ acute lymphoblastic leukemia (ALL) who had relapsed after Treg/Tcon haploidentical HSCT with blinatumomab and DLI. Despite the mismatch for one HLA haplotype, they did not develop GvHD and achieved complete remission with negative minimal residual disease. Consistently, we found that blinatumomab did not enhance T cell alloreactivity in vitro. Eventually, the two patients relapsed again because of their high disease risk. This study suggests that treatment with blinatumomab and DLI can be feasible to treat relapse after haploidentical transplantation, and its pre-emptive use should be considered to improve efficacy.

MYB rearrangements and over-expression in T-cell acute lymphoblastic leukemia.

We investigated MYB rearrangements (MYB-R) and the levels of MYB expression, in 331 pediatric and adult patients with T-cell acute lymphoblastic leukemia (T-ALL). MYB-R were detected in 17 cases and consisted of MYB tandem duplication (tdup) (= 14) or T cell receptor beta locus (TRB)-MYB (= 3). As previously reported, TRB-MYB was found only in children (1.6%) while MYB tdup occurred in both age groups, although it was slightly more frequent in children (5.2% vs 2.8%). Shared features of MYB-R T-ALL were a non-early T-cell precursor (ETP) phenotype, a high incidence of NOTCH1/FBXW7 mutations (81%) and CDKN2AB deletions (70.5%). Moreover, they mainly belonged to HOXA (=8), NKX2-1/2-2/TLX1 (=4), and TLX3 (=3) homeobox-related subgroups. Overall, MYB-R cases had significantly higher levels of MYB expression than MYB wild type (MYB-wt) cases, although high levels of MYB were detected in ~ 30% of MYB-wt T-ALL. Consistent with the transcriptional regulatory networks, cases with high MYB expression were significantly enriched within the TAL/LMO subgroup (P = .017). Interestingly, analysis of paired diagnosis/remission samples demonstrated that a high MYB expression was restricted to the leukemic clone. Our study has indicated that different mechanisms underlie MYB deregulation in 30%-40% of T-ALL and highlighted that, MYB has potential as predictive/prognostic marker and/or target for tailored therapy.

Rifaximin use favoured micafungin-resistant Candida spp. infections in recipients of allogeneic hematopoietic cell transplantation.

Damage to gut mucosa following conditioning regimens may favour bacterial infections that can trigger graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Rifaximin, an oral and non-absorbable antibiotic, has been recently proposed as effective prophylaxis to reduce bacterial infections in the gut and consequently acute GvHD in this setting. The present study evaluated safety and outcomes of HSCT patients that were treated with rifaximin prophylaxis at Perugia University Hospital. Rifaximin prophylaxis was introduced as standard of care in HSCT patients in May 2018. We retrieved data from 118 consecutive transplants, and we compared the outcomes of rifaximin-treated patients with historical controls that did not receive antibiotic prophylaxis. While incidences of neutropenic fever, documented bacterial infections, and aGvHD were similar in the two groups, we found an increased frequency of invasive candidiasis and clinically relevant Candida spp. infections in rifaximin-treated patients (5 patients vs 1 patient, 25% [± 0.99%] vs 1% [± 0.01%], p < .0001). Three rifaximin-treated patients experienced life-threating candidemia (2 C. krusei, 1 C. orthopsilosis). Rifaximin was the only factor that increased the risk of Candida spp. infections. Rifaximin could have contributed to microbiome disruption which favoured an outbreak of life-threatening Candida infections. This important complication forced us to halt its use. Larger, prospective studies are needed to assess the impact of rifaximin prophylaxis on incidence of bacterial infections, aGvHD, and survival of HSCT patients.

NK Cells in Chronic Lymphocytic Leukemia and Their Therapeutic Implications.

