Cytomegalovirus Reactivation Shapes NK Cells Diversity Following Allogeneic Hematopoietic Stem Cell Transplantation

Abstract Natural killer (NK) cells are the first lymphocyte population to reconstitute following allogeneic hematopoietic stem cell transplantation (HSCT) and are key players in immune defense against viral infections and malignant transformation. NK cell numbers generally recover within the first month post-transplant, but the acquisition of mature NK cell phenotype and full functional competency can take over 6 months and is influenced by various host and donor factors. Cytomegalovirus (CMV) infection has been shown to modulate NK cell maturation after HSCT. The diversity of the NK cell repertoire is dictated by a variety of combinatorially expressed activating and inhibitory receptors that dictate the NK activation status. Moreover, whereas the expression of inhibitory receptors is primarily genetically determined, environmental factors such as viral infections influence the expression of activating receptors to a great extent.. We propose that assessment of diversity could provide a different perspective for the evaluation of the NK cell compartment after HSCT, since it is a quantitative measure that takes into account both the number and evenness of the different NK subpopulations. To better understand the factors that influence NK cell recovery after cord blood (CB) transplant (CBT) and specifically the influence of cytomegalovirus (CMV) infection on NK cell maturity, we used 40-parameter mass cytometry (CyTOF) to interrogate the NK cell repertoire. A panel including 37 monoclonal antibodies was designed to recognize NK cells lineage markers and receptors as well as intracellular markers such as transcription factors and adaptor proteins. We first evaluated and compared the diversity of NK cells in 10 CB units and peripheral blood (PB) from 20 healthy donors. We then examined the diversity of NK cells before and after CBT in 22 serially collected blood samples from in 10 CBT recipients. NK cell diversity was significantly lower in CB (mean 574, range 417-891) compared to PB samples from healthy donors (mean 3792, range 1284-5079; P=0.001), indicating less diversification within the naive CB NK compartment. After CBT, NK cell diversity was lower at earlier time point (Day30) (mean 1129, range 428-1768) compared to PB from healthy donors; P=0.01. The diversity of NK cells increased gradually over time following CBT (Day 30 mean 1129 range 428-1768; Day 60, mean 1185, range 515-1864; 4 months, mean 1711 range 597-2640). We also compared the diversity of NK cells in the PB of healthy CMV seronegative (n=10) and seropositive adult donors (n=10). The diversity of NK cells was higher in CMV seropositive vs. CMV seronegative healthy donors (3887 vs 2473; P=0.04). This difference in NK diversity was even more pronounced within the KIR positive (mean 1701, range, 981-2152) compared to the KIR negative subset (mean 551, range 456-647; P=0.02), indicating that CMV infection increases the richness of mature NK cells. In keeping with these findings, CMV infection after CBT was associated with a significantly greater diversity of NK cells, especially within the KIR positive compartment (mean 604, range 207-1035) compared to the KIR negative subset (mean 283, 257-457; P=0.025). However, in CMV negative patients, we found no difference in diversity within the KIR positive and negative subsets (mean 1120 vs. 1366; P=0.28). Taken together, these data suggest that NK cell diversity reflects NK cells differentiation and maturation, and that CMV shapes NK cell diversity, especially within the KIR positive compartment. To further understand how CMV influences NK cells diversity, we examined the top 15 NK cell subsets and their distribution at multiple timepoints before and after CMV reactivation post-CBT. CMV infection post-CBT was associated with a significant change in the distribution of NK subsets within the KIR positive population, with the top 15 subsets prior to CMV reactivation being mostly replaced by the emergence of new subsets. In contrast, the top 15 subsets within the KIR negative NK population remained stable. These data suggest that CMV drives NK cell maturation by differentiating KIR positive NK cells. In summary, we used high-dimensional single-cell data to evaluate NK cell reconstitution following HSCT. These data can help us better understand the biology of NK cell recovery after HSCT and discover the functional significance of NK cell diversity in the setting of viral infections. Disclosures Champlin: Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties.

