Absolute Natural Killer Cell Counts Research Articles

Prognostic impact of absolute peripheral blood NK cell count after four cycles of R-CHOP-like regimen treatment in patients with diffuse large B cell lymphoma

As a subtype of lymphocyte, natural killer (NK) cell is the first line of defense that shows a strong function in tumor immunotherapy response and clinical outcomes. The current study aims to investigate the prognostic influence of peripheral blood absolute NK cell count after four cycles of rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) treatment (NKCC4) in diffuse large B cell lymphoma (DLBCL) patients. A total of 261 DLBCL patients treated with R-CHOP from January 2018 to September 2022 were enrolled. The low NKCC4 was observed in patients who died during the study period compared with survival individuals. A NKCC4 < 135 cells/μl had a remarkable negative influence in overall survival and progression-free survival (PFS) compared to a NKCC4 ≥ 135 cells/μl (p < 0.0001 and p < 0.0004, respectively). In addition, the OS and PFS were synergistically lower in a NKCC4 < 135 cells/μl group among DLBCL patients with GCB type or high IPI. In conclusion, this study indicates NCKK4 as a valuable marker in clinical practice and provides an insight for combination treatment of R-CHOP to improve outcomes of DLBCL patients.

Factors Affecting Natural Killer Cell Recovery after Autologous Stem Cell Transplantation in Multiple Myeloma

Introduction:Natural killer (NK) cells are large granular lymphocytes, very potent effector lymphocytes that can induce cytotoxicity against a vast array of tumors without the need for antigen sensitization or antigen presentation by MHC class I. NK cells are believed to play important role in immune surveillance for cancer, limiting neoplastic progression, and effectors of anti-tumor therapies -by introducing Abs that block NK cell inhibitory receptors yielding improved NK cell-mediated lysis-Retrospective studies demonstrated a significant correlation of NK cell recovery after autologous stem cell transplantation (ASCT) in multiple myeloma (MM) and disease outcomes. Higher absolute NK cell count one month after ASCT is associated with longer progression-free survival (PFS) and decreased risk of relapse. We aimed at investigating factors affecting NK cell recovery after ASCT in patients with multiple myeloma.Method:We designed a prospective cohort study, evaluating the potential factors that could affect NK cell recovery after ASCT. We included participants undergoing frontline ASCT for MM. We excluded participants with non-secretory myeloma or other malignancies that require active treatment, or with autoimmune disorders.All participants received conditioning regimen consisted of melphalan 200 mg/m2 on day -2. In patients older than 65 years old given 100mg/m2 for 2 days. Dose reduction to 140 mg/m2 for patients with serum creatinine > 2.0 mg/dL. Hematopoietic growth factor (peg-filgrastim 6 mg once subcutaneously) was given on day +1.Absolute NK cell count (CD3-, CD56+ cells) was determined using flow cytometric immunophenotyping of peripheral blood two-three months after ASCT. To determine variables that affected NK cell recovery after ASCT, absolute NK cell count was dichotomized to less than the lower limit of normal “NK < 76” and normal “NK > or equal 76” following institutional normal reference range which is consistent with ARUP NK cell enumeration reference. Statistical analysis was performed using Fisher-Exact test for categorical variable and Wilcoxon rank test for continuous variables.Results:We analyzed the results of fifteen patients treated with frontline ASCT for multiple myeloma. With a median age at diagnosis of 60 years, ranging from 47 - 70 years. Of which 53 percent females and 47 percent males. Patients' baseline characteristics are described in Table 1Our data showed that 63 percent of patients with post-transplantation ALC > 1000 (5 of 8 patients), and 73 percent for ALC > 500, (8 of 11 patients), had normal absolute NK cell count; with a correlation coefficient of 0.3 (Pearson Correlation Coefficients), suggesting a moderate linear correlation between absolute NK cell count and ALC two-three months after ASCT.Patients' gender, age at ASCT, and comorbidity index (SORROR score) did not differ between the two groups of patients, P-value= 0.6193, 0.8454, 0.1721, respectively. Additionally, disease burden at the time of diagnosis, presence of CRAB criteria, involved free light chain and monoclonal immunoglobulins; all did not differ between the two cohorts. (Table 2)Medications received before ASCT did affect the absolute NK cell count with a higher proportion of normal absolute NK cell count, and higher mean absolute NK cell in the cohort received double-class (PI plus IMiDs) when compared to triple-class (PI plus IMiDs plus Anti-CD 38 mAb) therapy before transplantation (mean NK cell count 125 and 53 Cell/μL, respectively), with marginal statistical significance P-value= 0.0667 (Fisher's Exact Test).Discussion:Our study showed that the addition of anti-CD38 mAb to myeloma induction regimen, associated with compromised NK cell recovery. Casneuf et al reported similar findings, in daratumumab-treated myeloma patients, total and activated NK-cell counts reduced rapidly in peripheral blood after the first dose, remained low throughout treatment, and recovered after treatment ended. Similar reductions were observed in the bone marrow.Conclusion:Medications received before transplantation potentially affect NK cell recovery in multiple myeloma patients. Although, the addition of anti-CD38 mAb to the myeloma induction regimen is associated with deeper response, its impact on immune reconstitution needs to be investigated on a larger sample size. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FC 129CHANGES IN PERIPHERAL NK CELLS IN KIDNEY TRANSPLANT RECIPIENTS WITH AND WITHOUT HLA DSA