Key features of chronic lymphocytic leukemia (CLL) are defects in the immune system and the ability of leukemic cells to evade immune defenses and induce immunosuppression, resulting in increased susceptibility to infections and disease progression. Several immune effectors are impaired in CLL, including T and natural killer (NK) cells. The role of T cells in defense against CLL and in CLL progression and immunotherapy has been extensively studied. Less is known about the role of NK cells in this leukemia, and data on NK cell alterations in CLL are contrasting. Besides studies showing that NK cells have intrinsic defects in CLL, there is a large body of evidence indicating that NK cell dysfunctions in CLL mainly depend on the escape mechanisms employed by leukemic cells. In keeping, it has been shown that NK cell functions, including antibody-dependent cellular cytotoxicity (ADCC), can be retained and/or restored after adequate stimulation. Therefore, due to their preserved ADCC function and the reversibility of CLL-related dysfunctions, NK cells are an attractive source for novel immunotherapeutic strategies in this disease, including chimeric antigen receptor (CAR) therapy. Recently, satisfying clinical responses have been obtained in CLL patients using cord blood-derived CAR-NK cells, opening new possibilities for further exploring NK cells in the immunotherapy of CLL. However, notwithstanding the promising results of this clinical trial, more evidence is needed to fully understand whether and in which CLL cases NK cell-based immunotherapy may represent a valid, alternative/additional therapeutic option for this leukemia. In this review, we provide an overview of the current knowledge about phenotypic and functional alterations of NK cells in CLL and the mechanisms by which CLL cells circumvent NK cell-mediated immunosurveillance. Additionally, we discuss the potential relevance of using NK cells in CLL immunotherapy.

Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35.

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-ß, granzyme B). TIM3+ cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

A novel mutation of indoleamine 2,3-dioxygenase 1 causes a rapid proteasomal degradation and compromises protein function.

Indoleamine 2,3-dioxygenase 1 (IDO1) - the enzyme catalyzing the rate-limiting step of tryptophan catabolism along the kynurenine pathway - belongs to the class of inhibitory immune checkpoint molecules. Such regulators of the immune system are crucial for maintaining self-tolerance and thus, when properly working, preventing autoimmunity. A dysfunctional IDO1 has recently been associated with a specific single nucleotide polymorphism (SNP) and with the occurrence of autoimmune diabetes and multiple sclerosis. Many genetic alterations of IDO1 have been proposed being related with dysimmune disorders. However, the molecular and functional meaning of variations in IDO1 exomes as well as the promoter region remains a poorly explored field. In the present study, we identified a rare missense variant (rs751360195) at the IDO1 gene in a patient affected by coeliac disease, thyroiditis, and selective immunoglobulin A deficiency. Molecular and functional studies demonstrated that the substitution of lysine (K) at position 257 with a glutamic acid (E) results in an altered IDO1 protein that undergoes a rapid protein turnover. This genotype-to-phenotype relation is produced by peripheral blood mononuclear cells (PBMCs) of the patient bearing this variation and is associated with a specific phenotype (i.e., impaired tryptophan catabolism and defective mechanisms of immune tolerance). Thus decoding functional mutations of the IDO1 exome may provide clinically relevant information exploitable to personalize therapeutic interventions.

Haploidentical hematopoietic transplantation for the cure of leukemia: from its biology to clinical translation.

The present review describes the biology of human leukocyte antigen haplotype mismatched ("haploidentical") transplantation, its translation to clinical practice to cure leukemia, and the results of current transplantation protocols. The 1990s saw what had been major drawbacks of haploidentical transplantation, ie, very strong host-versus-graft and graft-versus-host alloresponses, which led respectively to rejection and graft-versus-host disease (GVHD), being overcome through transplantation of a "mega-dose" of T cell-depleted peripheral blood hematopoietic progenitor cells and no posttransplant pharmacologic immunosuppression. The absence of posttransplant immunosuppression was an opportunity to discover natural killer cell alloreactions that eradicated acute myeloid leukemia and improved survival. Furthermore, it also unveiled the benefits of transplantation from mother donors, a likely consequence of the mother-to-child interaction during pregnancy. More recent transplantation protocols use unmanipulated (without ex vivo T-cell depletion) haploidentical grafts combined with enhanced posttransplant immunosuppression to help prevent GVHD. Unmanipulated grafts substantially extended the use of haploidentical transplantation with results than even rival those of matched hematopoietic transplantation. In T cell-depleted haploidentical transplantation, recent advances were made by the adoptive transfer of regulatory and conventional T cells.