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Activation Status Dictates the Function of Unlicensed Natural Killer Cells

Blood Advances ◽

10.1182/bloodadvances.2021004589 ◽

2021 ◽

Author(s):

Ethan G Aguilar ◽

Cordelia Dunai ◽

Sean J. Judge ◽

Anthony Elston Zamora ◽

Lam T. Khuat ◽

...

Keyword(s):

Viral Infection ◽

Nk Cells ◽

Natural Killer ◽

Viral Infections ◽

Nk Cell ◽

T Cell Response ◽

High Dose ◽

Hematopoietic Stem ◽

Innate Defense ◽

Activation Status

Natural Killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class I molecules resulting in differential responses upon activation in a process called "licensing" or "arming". NK cells expressing receptors that bind self-MHC are considered licensed due to augmented effector lytic function capability compared to unlicensed subsets. However, we demonstrated unlicensed NK subsets instead positively regulate the adaptive T cell response during viral infections due to localization and cytokine production. We demonstrate here that the differential effects of the two types of NK subsets is contingent on the environment using viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) MCMV leads to a loss of licensing-associated differences as compared to mice with low-dose infection, as the unlicensed NK subset no longer localized in lymph nodes (LN), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled with the phenotypes of both human and mouse NK cells in a HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to effects of subset depletion in T-replete models, the licensed NK cell subsets still dominated anti-viral responses post-HSCT. Overall, our results highlight the intricate tuning of the NK cells and how it impacts overall immune responses with regard to licensing patterns, as it is dependent on the level of stimulation and their activation status.

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Poor NKG2C+ Adaptive NK Cell Recovery at Early Stage after Allo-HSCT Increased the Occurrence of Refractory Cytomegalovirus Infection

Blood ◽

10.1182/blood-2018-99-115772 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 360-360

Author(s):