Abstract Background and Aims Antibody-mediated rejection (ABMR) is a frequent cause of renal allograft loss. There is increasing evidence of the role of Natural Killer (NK) cells in the establishment of ABMR damage. Our group described that patients with donor-specific antibodies (DSA) and ABMR present higher proportions of NKG2A+ NK cell subset in peripheral blood than those without HLA DSA or HLA antibodies. Method We selected 177 kidney transplant recipients (KT) with renal biopsies 2011-2017: 77 with ABMR (DSA+: 53, DSA-: 24) and 100 without ABMR (DSA+: 15, DSA-: 85). We assessed graft survival with a median time of follow-up since the renal biopsy of 53 months. In 138 KT we evaluated the peripheral blood NK cell immunophenotyping and its value as a prognostic biomarker. Results Graft survival was worse in ABMR-KT at the end of follow-up (p&amp;lt;0.001) independently of DSA detection (p=0.63). Regarding NK cell immunophenotyping, we observed a lower proportion and absolute NK cell count in ABMR+DSA+-KT and ABMR+DSA--KT compared with ABMR-DSA--KT (p=0.027, p=0.017). ABMR+DSA+-KT showed higher proportion of NKG2A+ NK cells compared with ABMR-DSA--KT (p=0.007). All ABMR+ patients, independently of DSA detection, presented lower absolute NKG2A- NK cell count in comparison with ABMR-DSA--KT (p=0.001, p=0.017). Finally, a proportion of NKG2A- &amp;lt;30% was associated with lower graft survival 36 months after graft biopsy with ABMR (p=0.067) (Figure). Conclusion Graft survival is worse in ABMR+ compared with ABMR- KT independently of DSA detection. Kidney transplant recipients with ABMR show reduced peripheral absolute numbers of NK cells and NKG2A- NK cells regardless of undetectable DSA. This NK cell phenotype associated with a worse medium-term graft survival in cases with ABMR.

A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation.

Background: Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay requires large amounts of blood and a rapid PBMC isolation step. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases.Methods: Peripheral blood of healthy volunteers (n = 28) and patients with liver diseases, including hepatocellular carcinoma (n = 19) and liver cirrhosis (n = 7), was analyzed for complete blood count, absolute NK cell count, and NK cell activity (NKA). NKA was evaluated in three assay types: an FC-based overnight WB NK cytotoxicity assay using carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells in the presence of various cytokine combinations [including interleukin (IL)-2, IL-18, and IL-21], an FC-based 4-h PBMC NK cytotoxicity assay, and an FC-based CD107a degranulation assay using WB and PBMCs.Results: Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). A good correlation was observed between WB and PBMC NK cytotoxicity assays; absolute NK cell counts were better correlated with the WB NK cytotoxicity assay than with the PBMC NK cytotoxicity assay. This WB NK cytotoxicity assay showed that patients with liver diseases had significantly lower NK cytotoxicity than healthy volunteers, under stimulation with various cytokines (p < 0.001).Conclusion: The proposed FC-based overnight WB NK cytotoxicity assay correlates well with the conventional 4-h PBMC NK cytotoxicity assay, demonstrating future potential as a supportive assay for clinical laboratory research and observational studies.