Clinical-Grade-Expanded Regulatory T Cells Prevent Graft-versus-Host Disease While Allowing a Powerful T Cell-Dependent Graft-versus-Leukemia Effect in Murine Models.

We developed a good manufacturing practices-compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons.

Haploidentical hematopoietic transplantation from KIR ligand-mismatched donors with activating KIRs reduces nonrelapse mortality.

Because activating killer cell immunoglobulinlike receptors (KIRs) are heterogeneously expressed in the population, we investigated the role of donor activating KIRs in haploidentical hematopoietic transplants for acute leukemia. Transplants were grouped according to presence vs absence of KIR-ligand mismatches in the graft-vs-host direction (ie, of donor-vs-recipient natural killer [NK]-cell alloreactivity). In the absence of donor-vs-recipient NK-cell alloreactivity, donor activating KIRs had no effects on outcomes. In the 69 transplant pairs with donor-vs-recipient NK-cell alloreactivity, transplantation from donors with KIR2DS1 and/or KIR3DS1 was associated with reduced risk of nonrelapse mortality, largely infection related (KIR2DS1 present vs absent: hazard ratio [HR], 0.25; P = .01; KIR3DS1 present vs absent: HR, 0.18; P = .006), and better event-free survival (KIR2DS1 present vs absent: HR, 0.31; P = .011; KIR3DS1 present vs absent: HR, 0.30; P = .008). Transplantation from donors with KIR2DS1 and/or KIR3DS1 was also associated with a 50% reduction in infection rate (P = .003). In vitro analyses showed that KIR2DS1 binding to its HLA-C2 ligand upregulated inflammatory cytokine production by alloreactive NK cells in response to infectious challenges. Because ∼40% of donors able to exert donor-vs-recipient NK-cell alloreactivity carry KIR2DS1 and/or KIR3DS1, searching for them may become a feasible, additional criterion in donor selection.

"Designed" grafts for HLA-haploidentical stem cell transplantation.

Today human leukocyte antigen-haploidentical transplantation is a feasible option for patients with high-risk acute leukemia who do not have matched donors. Whether it is T-cell replete or T-cell depleted, it is still, however, associated with issues of transplant-related mortality and posttransplant leukemia relapse. After reports that adoptive immunotherapy with T-regulatory cells controls the alloreactivity of conventional T lymphocytes in animal models, tomorrow's world of haploidentical transplantation will focus on new "designed" grafts. They will contain an appropriate ratio of conventional T lymphocytes and T-regulatory cells, natural killer cells, γ δ T cells, and other accessory cells. Preliminary results of ongoing clinical trials show the approach is feasible. It is associated with better immune reconstitution and a quite powerful graft-versus-leukemia effect with a low incidence of graft-versus-host disease and no need for posttransplant pharmacological prophylaxis. Future strategies will focus on enhancing the clinical benefit of T-regulatory cells by increasing their number and strengthening their function.

HLA-haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia relapse.