Xingxing Yu ◽

Xiangyu Zhao ◽

Xunhong Cao ◽

Xuefei Liu ◽

Xuying Pei ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Solid Organ ◽

Cmv Infection ◽

Cell Recovery ◽

Post Transplantation ◽

Cell Counts ◽

Ifn Γ ◽

The Absolute ◽

Cmv Reactivation

Abstract Backgroud: Refractory cytomegalovirus (CMV) infection remains important causes of morbidity and mortality after allogeneic hematopoietic stem cells transplantation (allo-HSCT). Previous researches reported that adaptive immunity, such as CD8+ CMV-CTL, plays an important role in the in the control of refractory CMV infection. In mouse, Lanier et al. found there existed subsets of adaptive NK cells with the features of expanding, contracting after control of mouse CMV, and generating long-lived "memory" NK cells. In human, these adaptive NK cells were initially identified based on the high expression of the NKG2C which against HCMV through their cytotoxic potential and the production of TNF-α and IFN-γ upon Ab-mediated stimuli in vitro. Meanwhile, the expression levels of the NKG2C+ adaptive NK cells has been positively correlated with the NKG2C copy number. Several researchers had found that NKG2C+ adaptive NK cells persistent expanded and were potent producers of IFN-γ during CMV reactivation after solid-organ transplant or allo-HSCT. However, the role of NKG2C+ adaptive NK cells on refractory CMV reactivation were still unknown. Whether the rapid reconstitution of NKG2C+ adaptive NK cells can reduce the refractory CMV reactivation merit to be investigated. Aims:In this research, we had investigated the impacts of the quantity and quality recovery of NKG2C+ adaptive NK cells on the occurrence of refractory CMV infection. Method: At first, continuous 364 patients underwent allo-HSCT since June 2012 to February 2016 were prospectively enrolled and we retrospectively analyzed the correlationship between their donor NKG2C genotype and refractory CMV infection occurrence post transplantation. Secondly, the second cohort comprising continuous 125 patients underwent allo-HSCT since May 2016 to April 2017 were prospectively enrolled to analyzed the effect of donor NKG2C genotype on NKG2C+ adaptive NK cell recovery as well as the effect of NKG2C+ adaptive NK cell recovery on refractory CMV infection. The cytotoxicity of reconstituting NKG2C+ adaptive NK cells were evaluated against K562 cells, AD169 CMV stain infected MRC-5 cells, and UL40 peptide pulsed 721.221 cells to detect the anti-tumor or anti-CMV function of NKG2C+ adaptive NK cells. Results: Firstly, from the first cohort, we found that donor NKG2C gene deletion was an independent prognostic factor for refractory CMV reactivation (P=0.010) through the multivariate analysis. Then, through in-depth investigation from the second cohort, we found that the absolute cell counts recovery and anti-tumor function of NKG2C+ adaptive NK cells were both significantly lower in patients accepting NKG2C+/del donor than those patients accepting NKG2C+/+ donors at day 30, 90, and180, respectively after transplantation. There was no NKG2C+ adaptive NK cell recovery post transplantation in the patients who accepted NKG2Cdel/del donors. Meanwhile, anti-CMV function recovery of NKG2C+ adaptive NK cells in patients with NKG2C+/del donors were significantly lower than those patients with NKG2C+/+ donors at day 30 post transplantation. Furthermore, we further analyzed the relationship between the early reconstitution of NKG2C+ adaptive NK cells and refractory CMV infection occurrence. The patients were divided into three groups: no CMV, CMV reactivation (persistent time of CMV infection <2 weeks), and refractory CMV infection (persistent time of CMV infection>2 weeks). We found that the absolute cell counts of NKG2C+ adaptive NK cells in refractory CMV group was significantly lower than that of other two groups at day 30 transplantation. When the patients were devided into high and low level groups based on the ROC cut-off percentage of NKG2C+ adaptive NK cells (1.42%), the result revealed that the patients with lower level of NKG2C+ adaptive NK cells at day 30 post-HSCT had an higher cumulative incidence rate of refractory CMV infection (81.1%) comparing with the higher one (40.5%) (P=0.0014). Moreover, Cox regression model further demonstrated that the lower level of NKG2C+ adaptive NK cells at day 30 post-HSCT was significantly associated with refractory CMV infection (HR=2.578, 95% CI 1.379-4.21, P=0.003). Summary/Conclusion: Our results indicated that donor NKG2C deletion damaged the reconstitution of NKG2C+ adaptive NK cells after allo-HSCT, therefore increased the occurrence of refractory CMV infection post transplantation. Disclosures No relevant conflicts of interest to declare.

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Phenotypic and Functional Characterization of NK Cells in αβT-Cell and B-Cell Depleted Haplo-HSCT to Cure Pediatric Patients with Acute Leukemia

Cancers ◽

10.3390/cancers12082187 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2187

Author(s):

Raffaella Meazza ◽

Michela Falco ◽

Fabrizio Loiacono ◽

Paolo Canevali ◽

Mariella Della Chiesa ◽

...

Keyword(s):