Successful treatment-free remission in chronic myeloid leukaemia and its association with reduced immune suppressors and increased natural killer cells.

There is currently no biomarker that reliably predicts treatment-free remission (TFR) in chronic myeloid leukaemia (CML). We characterised effector and suppressor immune responses at the time of tyrosine kinase inhibitor (TKI) cessation in patients from the CML8 and CML10 clinical studies. Natural killer (NK) cells with increased expression of activating NK receptors were higher in patients who achieved TFR. There was no difference in the proportion of CD4+ or CD8+ T cells. Furthermore, we found that FoxP3+ regulatory T cells (T reg) and monocytic myeloid-derived suppressor cells (Mo-MDSCs) were concomitantly decreased in TFR patients, suggesting that the effector and suppressor arms of the immune system work in concert to mediate TFR. A discovery cohort (CML10) was used to generate a predictive model, using logistic regression. Patients classified into the high-risk group were more likely to relapse when compared with the low-risk group (HR 7·4, 95% CI 2·9-19·1). The model was successfully validated on the independent CML8 cohort (HR 8·3, 95% CI 2·2-31·3). Effective prediction of TFR success may be obtained with an effector-suppressor score, calculated using absolute NK cell, T reg, and Mo-MDSC counts, at TKI cessation, reflecting the contribution of both immune suppressors and effectors in the immunobiology underlying successful TFR.

Distribution of Natural Killer Cell in Patients with Newly Diagnosed Myelodysplastic Syndromes and Its Correlation with Prognosis

To investigate the distribution of peripheral blood lymphocytes and natural killer (NK) cells, and its influence on the prognosis of patients with myelodysplastic syndromes (MDS). The lymphocytes proportion, absolute lymphocyte counts (ALC), NK cell proportion and absolute NK cell counts (ANKC) as well as the related data of 95 MDS patients diagnosed between 2013 and 2017 analyzed retrospectively. The correlation of ALC and ANKC with prognosis was also analyzed. As compared with low ALC patients, MDS patients with ALC≥0.885×109/L had a higher overall response rate (66.7% vs 35.8%) (P＜0.01). The ALC of effective patients after treatment significatitly increased in compaison of ALC at diagnosis. Multivariate analysis indicated that patients with ALC≥0.885×109/L had long overall survival (OS) time in comparison with patients with low level (16.4 vs 12.4 months) (P＜0.05). The OS time of patients with ANKC≥0.110×109/L was shorter in comparison with patients with low level (10.9 vs 16.3 months) (P＜0.01). Otherwise, blast, cytogenetic risks and treatment response were also independent risk factors of MDS (P＜0.05). Revised International Prognostic Scoring System (IPSS-R) combined with ANKC could improve predictive accuracy of IPSS-R alone (AUC 0.718 vs 0.674) (P＜0.05). Lymphocytes and NK cells are important for the prognosis evaluation of MDS patients.

Distribution of Natural Killer Cell in Patients with Newly Diagnosed Myelodysplastic Syndromes and Its Correlation with Prognosis

To investigate the distribution of peripheral blood lymphocytes and natural killer (NK) cells, and its influence on the prognosis of patients with myelodysplastic syndromes (MDS). The lymphocytes proportion, absolute lymphocyte counts (ALC), NK cell proportion and absolute NK cell counts (ANKC) as well as the related data of 95 MDS patients diagnosed between 2013 and 2017 analyzed retrospectively. The correlation of ALC and ANKC with prognosis was also analyzed. As compared with low ALC patients, MDS patients with ALC≥0.885×109/L had a higher overall response rate (66.7% vs 35.8%) (P＜0.01). The ALC of effective patients after treatment significatitly increased in compaison of ALC at diagnosis. Multivariate analysis indicated that patients with ALC≥0.885×109/L had long overall survival (OS) time in comparison with patients with low level (16.4 vs 12.4 months) (P＜0.05). The OS time of patients with ANKC≥0.110×109/L was shorter in comparison with patients with low level (10.9 vs 16.3 months) (P＜0.01). Otherwise, blast, cytogenetic risks and treatment response were also independent risk factors of MDS (P＜0.05). Revised International Prognostic Scoring System (IPSS-R) combined with ANKC could improve predictive accuracy of IPSS-R alone (AUC 0.718 vs 0.674) (P＜0.05). Lymphocytes and NK cells are important for the prognosis evaluation of MDS patients.

