CAR-engineered lymphocyte persistence is governed by a FAS ligand/FAS auto-regulatory circuit.

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG. To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor (ΔFAS). Following adoptive transfer, ΔFAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, ΔFAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.

Fatty acid oxidation fuels natural killer cell responses against infection and cancer.

Natural killer (NK) cells are a vital part of the innate immune system capable of rapidly clearing mutated or infected cells from the body and promoting an immune response. Here, we find that NK cells activated by viral infection or tumor challenge increase uptake of fatty acids and their expression of carnitine palmitoyltransferase I (CPT1A), a critical enzyme for long-chain fatty acid oxidation. Using a mouse model with an NK cell-specific deletion of CPT1A, combined with stable 13C isotope tracing, we observe reduced mitochondrial function and fatty acid-derived aspartate production in CPT1A-deficient NK cells. Furthermore, CPT1A-deficient NK cells show reduced proliferation after viral infection and diminished protection against cancer due to impaired actin cytoskeleton rearrangement. Together, our findings highlight that fatty acid oxidation promotes NK cell metabolic resilience, processes that can be optimized in NK cell-based immunotherapies.

Single-Cell Profiling Reveals a Naive-Memory Relationship between CD56 bright and Adaptive Human Natural Killer Cells.

Development of antigen-specific memory upon pathogen exposure is a hallmark of the adaptive immune system. While natural killer (NK) cells are considered part of the innate immune system, humans exposed to the chronic viral pathogen cytomegalovirus (CMV) often possess a distinct NK cell population lacking in individuals who have not been exposed, termed "adaptive" NK cells. To identify the "naïve" population from which this "memory" population derives, we performed phenotypic, transcriptional, and functional profiling of NK cell subsets. We identified immature precursors to the Adaptive NK cells that are equally present in both CMV+ and CMV-individuals, resolved an Adaptive transcriptional state distinct from most mature NK cells and sharing a common gene program with the immature CD56 bright population, and demonstrated retention of proliferative capacity and acquisition of superior IFNγ production in the Adaptive population. Furthermore, we distinguish the CD56 bright and Adaptive NK populations by expression of the transcription factor CXXC5, positioning these memory NK cells at the inflection point between innate and adaptive lymphocytes.

Control of nutrient uptake by IRF4 orchestrates innate immune memory.

Natural killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen specificity, clonal expansion and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8+ T cell siblings. Various signals ranging from antigen, co-stimulation and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified that the transcription factor IRF4 integrates signals to coordinate the NK cell response during mouse cytomegalovirus infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest that IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection.

Immune profiling after allogeneic hematopoietic cell transplantation in pediatric acute myeloid leukemia.

Although allogeneic hematopoietic cell transplant (allo-HCT) is curative for high-risk pediatric acute myeloid leukemia (AML), disease relapse remains the primary cause of posttransplant mortality. To identify pressures imposed by allo-HCT on AML cells that escape the graft-versus-leukemia effect, we evaluated immune signatures at diagnosis and posttransplant relapse in bone marrow samples from 4 pediatric patients using a multimodal single-cell proteogenomic approach. Downregulation of major histocompatibility complex class II expression was most profound in progenitor-like blasts and accompanied by correlative changes in transcriptional regulation. Dysfunction of activated natural killer cells and CD8+ T-cell subsets at relapse was evidenced by the loss of response to interferon gamma, tumor necrosis factor α signaling via NF-κB, and interleukin-2/STAT5 signaling. Clonotype analysis of posttransplant relapse samples revealed an expansion of dysfunctional T cells and enrichment of T-regulatory and T-helper cells. Using novel computational methods, our results illustrate a diverse immune-related transcriptional signature in posttransplant relapses not previously reported in pediatric AML.

Emergence of human CMV-induced NKG2C+ NK cells is associated with CD8+ T-cell recovery after allogeneic HCT.