Acute Leukemia ◽

Nk Cells ◽

Nk Cell ◽

Functional Characterization ◽

Cell Subset ◽

Cell Phenotype ◽

Cell Repertoire ◽

Hematopoietic Stem ◽

Γδt Cells ◽

Effector Cells

NK cells can exert remarkable graft-versus-leukemia (GvL) effect in HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Here, we dissected the NK-cell repertoire of 80 pediatric acute leukemia patients previously reported to have an excellent clinical outcome after αβT/B-depleted haplo-HSCT. This graft manipulation strategy allows the co-infusion of mature immune cells, mainly NK and γδT cells, and hematopoietic stem cells (HSCs). To promote NK-cell based antileukemia activity, 36/80 patients were transplanted with an NK alloreactive donor, defined according to the KIR/KIR-Ligand mismatch in the graft-versus-host direction. The analysis of the reconstituted NK-cell repertoire in these patients showed relatively high proportions of mature and functional KIR+NKG2A−CD57+ NK cells, including the alloreactive NK cell subset, one month after HSCT. Thus, the NK cells adoptively transfused with the graft persist as a mature source of effector cells while new NK cells differentiate from the donor HSCs. Notably, the alloreactive NK cell subset was endowed with the highest anti-leukemia activity and its size in the reconstituted repertoire could be influenced by human cytomegalovirus (HCMV) reactivation. While the phenotypic pattern of donor NK cells did not impact on post-transplant HCMV reactivation, in the recipients, HCMV infection/reactivation fostered a more differentiated NK-cell phenotype. In this cohort, no significant correlation between differentiated NK cells and relapse-free survival was observed.

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Relevance of Polymorphic KIR and HLA Class I Genes in NK-Cell-Based Immunotherapies for Adult Leukemic Patients

Cancers ◽

10.3390/cancers13153767 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3767

Author(s):

Léa Dubreuil ◽

Patrice Chevallier ◽

Christelle Retière ◽

Katia Gagne

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Current Knowledge ◽

Killer Cell ◽

Hla Class I ◽

Class I ◽

Cell Repertoire ◽

Hematopoietic Stem ◽

Structural Formation ◽

The Impact

Since the mid-1990s, the biology and functions of natural killer (NK) cells have been deeply investigated in healthy individuals and in people with diseases. These effector cells play a particularly crucial role after allogeneic hematopoietic stem-cell transplantation (HSCT) through their graft-versus-leukemia (GvL) effect, which is mainly mediated through polymorphic killer-cell immunoglobulin-like receptors (KIRs) and their cognates, HLA class I ligands. In this review, we present how KIRs and HLA class I ligands modulate the structural formation and the functional education of NK cells. In particular, we decipher the current knowledge about the extent of KIR and HLA class I gene polymorphisms, as well as their expression, interaction, and functional impact on the KIR+ NK cell repertoire in a physiological context and in a leukemic context. In addition, we present the impact of NK cell alloreactivity on the outcomes of HSCT in adult patients with acute leukemia, as well as a description of genetic models of KIRs and NK cell reconstitution, with a focus on emergent T-cell-repleted haplo-identical HSCT using cyclosphosphamide post-grafting (haplo-PTCy). Then, we document how the immunogenetics of KIR/HLA and the immunobiology of NK cells could improve the relapse incidence after haplo-PTCy. Ultimately, we review the emerging NK-cell-based immunotherapies for leukemic patients in addition to HSCT.

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The Impact of Immunosuppressive Therapy on an Early Quantitative Reconstitution of NK Cells after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v104.11.4946.4946 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4946-4946

Author(s):

Sebastian Giebel ◽

Joanna Dziaczkowska ◽

Iwona Wylezol ◽

Jerzy Wojnar ◽

Malgorzata Krawczyk-Kulis ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Nk Cell ◽