Low absolute NK cell counts in peripheral blood are associated with inferior survival in patients with mantle cell lymphoma.

Although risk stratification of mantle cell lymphoma (MCL) is most frequently performed using the simplified MCL International Prognostic Index (sMIPI), the identification of host-related factors and tumor microenvironment, including absolute monocyte counts (AMC) and peripheral blood T lymphocyte subsets, especially absolute natural killer cell counts (ANKC) has been suggested to be critical in the prediction of prognosis and the guidance of treatment. This study was aimed at investigating whether peripheral blood ANKC and AMC at diagnosis had an impact on MCL prognosis. A total of 92 newly diagnosed MCL patients was enrolled in this retrospective study. Flow cytometric analysis was conducted on fresh peripheral blood samples with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The median follow-up was 42 months (range, 2-144 months) and the median overall survival (OS) of all cases was 45 months. High AMC (> 0.6 × 109/L) was the parameter associated with inferior progression free survival (PFS) (P= 0.044) and poor OS (P= 0.028) while low ANKC (⩽ 0.1 × 109/L) was associated with unfavorable OS (P= 0.023) by univariable analysis. Multivariable analysis revealed that only low ANKC (⩽ 0.1 × 109/L) was statistically significant in worse OS (P= 0.009) independent of sMIPI. Low ANKC (⩽ 0.1 × 109/L) proved to be a significant predictor of inferior OS in patients with MCL.

Elevated absolute NK cell counts in peripheral blood predict good prognosis in chronic lymphocytic leukemia.

The aim of this study was to investigate the prognostic significance of the absolute natural killer (NK) cell counts in peripheral blood in patients with chronic lymphocytic leukemia (CLL). A total of 273 previously untreated patients with CLL from April 2004 and October 2015 were enrolled into this retrospective study. We analysed the T cell subsets of all patients and figured out the number of NK cells. Comparisons of NK cell count as continuous parameter in different groups were described using Mann-Whitney U test and the Kruskal-Wallis test. Kaplan-Meier method was used to survival analysis, and the Cox proportional hazards models were used for the estimation of prognostic factors. NK cell counts were calculated in 273 therapy-naive CLL patients, and higher number of NK cell was observed in those with Binet stage A/B, ZAP-70 < 20%, normal serum albumin and β2-microglobulin levels. Using a NK cell count cut-off of 0.40 × 109/L, patients with lower NK cell count (< 0.40 × 109/L) had a significantly shorter overall survival (OS) than those with higher NK cell count (≥ 0.40 × 109/L) (P = 0.0014). Multivariate analysis showed that NK cell counts remained its prognostic value. However, the effect of NK cell count on time to treatment was not significant. Our results suggest that NK cell count is an independent prognostic marker for OS in patients with CLL and NK cell counts ≥ 0.40 × 109/L can routinely be used to identify patients with favorable survival.

Sustained Natural Killer Cell Recovery Post-Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation Predicts Survival in Non-Hodgkin's Lymphoma

AbstractOur group recently reported a Phase III clinical trial (Porrata et al. Biology of Blood and Marrow Transplantation 2016; 22: 1017-1023) demonstrating that the infusion of the autograft-absolute lymphocyte count (A-ALC) and autograft natural killer cells (A-NKC) are survival prognostic factors in non-Hodgkin lymphoma (NHL) patients treated with autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). A limitation of our Phase III clinical trial was the short-term follow-up of just two years. Therefore, we evaluated longer follow-up and assessed the role of the infused A-ALC, infused A-NKC, and peripheral blood absolute natural killer cell count (ANKC) recovery on survival post-APBHSCT. Of the 122 patients that participated in the Phase III trial, 111 patients were able to complete APBHSCT and included in the current study. With a median follow-up of 57.2 months (range: 2.1-84.6 months), superior overall survival (OS) and progression-free survival (PFS) was observed in patients infused with an A-ALC ≥ 0.5 x 109 lymphocytes/kg [OS: HR = 0.429, 95%CI, 0.128-0.828, p < 0.01; and PFS: HR = 0.456, 95% CI, 0.177-0.863, p < 0.01] and an A-NKC ≥ 0.09 x 109 cells/kg [OS: HR = 1.58e-3, 95% CI, 1.046e-6-0.179, p < 0.003; and PFS: HR= 1.58e-3, 95% CI, 7.81e-7-0.064, p < 0.003]. Furthermore, patients that maintained an ANKC ≥ 250 cells/µl at 3 months, 6 months, 9 months and 12 months post-APBHSCT also experienced superior OS (Figure 1A) and PFS (Figure 1B)[OS and PFS times in months were evaluated from infusion date of stem cells]. In the multivariate analysis a sustained ANKC ≥ 250 cells/µl was an independent predictor for OS (HR = 0.013, 95% CI, 0.001-0.063, p < 0.0001) and PFS (HR = 0.014, 95% CI, 0.001-0.067, p < 0.0001). This study is an extension of our Phase III clinical trial showing that sustained innate immunity by measuring the ANKC correlated with superior clinical outcomes; thus providing a platform to develop innate immunity immunotherapeutic strategists directing to improve clinical outcomes post-APBHSCT in NHL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Acute Intermittent Exercise on Natural Killer Cells in Breast Cancer Survivors.