Cytomegalovirus (CMV) infection is associated with the expansion of a mature NKG2C+FcεR1γ- natural killer (NK) cell population. The exact mechanism underlying the emergence of NKG2C+ NK cells, however, remains unknown. Allogeneic hematopoietic cell transplantation (HCT) provides an opportunity to longitudinally study lymphocyte recovery in the setting of CMV reactivation, particularly in patients receiving T-cell-depleted (TCD) allografts. We analyzed peripheral blood lymphocytes from 119 patients at serial time points after infusion of their TCD allograft and compared immune recovery with that in samples obtained from recipients of T-cell-replete (T-replete) (n = 96) or double umbilical cord blood (DUCB) (n = 52) allografts. NKG2C+ NK cells were detected in 92% (45 of 49) of recipients of TCD HCT who experienced CMV reactivation. Although NKG2A+ cells were routinely identifiable early after HCT, NKG2C+ NK cells were identified only after T cells could be detected. T-cell reconstitution occurred at variable times after HCT among patients and predominantly comprised CD8+ T cells. In patients with CMV reactivation, recipients of TCD HCT expressed significantly higher frequencies of NKG2C+ and CD56neg NK cells compared with patients who received T-replete HCT or DUCB transplantation. NKG2C+ NK cells after TCD HCT were CD57+FcεR1γ+ and degranulated significantly more in response to target cells compared with the adaptive the NKG2C+CD57+FcεR1γ- NK cell population. We conclude that the presence of circulating T cells is associated with the expansion of a CMV-induced NKG2C+ NK cell population, a potentially novel example of developmental cooperation between lymphocyte populations in response to viral infection.

Role of NKG2D ligands and receptor in haploidentical related donor hematopoietic cell transplantation.

The recurrence of malignancy after hematopoietic cell transplantation (HCT) is the primary cause of transplantation failure. The NKG2D axis is a powerful pathway for antitumor responses, but its role in the control of malignancy after HCT is not well-defined. We tested the hypothesis that gene variation of the NKG2D receptor and its ligands MICA and MICB affect relapse and survival in 1629 patients who received a haploidentical HCT for the treatment of a malignant blood disorder. Patients and donors were characterized for MICA residue 129, the exon 5 short tandem repeat (STR), and MICB residues 52, 57, 98, and 189. Donors were additionally defined for the presence of NKG2D residue 72. Mortality was higher in patients with MICB-52Asn relative to those with 52Asp (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.24-2.71; P = .002) and lower in those with MICA-STR mismatch than in those with STR match (HR, 0.66; 95% CI, 0.54-0.79; P = .00002). Relapse was lower with NKG2D-72Thr donors than with 72Ala donors (relapse HR, 0.57; 95% CI, 0.35-0.91; P = .02). The protective effects of patient MICB-52Asp with donor MICA-STR mismatch and NKG2D-72Thr were enhanced when all 3 features were present. The NKG2D ligand/receptor pathway is a transplantation determinant. The immunobiology of relapse is defined by the concerted effects of MICA, MICB, and NKG2D germ line variation. Consideration of NKG2D ligand/receptor pairings may improve survival for future patients.

Dominant epitopes presented by prevalent HLA alleles permit wide use of banked CMVpp65 T cells in adoptive therapy.

We established and characterized a bank of 138 CMVpp65 peptide-specific T-cell (CMVpp65CTLs) lines from healthy marrow transplant donors who consented to their use for treatment of individuals other than their transplant recipient. CMVpp65CTL lines included 131 containing predominantly CD8+ T cells and 7 CD4+ T cells. CD8+ CMVpp65CTLs were specific for 1 to 3 epitopes each presented by one of only 34 of the 148 class I alleles in the bank. Similarly, the 7 predominantly CD4+ CMVpp65CTL lines were each specific for epitopes presented by 14 of 40 HLA DR alleles in the bank. Although the number of HLA alleles presenting CMV epitopes is low, their prevalence is high, permitting selection of CMVpp65CTLs restricted by an HLA allele shared by transplant recipient and hematopoietic cell transplant donor for >90% of an ethnogeographically diverse population of hematopoietic cell transplant recipients. Within individuals, responses to CMVpp65 peptides presented by different HLA alleles are hierarchical. Furthermore, within groups, epitopes presented by HLA B*07:02 and HLA A*02:01 consistently elicit immunodominant CMVpp65CTLs, irrespective of other HLA alleles inherited. All dominant CMVpp65CTLs exhibited HLA-restricted cytotoxicity against epitope loaded targets and usually cleared CMV infections. However, immunodominant CMVpp65CTLs responding to epitopes presented by certain HLA B*35 alleles were ineffective in lysing CMV-infected cells in vitro or controlling CMV infections post adoptive therapy. Analysis of the hierarchy of T-cell responses to CMVpp65, the HLA alleles presenting immunodominant CMVpp65 epitopes, and the responses they induce may lead to detailed algorithms for optimal choice of third-party CMVpp65CTLs for effective adoptive therapy.