The Other ◽

Cell Recovery ◽

Hematopoietic Stem ◽

Preparative Regimen ◽

The Impact

Abstract In a previous study we demonstrated that in case of KIR-ligand incompatibility, NK cells may mediate graft-versus-leukemia effect resulting in improved outcome in a setting of unrelated donor-hematopoietic stem cell transplantation (URD-HSCT). This effect, however, varies and depends on the preparative regimen including the use of pre-transplant antithymocyte globulin (ATG) as an in vivo T-depletion. On the other hand, the impact of ATG on NK cell recovery has not been determined so far. The goal of this study was to analyze the influence of various immunosuppressive modalities used for acuteGVHD prophylaxis or treatment on the early NK cell reconstitution after allogeneic HSCT. METHODS: We analyzed the number of peripheral blood NK cells with the use of flow cytometry on day +30 (+/−2) after alloHSCT. NK cells were defined as CD3-CD56+ cells. Eighty alloHSCT recipients were included in the study; 43 patients were given transplant from an HLA-identical sibling (sibHSCT) whereas in 37 cases the donor was an unrelated vulunteer. ATG (6–10 mg/kg) as a part of preparative regimen was used on days −3, −2, −1 in case of URD-HSCT, but not sibHSCT. Besides, acuteGVHD prophylaxis consisted of Cyclospirin A (3 mg/kg) since day −1 and short-course Methotrexate (three or four doses). For patients transplanted before year 2002 (38/80 analyzed cases) - prednisolone 0.5 mg/kg since day +1 until day +28 was additionally administered. Metylprednisolone (2 mg/kg) was used as a first-line therapy of acuteGVHD. RESULTS: We did not find any difference regarding the number of NK cells on day +30 after alloHSCT between patients given pre-transplant ATG and those in whom no form of T-depletion has been administered: 130 (11–841) x106/L vs. 116 (65–841) x106/L (p=NS). In contrast, the use of steroids for acuteGVHD prophylaxis was associated with significant impairment of the quantitative NK cell reconstitution: 106 (10–694) x106/L vs. 211 (32–890) x106/L NK cells on day +30 (p=0.004). The incidence of acuteGVHD as well as the use of steroids for acuteGVHD treatment had no impact on the number of circulating NK cells. None of the other factors analyzed including the number of methotrexate doses, source of stem cells, the number of CD34+ cells transplanted, and age was found to influence an early NK cell recovery. CONCLUSIONS: Although ATG used as a part of pre-transplant conditioning regimen circulates and remains active for several weeks after alloHSCT, it does not seem to have any impact on an early NK cell quantitative reconstitution. In contrast, prolonged administration of low-dose steroids may impair the NK cell recovery. Since NK cells are considered a potential tool for a cellular therapy of hematologic and other malignancies, our findings should be taken into account when planning this kind of treatment in the context of allotransplantation. In addition, our results support the earlier hypothesis that the preparative regimens containing ATG may enable elucidation of a potential benefit from KIR-ligand incompatibility.

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Temporal, Quantitative and Functional Characteristics of Single-KIR Positive Alloreactive NK Cell Recovery Account for Impaired Graft Versus Leukemia Activity after Haploidentical HSCT.

Blood ◽

10.1182/blood.v110.11.3274.3274 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3274-3274

Author(s):

Luca Vago ◽

Barbara Forno ◽

Elisabetta Zino ◽

Simona Di Terlizzi ◽

Maria T. Lupo Stanghellini ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Therapeutic Option ◽

Flow Cytometric Analysis ◽

Cell Activity ◽

Biological Data ◽

Cell Recovery ◽

Hematopoietic Stem

Abstract Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT) is a promising therapeutic option for patients lacking a fully compatible donor. Due to extensive T cell depletion, Natural Killer (NK) cell activity represents the only immunological protection against disease relapse for the first months after haplo-HSCT. Clinical studies have associated donor-recipient incompatibility for Human Leukocyte Antigen (HLA) ligands of Killer Immunoglobulin-like Receptors (KIR), with a marked anti-leukemic activity. Alloreactive donor NK cells carrying a single KIR whose ligand is missing in the recipient mediate a potent graft vs. leukemia effect, resulting in reduced incidence of relapse and increased Overall Survival (OS). These exciting results have recently been challenged by conflicting clinical and biological data from different groups. In the present study, we have characterized reconstitution of NK cells, in particular of alloreactive single-KIR+ NK cells, in 58 patients who received CD34+ selected haplo-HSCT for high-risk hematologic malignancies. One month after haplo-HSCT CD56bright/CD56dim NK cell subsets were subverted in their proportions and phenotypic features, accounting for enrichment in maturation intermediates. We show that CD25 and CD117 deregulation by CD56bright, and NKG2A and CD62L by CD56dim, are intrinsic to NK cell physiologic differentiation and support a sequential CD56bright-to-CD56dim NK cell maturation. Consistently, the in vitro functional potential of these maturation intermediates against leukemic blasts was heavily impaired, both in terms of cytotoxicity and of cytokine release. Full mature receptor repertoire reconstitution took at least three months. Alloreactive single-KIR+ NK cells had highly variable frequency ranging from less than 1% to more than 30% of NK cells circulating at 90–120 days after transplantation, independently from predicted NK alloreactivity. Importantly, out of three patients with predicted NK alloreactivity, none had a relative expansion of alloreactive single-KIR+ cells, accounting for less than 1% of circulating NK cells in two of them. As demonstrated by flow cytometric analysis of NK cell CD107a mobilization in response to the HLA class I negative target 721.221, single-KIR+ NK cells at three months after haplo-HSCT showed a not yet fully developed functional reactivity, which was recovered to donor-levels only at later time-points. In line with these observations, clinical outcome of haplo-HSCT was not affected in any way by the presence of donor NK alloreactivity. The incidence of relapse was virtually identical in patients transplanted from alloreactive or non-alloreactive donors. Taken together, our data shed new light onto the kinetics of NK cell differentiation in vivo and suggest that NK alloreactivity could be best exploited by the use of mature donor single-KIR+ selected alloreactive NK cells.