Current research examining the effect of exercise on immune responses in cancer survivors is limited. The aim of this pilot study was to examine the effect of 1 bout of intermittent exercise on natural killer (NK) cell numbers in breast cancer survivors. A total of 9 women with stage I to III invasive breast cancer who were 3 to 6 months posttreatment and 9 sedentary women without a history of cancer completed 10 three-minute intervals of aerobic exercise on the cycle ergometer at 60% of VO2peak (peak oxygen uptake). Whole blood samples were taken pre-exercise, immediately postexercise, and at 2 hours and 24 hours postexercise. NK cell counts were assessed using flow cytometry. In both groups, NK cell counts significantly increased immediately postexercise compared with pre-exercise (P = .004-.008) and returned to near pre-exercise levels during recovery (P = .129-.547). Absolute NK cell counts were significantly lower in breast cancer survivors immediately postexercise when compared with controls (P = .046). The breast cancer survivor group exhibited NK cell responses to 30 minutes of moderate-intensity intermittent aerobic exercise that were comparable with that in the group of physically similar women without a history of cancer. Immune changes related to cancer treatments may be related to the lower absolute NK cell counts observed in the breast cancer survivor group. Although the results of this study are preliminary in nature, they suggest that this type of exercise does not disrupt this aspect of innate immunity in recent breast cancer survivors, thereby supporting current exercise recommendations for this population.

Abstract CT304: Anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 plus low-dose interleukin-2 (IL-2) in patients with recurrent prostate cancer (PC)

Abstract Purpose J591 is a deimmunized monoclonal antibody that recognizes the extracellular domain of prostate specific membrane antigen and was engineered to induce antibody-dependent cellular cytotoxicity (ADCC). Low dose IL-2 may enhance ADCC by the clonal expansion of natural killer (NK) cells. Immunotherapy may result in survival benefits independent of immediate response. This study was designed to evaluate the efficacy of J591 plus low-dose IL-2 in men with recurrent PC. Experimental Design Men with non-castrate PSA relapse or progressive metastatic castration-resistant PC (mCRPC) received low-dose subcutaneous IL-2 (1.2 x 106 IU/m2/day) daily for 8 weeks with weekly intravenous infusions of J591 (25 mg/m2) on weeks 4-6 (considered 1 cycle). A maximum of 3 cycles of therapy was delivered. Flow cytometry was used to assess NK cell fraction and PSA changes and survival were assessed. Results Seventeen men were treated between 2001-03 with at least 1 cycle (11 PSA relapse, 6 mCRPC) with 1 mCRPC patient invaluable for PSA response due to a protocol violation (9 received 2 cycles, 2 received 3 cycles). Toxicity was mild, mainly limited to fatigue and injection site reactions. PSA declined following therapy in 7 of 16 evaluable men (2 of 5 mCRPC). Flow cytometry revealed an average increase in absolute NK cell count of 107% at week 4 and 117% at the end of cycle 1. NK cell percent change had an inverse relationship with PSA (2-tailed Pearson correlation coefficient r = -0.46; P= 0.13). The actual vs. predicted survival for 5 of the 6 men with mCRPC disease was greater than predicted by the Halabi nomogram (Spearman correlation analysis r=0.29; P=0.57, i.e. nomogram did not predict survival). Conclusions The combination of J591 with low-dose IL-2 was well tolerated in patients with biochemical relapse and mCRPC. In addition, combination therapy expanded NK cell fraction which correlated with PSA decline. Interestingly, long term follow up reveals that 83% of the mCRPC group lived significantly longer than predicted, mostly in an era prior to the approval of life-prolonging therapy. These data support further testing of the hypothesis that PSMA-directed immunotherapy influences outcome in men with advanced PC. Citation Format: Yulian Khagi, Gurveen Kaur, Paul Christos, Naveed H. Akhtar, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 plus low-dose interleukin-2 (IL-2) in patients with recurrent prostate cancer (PC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT304. doi:10.1158/1538-7445.AM2014-CT304