Posttransplant relapse is still the major cause of treatment failure in high-risk acute leukemia. Attempts to manipulate alloreactive T cells to spare normal cells while killing leukemic cells have been unsuccessful. In HLA-haploidentical transplantation, we reported that donor-derived T regulatory cells (Tregs), coinfused with conventional T cells (Tcons), protected recipients against graft-versus-host disease (GVHD). The present phase 2 study investigated whether Treg-Tcon adoptive immunotherapy prevents posttransplant leukemia relapse. Forty-three adults with high-risk acute leukemia (acute myeloid leukemia 33; acute lymphoblastic leukemia 10) were conditioned with a total body irradiation-based regimen. Grafts included CD34(+) cells (mean 9.7 × 10(6)/kg), Tregs (mean 2.5 × 10(6)/kg), and Tcons (mean 1.1 × 10(6)/kg). No posttransplant immunosuppression was given. Ninety-five percent of patients achieved full-donor type engraftment and 15% developed ≥grade 2 acute GVHD. The probability of disease-free survival was 0.56 at a median follow-up of 46 months. The very low cumulative incidence of relapse (0.05) was significantly better than in historical controls. These results demonstrate the immunosuppressive potential of Tregs can be used to suppress GVHD without loss of the benefits of graft-versus-leukemia (GVL) activity. Humanized murine models provided insights into the mechanisms underlying separation of GVL from GVHD, suggesting the GVL effect is due to largely unopposed Tcon alloantigen recognition in bone marrow.

Effects of anti-NKG2A antibody administration on leukemia and normal hematopoietic cells.

Natural killer cells are key cells of the innate immune system. Natural killer cell receptor repertoires are diversified by a stochastic expression of killer-cell-immunoglobulin-like receptors and lectin-like receptors such as NKG2 receptors. All individuals harbor a subset of natural killer cells expressing NKG2A, the inhibitory checkpoint receptor for HLA-E. Most neoplastic and normal hematopoietic cells express HLA-E, the inhibitory ligand of NKG2A. A novel anti-human NKG2A antibody induced tumor cell death, suggesting that the antibody could be useful in the treatment of cancers expressing HLA-E. We found that immunodeficient mice, co-infused with human primary leukemia or Epstein-Barr virus cell lines and NKG2A(+) natural killer cells, pre-treated with anti-human NKG2A, were rescued from disease progression. Human NKG2A(+) natural killer cells reconstituted in immunodeficient mice after transplantation of human CD34(+) cells. These natural killer cells are able to kill engrafted human primary leukemia or Epstein-Barr virus cell lines by lysis after intraperitoneal administration of anti-human NKG2A. Thus, this anti-NKG2A may exploit the anti-leukemic action of the wave of NKG2A(+) natural killer cells recovering after hematopoietic stem cell transplants or adoptive therapy with natural killer cell infusions from matched or mismatched family donors after chemotherapy for acute leukemia, without the need to search for a natural killer cell alloreactive donor.

How adoptive transfer of components of the donor immune system boosts GvL and prevents GvHD in HLA-haploidentical hematopoietic transplantation for acute leukemia.

Why a new Perspective in allogeneic hematopoietic transplantation? A summary. Nowadays, for high-risk acute leukemia patients without an HLA-matched donor (sibling or volunteer), hematopoietic transplants that use HLA-haploidentical grafts combined with enhanced post transplant immune suppression (i.e., high-dose cyclophosphamide) are widely used. They are associated with low TRM rates. However, they are also associated with significant chronic GvHD while they only partially abrogate leukemia relapse rates. One may speculate that post-transplant immune suppression, required for GvHD prophylaxis, weakens the anti-leukemic potential of the graft. Historically, haploidentical transplants became feasible for the first time through transplantation of T cell-depleted peripheral blood hematopoietic progenitor cells. Lack of post-transplant immune suppression allowed the emergence of donor-versus-recipient NK-cell alloreactions that eradicated AML. In an attempt to improve these results we recently combined an age-adapted, irradiation-based conditioning regimen with transplant of T-cell-depleted grafts and infusion of regulatory and conventional T cells, without any post transplant immune suppression. With the obvious limitations of a single center experience, this protocol resulted in extremely low relapse and chronic GvHD rates and, consequently, in a remarkable 75% chronic GvHD/relapse-free survival in over 50 AML patients up to the age of 65 many of whom at high risk of relapse.

Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.

Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.

Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients.