Dynamic variability in SHP-1 abundance determines natural killer cell responsiveness.

Interactions between human leukocyte antigen (HLA) molecules on target cells and the inhibitory killer cell immunoglobulin-like receptors (KIRs) and heterodimeric inhibitory receptor CD94-NKG2A on human natural killer (NK) cells shape and program various response capacities. A functionally orthologous system exists in mice, consisting of major histocompatibility complex (MHC) molecules on target cells and the inhibitory Ly49 and CD94-NKG2A receptors on NK cells. Here, we found that the abundance of Src homology 2 domain­containing phosphatase-1 (SHP-1) in NK cells was established by interactions between MHCs and NK cell inhibitory receptors, although phenotypically identical NK cell populations still showed substantial variability in endogenous SHP-1 abundance and NK cell response potential. Human and mouse NK cell populations with high responsiveness had low SHP-1 abundance, and a reduction in SHP-1 abundance in NK cells enhanced their responsiveness. Computational modeling of NK cell activation by membrane-proximal signaling events identified SHP-1 as a negative amplitude regulator, which was validated by single-cell analysis of human NK cell responsiveness. The amount of mRNA and protein varied among responsive NK cells despite their similar chromatin accessibility to that of unresponsive cells, suggesting dynamic regulation of SHP-1 abundance. Low intracellular SHP-1 abundance was a biomarker of responsive NK cells. Together, these data suggest that enhancing NK cell function through the acute loss of SHP-1 abundance or activity may enhance the tumoricidal capacity of NK cells.

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8ß, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rß, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Human cytomegalovirus expands a CD8+ T cell population with loss of BCL11B expression and gain of NK cell identity.

CD8+ T cells not only are critical mediators of adaptive immunity but also may exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with natural killer (NK) cells. We demonstrate that, similar to NK cells, NKG2C+TCRαß+CD8+ T cells are associated with prior human cytomegalovirus (HCMV) exposure. In addition to expressing several NK cell markers such as CD56 and KIR, NKG2C+CD8+ T cells are oligoclonal and do not up-regulate PD-1 even in response to persistent activation. Furthermore, we found that NKG2C+CD8+ T cells from some individuals exhibited strong effector function against leukemia cells and HCMV-infected fibroblasts, which was dictated by both NKG2C and TCR specificity. Transcriptomic analysis revealed that the transcription factor BCL11B, a regulator of T cell developmental fate, is down-regulated in NKG2C+CD8+ T cells when compared with conventional NKG2C−CD8+ T cells. BCL11B deletion in conventional CD8+ T cells resulted in the emergence of a similar innate-like CD56+CD94+DAP12+NKG2C+CD45RA+CCR7−PD-1−/low T cell population with activity against HLA-E+ targets. On the basis of their intrinsic capacity to recognize diseased cells coupled with lack of PD-1 induction, NKG2C+CD8+ T cells represent a lymphocyte population that resides at the boundary between innate and adaptive immunity, presenting an attractive alternative for cellular therapy, including CAR T cell­based therapies.

Deconvoluting global cytokine signaling networks in natural killer cells.

Cytokine signaling via signal transducer and activator of transcription (STAT) proteins is crucial for optimal antiviral responses of natural killer (NK) cells. However, the pleiotropic effects of both cytokine and STAT signaling preclude the ability to precisely attribute molecular changes to specific cytokine-STAT modules. Here, we employed a multi-omics approach to deconstruct and rebuild the complex interaction of multiple cytokine signaling pathways in NK cells. Proinflammatory cytokines and homeostatic cytokines formed a cooperative axis to commonly regulate global gene expression and to further repress expression induced by type I interferon signaling. These cytokines mediated distinct modes of epigenetic regulation via STAT proteins, and collective signaling best recapitulated global antiviral responses. The most dynamically responsive genes were conserved across humans and mice, which included a cytokine-STAT-induced cross-regulatory program. Thus, an intricate crosstalk exists between cytokine signaling pathways, which governs NK cell responses.