Composition of PBMC Population and Functionality of T- and NK-Cells in AML Patients Receiving Cytotoxic Maintenance Therapy,

Abstract 3601Novel therapeutic venues are following immunotherapeutic strategies by translating the successes of allogeneic stem cell transplantation to immunotherapy in an autologous setting. Several phase I/II clinical trials have been published using AML-associated peptides or RNA-transfected dendritic cells for vaccination of AML patients. The success of immunotherapy is hampered by negative regulatory mechanisms that inhibit anti-leukemic T cell function, leading to evasion from immune attack. A promising approach to overcome negative regulatory immune mechanisms is the combination of immunotherapy with cytotoxic chemotherapy. Within the AML-CG study group, we regularly apply cytarabine in an alternating combination with daunorubicin, 6-thioguanine and cyclophosphamide every 28 days as a maintenance postremission therapy in patients not eligible for stem cell transplantation. We set out to assess the immune status of patients prior to and during this maintenance therapy in order to define a potentially beneficial point of time for active immunotherapies, e.g. dendritic cell vaccination.We analyzed PBMC subpopulations (CD3+CD8+ cytotoxic T cells, CD3+CD4+ T helper cells, CD4+CD25hiCD127loFoxP3+ regulatory T cells, and CD3−CD16+CD56+ natural killer cells) in 8 AML patients at several time-points (days 0, 3, 9, 14, and 21) throughout early maintenance therapy cycles by flow cytometry. Numbers of regulatory T (Treg) cells were additionally determined by a DNA methylation assay for selected samples. In order to test the proliferative capacity of T helper (Th) cells, CFSE assays with PHA as stimulus were performed in 3 patients. T cell polarization was analyzed by determination of secreted cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, IFN-γ) using a cytometric bead array after IL-2 stimulation. Natural killer (NK) cell functionality was assessed in 4 patients using CD69 upregulation after stimulation with IL-2. The resulting data was compared with 15 healthy donors subjected to the same analyses.The absolute Th cell counts, absolute numbers of Treg cells and absolute NK cell counts were significantly lower in AML patients prior to the start of maintenance therapy compared to healthy donors (p < 0.05), while the relative Treg numbers in relation to all lymphocytes were similar. In the course of maintenance therapy, several dynamics were observed: Total leukocyte numbers decreased, with the nadir around 1 G/l on day 21. The proportion of cytotoxic T cells did not vary significantly throughout the course of therapy. However, we observed a temporary increase in Th cells (p < 0.05 for days 9 and 14 compared to day 0). Interestingly, the fraction of Treg cells more than doubled in the course of the therapy, with a maximum on day 14 (p < 0.05), explaining part of the relative increase in CD4+ T cells. In contrast, relative numbers of NK cells declined in the course of maintenance therapy (p < 0.05 for days 9, 14 and 21 compared to day 0).Functionally, we did not find differences in Th cell proliferation between healthy donors and AML patients before the start of maintenance chemotherapy, while the response was substantially reduced two weeks after the start of the therapy (p < 0.05). When comparing cytokine secretion after stimulation with IL-2, we found that the Th cells isolated from AML patients tended to secrete higher concentrations of IL-4 and IL-17, but less IFN-γ (not statistically significant). This trend did not change in the course of maintenance therapy. Similarly, the activation capacity of NK cells was generally reduced in AML patients relative to healthy donors (p < 0.05), but was not further diminished in the course of the therapy.In our ongoing study of AML patients, we found that T and NK cell subsets after intense polychemotherapy significantly differed from those of healthy donors. Prior to the start of maintenance chemotherapy, we prominently saw a diminished number and function of NK cells. This is in accordance with previous reports on NK cell dysfunction in AML patients. In the course of maintenance therapy, we observed a significant relative increase in Treg cells and a decrease in the functional response of Th cells. These results urge to intensively monitor cell populations and functionality during chemotherapy and immunotherapy. Data generated from close immunomonitoring is the basis for creating synergistic combinations of chemotherapy and immunotherapy. Disclosures:Hoffmueller:Epiontis: Employment.