Thirteen patients with acute myeloid leukemia, 5 with active disease, 2 in molecular relapse, and 6 in morphologic complete remission (CR; median age, 62 years; range, 53-73 years) received highly purified CD56(+)CD3(-) natural killer (NK) cells from haploidentical killer immunoglobulin-like receptor-ligand mismatched donors after fludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by IL-2. The median number of infused NK cells was 2.74 × 10(6)/Kg. T cells were < 10(5)/Kg. No NK cell-related toxicity, including GVHD, was observed. One of the 5 patients with active disease achieved transient CR, whereas 4 of 5 patients had no clinical benefit. Both patients in molecular relapse achieved CR that lasted for 9 and 4 months, respectively. Three of 6 patients in CR are disease free after 34, 32, and 18 months. After infusion, donor NK cells were found in the peripheral blood of all evaluable patients (peak value on day 10). They were also detected in BM in some cases. Donor-versus-recipient alloreactive NK cells were shown in vivo by the detection of donor-derived NK clones that killed recipient's targets. Adoptively transferred NK cells were alloreactive against recipient's cells, including leukemia. In conclusion, infusion of purified NK cells is feasible in elderly patients with high-risk acute myeloid leukemia. This trial was registered at www.clinicaltrial.gov as NCT00799799.

Killer-cell immunoglobulin-like receptors reactivity and outcome of stem cell transplant.

PURPOSE OF REVIEW: This review focuses on advances in the field of natural killer (NK) cell alloreactivity as a form of immunotherapy in the setting of allogeneic haematopoietic transplantation. RECENT FINDINGS: Recent research has focused on mechanisms underlying the acquisition of function by NK cells after haematopoietic transplantation, on the therapeutic impact of NK-cell alloreactivity in various forms of transplantation and in adoptive immunotherapy. Finally, studies have highlighted the role of NK-cell responses in viral infections after transplantation. SUMMARY: Donor-versus-recipient NK-cell alloreactivity is established as a key therapeutic element in human leukocyte antigen haplotype-mismatched haematopoietic transplants in adult acute myeloid leukaemia (AML) and paediatric acute lymphoblastic leukaemia (ALL). NK-cell allotherapy for leukaemia is deployed through stem cell transplantation (and ensuing NK-cell reconstitution) across killer-cell immunoglobulin-like receptor ligand mismatches. Donor-derived NK cells were also reported to respond to cytomegalovirus by acquiring features that are reminiscent of the specificity and memory of adaptive (T-cell) immune responses. As NK cells are the earliest immune cells to recover after transplant, this observation suggests they may contribute to controlling viral reactivation early after transplant.

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

Case Report: Subtotal Lymphoid and Total Marrow Irradiation as Bridge Therapy to CD19-Directed CAR T Cells in a Chemorefractory DLBCL With Leukemic Involvement.

CAR T cell therapy has transformed the salvage approach for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). Maintaining disease control before CAR T cell infusion during product manufacturing (so-called bridging therapy) is an important step to optimizing outcome. Among possible bridging therapies, radiation therapy (RT) represents a valuable option, particularly when the disease is limited. Here, we report for the first time on a patient with chemorefractory-transformed DLBCL showing nodal, extranodal, and massive bone marrow (BM) lymphoma infiltration associated with leukemic involvement, a successful bridge therapy to CD19-directed CAR T cell therapy by subtotal lymphoid/total marrow irradiation plus thiothepa followed by reinfusion of CD34+ autologous hematopoietic stem cells. Such a novel bridging regimen allowed a significant reduction of nodal and BM tumor volume while improving blood cell count before CAR T cell infusion. The PET-CT scan and BM evaluation performed at 1, 3, and 6 months after treatment showed complete remission of the disease. A relapse occurred at almost 1 year in lymph nodes because of CD19 antigen escape while the BM remained free of disease. This extended radiotherapy approach may be an effective bridging therapy for chemorefractory DLBCL patients eligible for CAR T cells who present with a high tumor burden, including massive BM involvement associated with leukemic involvement. This preliminary evidence is worth confirming in additional patients.