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Genetic and Molecular Basis of Heterogeneous NK Cell Responses against Acute Leukemia

Cancers ◽

10.3390/cancers12071927 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1927 ◽

Cited By ~ 2

Author(s):

Dhon Roméo Makanga ◽

Francesca Da Rin de Lorenzo ◽

Gaëlle David ◽

Catherine Willem ◽

Léa Dubreuil ◽

...

Keyword(s):

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Lymphoblastic Leukemia ◽

Killer Cell ◽

Cell Repertoire ◽

Hematopoietic Stem ◽

Cell Responses ◽

Cmv Status ◽

Selection Of

Natural killer (NK) cells are key cytotoxic effectors against malignant cells. Polygenic and polymorphic Killer cell Immunoglobulin-like Receptor (KIR) and HLA genes participate in the structural and functional formation of the NK cell repertoire. In this study, we extensively investigated the anti-leukemic potential of NK cell subsets, taking into account these genetic parameters and cytomegalovirus (CMV) status. Hierarchical clustering analysis of NK cell subsets based on NKG2A, KIR, CD57 and NKG2C markers from 68 blood donors identified donor clusters characterized by a specific phenotypic NK cell repertoire linked to a particular immunogenetic KIR and HLA profile and CMV status. On the functional side, acute lymphoblastic leukemia (ALL) was better recognized by NK cells than acute myeloid leukemia (AML). However, a broad inter-individual disparity of NK cell responses exists against the same leukemic target, highlighting bad and good NK responders. The most effective NK cell subsets against different ALLs expressed NKG2A and represented the most frequent subset in the NK cell repertoire. In contrast, minority CD57+ or/and KIR+ NK cell subsets were more efficient against AML. Overall, our data may help to optimize the selection of hematopoietic stem cell donors on the basis of immunogenetic KIR/HLA for ALL patients and identify the best NK cell candidates in immunotherapy for AML.

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Adaptive Subsets Limit the Anti-Tumoral NK-Cell Activity in Hepatocellular Carcinoma

Cells ◽

10.3390/cells10061369 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1369

Author(s):

Charlotte Rennert ◽

Catrin Tauber ◽

Pia Fehrenbach ◽

Kathrin Heim ◽

Dominik Bettinger ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cirrhosis ◽