Phenotype and function of natural killer cells in systemic lupus erythematosus: Excess interferon-γ production in patients with active disease

To determine the phenotype and the functionality of natural killer (NK) cells in patients with systemic lupus erythematosus (SLE). A total of 94 patients with SLE (91 women and 3 men) were compared with 26 healthy controls. Active SLE was defined by an SLE Disease Activity Index score≥4. Immunologic tests were performed using nonactivated and/or interleukin-2 (IL-2)-activated peripheral blood mononuclear cells. NK cell phenotype was determined by flow cytometry. NK cell natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) were determined by 51Cr release and CD107a degranulation experiments. Intracellular interferon-γ (IFNγ) production by NK cells was evaluated after overnight stimulation with IL-12 and IL-18. IFNα levels were assessed using an antiviral cytopathic bioassay. The absolute NK cell count was decreased in patients with active SLE, but the relative frequencies of total CD3-CD56bright NK cells and CD3-CD56dim NK cells were unaffected. The CD3-CD56dim NK cells in patients with active SLE displayed unique phenotypic characteristics, including significant increases in CD69 and NKG2A and decreased expression of Fcγ receptor type IIIa/CD16, CD8α, and the killer cell immunoglobulin-like receptor (KIR) KIR2DL1/KIR2DS1. Concomitant with these findings, NK cells from SLE patients had lower cytotoxicity but a normal level of ADCC compared with NK cells from healthy controls. There was a significant positive correlation between the increased level of IFNα in the serum and the enhanced frequency of IFNγ+ cells in patients with active SLE (r=0.370, P=0.04). NK cells in patients with active SLE display phenotypic and functional features associated with activation. Furthermore, NK cells from patients with active SLE have the capacity to produce large amounts of IFNγ. This could contribute to the dysregulation of the link between innate and adaptive immunity seen in SLE.

Characterization of Early Natural Killer Cell Reconstitution Following Autologous Transplantation in Multiple Myeloma.