Prospective KIR genotype evaluation of hematopoietic cell donors is feasible with potential to benefit patients with AML.

Donor KIR and recipient HLA combinations that minimize inhibition and favor activation of the NK repertoire are associated with improved outcomes after allogeneic hematopoietic cell transplantation (HCT) in patients with myeloid neoplasia. We prospectively evaluated a weighted donor ranking algorithm designed to prioritize HLA-compatible unrelated donors (URDs) with weak inhibitory KIR3DL1/HLA-Bw4 interaction, followed by donors with nontolerized activating KIR2DS1, and finally those with KIR centromeric B haplotype. During donor evaluation, we performed KIR genotyping and ranked 2079 URDs for 527 subjects with myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). Among all patients, 394 (75%) had at least 1 KIR-advantageous donor, and 263 (50%) underwent HCT. In patients with AML, KIR3DL1 weak inhibition provided protection from relapse. Compared with KIR3DL1-Weak Inhibiting donors, KIR3DL1-Noninteracting donors were associated with increased risk of relapse (HR, 2.97; 95% CI, 1.33-6.64; P = .008) and inferior event-free survival (EFS; HR, 2.14; 95% CI, 1.16-3.95; P = .015). KIR3DL1-Strong Inhibiting donors were associated with HR, 1.65 (95% CI, 0.66-4.08; P = .25) for AML relapse and HR, 1.6 (95% CI, 0.81-3.17; P = .1) for EFS when compared with the use of KIR3DL1-weak inhibiting donors. Donor KIR2DS1/HLA-C1 status and centromeric KIR haplotype-B content were not associated with decreased risk of AML relapse. There was no benefit to KIR-based donor selection in patients with MDS. This study demonstrates that donor KIR typing is feasible, and prioritization of donors with certain KIR3DL1 genotypes may confer a protection from relapse after HCT in patients with AML.

Cytotoxic lymphocytes target characteristic biophysical vulnerabilities in cancer.

Immune cells identify and destroy tumors by recognizing cellular traits indicative of oncogenic transformation. In this study, we found that myocardin-related transcription factors (MRTFs), which promote migration and metastatic invasion, also sensitize cancer cells to the immune system. Melanoma and breast cancer cells with high MRTF expression were selectively eliminated by cytotoxic lymphocytes in mouse models of metastasis. This immunosurveillance phenotype was further enhanced by treatment with immune checkpoint blockade (ICB) antibodies. We also observed that high MRTF signaling in human melanoma is associated with ICB efficacy in patients. Using biophysical and functional assays, we showed that MRTF overexpression rigidified the filamentous actin cytoskeleton and that this mechanical change rendered mouse and human cancer cells more vulnerable to cytotoxic T lymphocytes and natural killer cells. Collectively, these results suggest that immunosurveillance has a mechanical dimension, which we call mechanosurveillance, that is particularly relevant for the targeting of metastatic disease.

Specific Class I HLA Supertypes but Not HLA Zygosity or Expression Are Associated with Outcomes following HLA-Matched Allogeneic Hematopoietic Cell Transplant: HLA Supertypes Impact Allogeneic HCT Outcomes.