Nk Cells ◽

Nk Cell ◽

Cell Activity ◽

Cell Subset ◽

Cell Repertoire ◽

Healthy Donors ◽

B Virus ◽

The Impact

Hepatocellular carcinoma (HCC) is a global health burden with increasing incidence, poor prognosis and limited therapeutic options. Natural killer (NK) cells exhibit potent anti-tumoral activity and therefore represent potential targets for immunotherapeutic approaches in HCC treatment. However, the anti-tumoral activity of NK cells in HCC associated with different etiologies, and the impact of the heterogeneous NK cell subset, e.g., adaptive and conventional subsets, are not understood in detail. By comparatively analyzing the NK-cell repertoire in 60 HCC patients, 33 liver cirrhosis patients and 36 healthy donors (HD), we show in this study that the NK-cell repertoire is linked to HCC etiology, with increased frequencies of adaptive NK cells in Hepatitis B virus (HBV)-associated HCC. Adaptive NK cells exhibited limited anti-tumoral activity toward liver cancer cells; however, this was not a result of a specific NK-cell impairment in HCC but rather represented an intrinsic feature, since the characteristics of circulating and intra-tumoral adaptive NK cells were conserved between HD, HCC and liver cirrhosis patients. Hence, the expansion of adaptive NK cells with reduced anti-tumoral activity, detectable in HBV-associated HCC, may have implications for tumor surveillance and therapy.

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Adaptive subsets limit the anti-tumoral NK-cell activity in Hepatocellular Carcinoma

10.1101/2021.04.16.440140 ◽

2021 ◽

Author(s):

Charlotte Rennert ◽

Catrin Tauber ◽

Pia Fehrenbach ◽

Kathrin Heim ◽

Dominik Bettinger ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cirrhosis ◽

Nk Cells ◽

Nk Cell ◽

Cell Activity ◽

Cell Subset ◽

Cell Repertoire ◽

Healthy Donors ◽

B Virus ◽

Infiltrating Lymphocytes

Background and Aims: Hepatocellular carcinoma (HCC) is a global health burden with increasing incidence, poor prognosis and limited therapeutic options. Natural killer (NK) cells exhibit potent anti-tumoral activity and therefore represent potential targets for immunotherapeutic approaches in HCC treatment. However, the human NK-cell repertoire is highly diverse including conventional and adaptive NK cells that differ in phenotype and effector function. Adaptive NK-cell frequencies are increased in association with HCMV (human cytomegalovirus) seropositivity that is also common in HCC patients. In this study, we aimed to gain a better understanding of the NK-cell repertoire and the associated anti-tumoral activity in HCC patients. Methods: In-depth phenotypic and functional flow-cytometry analyses of the HCMV-associated NK cell-repertoire obtained from 57 HCC patients, 33 liver cirrhosis patients and 36 healthy donors (HD). Results: First, adaptive subsets are present in all three cohorts with conserved characteristics in patients with liver cirrhosis and HCC. Second, adaptive NK cells can be isolated from HCC tissue however lack features of tissue-residency and thus probably represent circulating/infiltrating lymphocytes. Third, the anti-tumoral activity by adaptive NK cells is reduced compared to conventional NK-cell subsets, also in HCC. Lastly, frequencies of adaptive NK cells were increased in patients suffering from Hepatitis B virus-associated HCC providing a link between etiology and the NK-cell repertoire in HCC. Conclusion: Adaptive NK cells limit the anti-tumoral activitity of NK cells in HCC, especially in association with HBV infection that is accompanied by an expansion of this NK cell subset.

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Impact of Graft Source on Immune Recovery: Comparions Between Unrelated Umbilical Cord Blood (UCB), HLA Matched Sibling (Sib) Donor and Autologous (Auto) Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.3731.3731 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3731-3731

Author(s):