Maximizing the probability of antigen presentation to T cells through diversity in HLAs can enhance immune responsiveness and translate into improved clinical outcomes, as evidenced by the association of heterozygosity and supertypes at HLA class I loci with improved survival in patients with advanced solid tumors treated with immune checkpoint inhibitors. We investigated the impact of HLA heterozygosity, supertypes, and surface expression on outcomes in adult and pediatric patients with acute myeloid leukemia (AML), myelodysplastic syndrome, acute lymphoblastic leukemia, and non-Hodgkin lymphoma who underwent 8/8 HLA-matched, T cell replete, unrelated, allogeneic hematopoietic cell transplant (HCT) from 2000 to 2015 using patient data reported to the Center for International Blood and Marrow Transplant Research. HLA class I heterozygosity and HLA expression were not associated with overall survival, relapse, transplant-related mortality (TRM), disease-free survival (DFS), and acute graft-versus-host disease following HCT. The HLA-B62 supertype was associated with decreased TRM in the entire patient cohort (hazard ratio [HR], 0.79; 95% CI, 0.69 to 0.90; P = .00053). The HLA-B27 supertype was associated with worse DFS in patients with AML (HR = 1.21; 95% CI, 1.10 to 1.32; P = .00005). These findings suggest that the survival benefit of HLA heterozygosity seen in solid tumor patients receiving immune checkpoint inhibitors does not extend to patients undergoing allogeneic HCT. Certain HLA supertypes, however, are associated with TRM and DFS, suggesting that similarities in peptide presentation between supertype members play a role in these outcomes. Beyond implications for prognosis following HCT, these findings support the further investigation of these HLA supertypes and the specific immune peptides important for transplant outcomes.

Selection of allogeneic hematopoietic cell transplant donors to optimize natural killer cell alloreactivity.

Natural killer (NK) cells are potent mediators of the graft versus leukemia phenomenon critical to the success of allogeneic hematopoietic cell transplantation. Central to calibrating NK effector function via their interaction with class I human leukocyte antigens are the numerous inhibitory killer Ig-like receptors (KIR). The KIR receptors are encoded by a family of polymorphic genes, whose expression is largely stochastic and uninfluenced by human leukocyte antigens genotype. These features provide the opportunity to select hematopoietic cell donors with favorable KIR genotypes that confer enhanced protection from relapse via NK-mediated graft versus leukemia. Over the last 2 decades, a large body of work has emerged examining the use of KIR genotyping to stratify potential donors based on anticipated NK alloreactivity. Overall, these results support KIR-based donor selection for patients undergoing allogeneic hematopoietic cell transplantation for a diagnosis of acute myelogenous leukemia. Despite this, the underlying factors that control NK cell responsiveness are not completely understood, and opportunities remain to refine donor selection using NK cell receptor genotyping. In this review, we will summarize the relevant findings with respect to KIR genotyping as a selection parameter for allogeneic hematopoietic cell donors and address practical considerations with respect to KIR-based selection of donors for patients with myeloid neoplasia.

HLA-A alleles influencing NK cell function impact AML relapse following allogeneic hematopoietic cell transplantation.

HLA-B allotypes exhibiting the Bw4 epitope trigger variable inhibitory signaling of KIR3DL1 receptor types, where strong inhibitory HLA-B and KIR3DL1 allele combinations are associated with increased risk for relapse of acute myelogenous leukemia (AML) following allogeneic hematopoietic cell transplantation (HCT). Several HLA-A allotypes also exhibit the Bw4 epitope. Studies with natural killer (NK) cell clones have demonstrated NK inhibition via KIR3DL1 by HLA-A Bw4+ allotypes, but did not delineate strengths of inhibition or hierarchies of NK education. Using primary NK cells from healthy donors, we demonstrate that HLA-A*23, HLA-A*24, and HLA-A*32 proteins are expressed at different densities and exhibit different capacities to educate and inhibit KIR3DL1-expressing NK cells in vitro. Among the HLA-A Bw4+ allotypes, HLA-A*24 and HLA-A*32 demonstrate the strongest inhibitory capacity. To determine if HLA-A allotypes with strong inhibitory capacity have similar negative impact in allogeneic HCT as HLA-B Bw4+ allotypes, we performed a retrospective analysis of 1729 patients with AML who received an allogeneic HCT from a 9/10 or 10/10 HLA allele-matched unrelated donor. Examination of the donor-recipient pairs whose Bw4 epitope was exclusively contributed from HLA-A*24 and A*32 allotypes revealed that patients with HLA-A*24 who received an allograft from a KIR3DL1+ donor experienced a higher risk of disease relapse (hazard ratio, 1.65; 95% confidence interval, 1.17-2.32; P = .004) when compared with patients without a Bw4 epitope. These findings indicate that despite weak affinity interactions with KIR3DL1, common HLA-A allotypes with the Bw4 epitope can interact with KIR3DL1+ donor NK cells with clinically meaningful impact and provide additional insight to donor NK alloreactivity in HLA-matched HCT.

Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation.

BACKGROUNDAdoptive transfer of donor-derived EBV-specific cytotoxic T-lymphocytes (EBV-CTLs) can eradicate EBV-associated lymphomas (EBV-PTLD) after transplantation of hematopoietic cell (HCT) or solid organ (SOT) but is unavailable for most patients.METHODSWe developed a third-party, allogeneic, off-the-shelf bank of 330 GMP-grade EBV-CTL lines from specifically consented healthy HCT donors. We treated 46 recipients of HCT (n = 33) or SOT (n = 13) with established EBV-PTLD, who had failed rituximab therapy, with third-party EBV-CTLs. Treatment cycles consisted of 3 weekly infusions of EBV-CTLs and 3 weeks of observation.RESULTSEBV-CTLs did not induce significant toxicities. One patient developed grade I skin graft-versus-host disease. Complete remission (CR) or sustained partial remission (PR) was achieved in 68% of HCT recipients and 54% of SOT recipients. For patients who achieved CR/PR or stable disease after cycle 1, one year overall survival was 88.9% and 81.8%, respectively. In addition, 3 of 5 recipients with POD after a first cycle who received EBV-CTLs from a different donor achieved CR or durable PR (60%) and survived longer than 1 year. Maximal responses were achieved after a median of 2 cycles.CONCLUSIONThird-party EBV-CTLs of defined HLA restriction provide safe, immediately accessible treatment for EBV-PTLD. Secondary treatment with EBV-CTLs restricted by a different HLA allele (switch therapy) can also induce remissions if initial EBV-CTLs are ineffective. These results suggest a promising potential therapy for patients with rituximab-refractory EBV-associated lymphoma after transplantation.TRIAL REGISTRATIONPhase II protocols (NCT01498484 and NCT00002663) were approved by the Institutional Review Board at Memorial Sloan Kettering Cancer Center, the FDA, and the National Marrow Donor Program.FUNDINGThis work was supported by NIH grants CA23766 and R21CA162002, the Aubrey Fund, the Claire Tow Foundation, the Major Family Foundation, the Max Cure Foundation, the Richard "Rick" J. Eisemann Pediatric Research Fund, the Banbury Foundation, the Edith Robertson Foundation, and the Larry Smead Foundation. Atara Biotherapeutics licensed the bank of third-party EBV-CTLs from Memorial Sloan Kettering Cancer Center in June 2015.

Publisher Correction: Novel multiplex PCR-SSP method for centromeric KIR allele discrimination.

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Donor HLA-E Status Associates with Disease-Free Survival and Transplant-Related Mortality after Non In Vivo T Cell-Depleted HSCT for Acute Leukemia.

Previous studies have suggested that HLA-E may have a significant role in the outcome of matched unrelated hematopoietic stem cell transplantation (HSCT), especially for patients with acute leukemia. We used Center for International Blood and Marrow Transplant Research data and samples of 1840 adult patients with acute leukemia and their 10/10 HLA-matched unrelated donors to investigate the impact of HLA-E matching status as well as of donor/recipient (D/R) HLA-E genotype on post-HSCT outcome. Both patients and donors were HLA-E genotyped by next-generation sequencing. All patients received their first transplant in complete remission between 2000 and 2015. Median follow-up time was 90 months. Overall survival, disease-free survival (DFS), transplant-related mortality (TRM), and relapse incidence were primary endpoints with statistical significance set at .01. D/R HLA-E genotype analysis revealed a significant association of donor HLA-E*01:03/01:03 genotype with DFS (hazard ratio [HR] = 1.35, P = .0006) and TRM (HR = 1.41, P = .0058) in patients who received T cell replete (ie, without in vivo T cell depletion) transplants (n = 1297). As for D/R HLA-E matching, we did not identify any significant effect on any of the clinical outcome endpoints. In conclusion, this is the largest study to date reporting an improvement of DFS and TRM after matched unrelated HSCT by avoidance of HLA-E*01:03 homozygous donors in patients transplanted with T cell replete grafts for acute leukemia.