Sarah Cooley ◽

John E. Wagner ◽

Claudio Brunstein ◽

Mie Hagiwara ◽

Giordi Orreggio ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Nk Cells ◽

Clinical Outcomes ◽

Nk Cell ◽

Cd4 Count ◽

Cell Recovery ◽

Hematopoietic Stem ◽

The Absolute

Abstract Abstract 3731 Both T cell and natural killer (NK) cell reconstitution have been shown to affect clinical outcomes after hematopoietic stem cell transplantation (HSCT). Killer immunoglobulin-like receptor (KIR) interactions between alloreactive NK cells and their targets can prevent relapse, but may be dysregulated, especially after T cell replete HSCT. T cell recovery is also affected by the stem cell source and T cell content of the graft. To better understand the effects of various NK and T cell subsets we evaluated lymphocyte recovery in 304 adult patients who received either UCB (n=116), Sib (n=84) or Auto (n=94) HSCT for hematologic malignancies between 2003 and 2010 at the University of Minnesota. Peripheral blood mononuclear cells obtained at 3 months after HSCT were stained with CD56, CD3, CD4, CD8, and a cocktail of anti-NK cell KIR antibodies to determine the relative percentage of lymphocyte subsets by flow cytometry. The absolute lymphocyte count (ALC) was measured and used to calculate the absolute (Abs) number of T and NK cells and their subsets. ALC recovery at 3 months was similar among groups (UCB: 901.9 ± 74.5, Sib 890.2 ± 73.0 and Auto 1076.7± 69.4 cells/ul). Abs NK cells were highest in the UCB cohort (375.4 ± 24.9) vs. Sib (183.8 ± 15.4; p<0.0001) or Auto (160.7 ± 11.0; p<0.0001), as were the CD56bright and KIR+ subsets (data not shown). In contrast, Abs T cell recovery was lowest in the UCB group (300.8 ± 39.6) vs. Sib (578.5 ± 57.9; p<0.0001) or Auto (737.3 ± 60.4; p<0.0001). Accordingly, the lowest Abs CD4 count was in the UCB group (158.8 ± 14.7) vs. Sib (272.5 ± 23.5; p<0.0001) or Auto (223.6 ± 20.2; p=0.01), with a similar pattern observed for Abs CD8 counts. We then examined the effect of lymphocyte recovery on clinical outcomes. Multivariate models were constructed for each transplant group with relevant covariates (risk status, conditioning, sex, age, number of UCB units, CMV status, HLA matching (4/6, 5/6, or 6/6), and ABO matching). The most significant effect of lymphocyte recovery on outcomes was observed specifically in the UCB group, where higher ALC was associated with improved OS with a hazard ratio (HR) of 0.86 (95% CI 0.78–0.95) for each unit increase in ALC of 100 cells/ul (p <0.01). A similar trend was observed in Sib recipients but not in the Auto group. Specifically, increases in Abs T cells (HR 0.75 [95% CI 0.58–0.98]; p=0.034), Abs CD4 count (HR 0.63 [95% CI 0.42–0.95]; p=0.03), Abs CD8 count (HR 0.31 [95% CI 0.13–0.73]; p=0.01) and to a lesser extent Abs NK cells (HR 0.85 [95% CI 0.71–1.02]; p=0.085) were associated with improved OS. In the Sib cohort, higher Abs CD4 count was associated with improved OS (HR 0.43 [95% CI 0.20–0.92]; p=0.03) and decreased relapse (HR 0.37 [95% CI 0.37–1.00]; p=0.02), with no other factor having a significant impact. In the Auto group, only Abs NK (HR 0.40 [95% CI 0.16–0.99]; p=0.05) and to a lesser extent Abs KIR+ NK cells (HR 0.17 [95% CI 0.02–1.36]; p=0.09) were associated with improved OS but no other outcomes. The effect of Abs CD4 count on OS in all groups is shown in Figure 1 with survival stratified by quartiles. Figure 1: Figure 1:. In summary, rapid recovery of T cells predicts significantly better survival in patients undergoing UCB and Sib HSCT, while the NK cell effects are less pronounced. In contrast, NK cell effects predominate after Auto HCT. This suggests that more rapid T cell recovery is critical for survival and that defects in NK cell education after allogeneic HSCT may affect their function such that just increasing numbers may not be sufficient for clinical benefit. Appropriate modifications to immune suppression or the use of agents that promote T cell (IL-7) and/or NK cell (IL-15) function and survival may positively influence survival outcomes. Disclosures: No relevant conflicts of interest to declare.

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