Abstract 4641 BackgroundDespite the advent of novel therapies, multiple myeloma (MM) remains incurable and high-dose chemotherapy (HDC) with autologous stem cell transplantation (SCT) remains an important therapy for this incurable malignancy. Prior research has suggested that early lymphocyte immune reconstitution following HDC/SCT may be associated with improved outcomes; however, little is known regarding the biologic mechanisms underlying this phenomenon. A potent natural killer (NK) cell versus MM effect has also been demonstrated by our group and others previously, and NK cells are the first lymphocyte subset to recover following HDC/SCT. Herein, we characterize the nature of this recovery to suggest that the early post-transplant period may be an especially favorable environment in which to augment the NK cell versus MM effect to improve outcomes for patients with MM undergoing HDC/SCT. MethodsWith informed consent and under institutional review board approval, peripheral blood samples from patients with multiple myeloma undergoing HDC/SCT were collected on the day of first peripheral white cell count > 1500/uL post- HDC/SCT. All patients received intravenous melphalan (200mg/m2) as the preparative regimen. Mononuclear cells were isolated and, by flow cytometry, CD56(+),CD3(-) NK cells were comprehensively immunophenotyped for expression of inhibitory and activating ligands. NK cell activation and cytotoxicity were assessed by ELISPOT and chromium release assays. Clinical outcome was assessed as a function of early NK cell recovery. Results11 patients (median age 57, 8 male, 3 female) with a median of 1 prior regimen to transplant (range 1-9) have been assessed to date. 1 patient was mobilized with AMD-3100, 3 had cyclophosphamide mobilizations and 7 were mobilized with neupogen alone. The average CD34(+) cell dose received was 5.22 (+/- SD 2.39). The median day of peripheral white cell recovery was day +12 (range 11-18). The median absolute lymphocyte count on sample procurement day was 410 (range 340 – 820), of which an average of 37% (range 13 – 70) were NK cells. Although total white count on procurement day correlated with CD34(+) cell dose (p = 0.02), neither absolute lymphocyte count or NK cell count correlated significantly with CD34(+) cell dose and mobilization strategy did not affect NK cell count. NK cell reconstitution correlated with best response to HDC/SCT (p=0.02), the average NK cell count for patients ultimately achieving CR was 389 (+/- 131) as compared to those achieving PR or less (171 +/- 101). Interestingly, nearly half of reconstituting NK cells (46% +/- SD 20) expressed CD117 (c-kit receptor), a marker shown previously to be associated with earlier stages of NK cell development. Inhibitory KIR (CD158a on 36% +/-17 and CD158b on 36% +/- 24) and NKG2A (76% +/- 10) expression also seemed restricted to the CD117(+) NK cells. This population of NK cells also displayed NKG2D (25% +/- 22) and CD16 (73% +/-8). Reconstituting NK cells were capable of interferon gamma production against MM tumor targets similar to NK cells isolated from healthy donors (E:T = 20:1, 956 +/- 98 spots/well versus 896 +/-85, p = n/s). Granzyme B production against MM tumor targets also compared to normal controls (E:T = 20:1, 547 +/- 66 versus 573 +/- 50, p= n/s). Moreover, reconstituting NK cells showed enhanced cytotoxicity against MM tumor targets when pre-treated with an inhibitory KIR blocking antibody (IPH2101/1-7F9) similar to healthy donor NK cells. DiscussionTaken together, we provide quantitative and qualitative data to suggest that early NK cell recovery post-SCT may contribute to outcome in patients with MM. This may be an optimal period to implement immunomodulatory strategies to augment NK cell function to improve outcomes for patients with MM undergoing HDC/SCT. Reconstituting NK cells, although exhibiting a relatively unique immunophenotype compared to mature NK cells in healthy, normal donors, appear to retain properties of activation and cytotoxicity. Given the favorable effector:target ratio in vivo following SCT, these results support further research into immunotherapeutic manipulation of the reconstituting NK cell clone to improve efficacy of HDC/SCT in patients with MM. Disclosures:Andre:Innate Pharma: Employment. Squiban:Innate Pharma: Employment. Romagne:Innate Pharma: Employment.

The relationship between circulating natural killer cells after reduced intensity conditioning hematopoietic stem cell transplantation and relapse-free survival and graft-versus-host disease

Natural killer cells are known to have anti-tumor activity in haploidentical hematopoietic stem cell transplantation. We hypothesized that reconstituted circulating natural killer cells may be associated with improved relapse-free survival after HLA-matched hematopoietic stem cell transplantation. Serial peripheral blood absolute natural killer cell counts were prospectively measured by flow cytometry of lymphocytes expressing CD56 and CD16 in 167 patients. Cluster analysis was used at engraftment and 60 days post-transplant to distinguish patients with high and low absolute natural killer cell counts. At engraftment 80 patients had high counts (> 22.2/mm3) and 43 had low counts. At 60 days post-transplant 84 patients had high counts (> 18.2/mm3) and 38 had low counts. The primary study end-points were death, relapse and acute graft-versus-host disease. The median follow-up was 373 days (range, 67-1767). Among patients given reduced intensity conditioning, a low absolute natural killer cell count at 60 days post-transplant was independently associated with relapse [adjusted hazard ratio (AHR) = 28.4, 95% confidence interval (CI) 4.3-186.4] and death (AHR = 17.5, 95% CI 4.3-71.3). Furthermore, patients given reduced intensity conditioning who had a high absolute natural killer cell count at 60 days had a significantly better 1-year survival than those with a low count by Kaplan-Meier analysis (83% vs. 11%, p<0.001). Multivariate analysis confirmed that low 60-day absolute natural killer count in patients given reduced intensity conditioning was independently associated with an increase in relapse or death (AHR = 20.22, 95% CI 4.76-85.40). In contrast, there was no significant association between 60-day absolute natural killer cell counts and clinical outcomes in patients receiving myeloablative conditioning. There was no significant association between absolute natural killer cell count and graft-versus-host disease. High natural killer cell reconstitution is associated with reduced relapse and death without an increased incidence of graft-versus-host-disease after reduced intensity conditioning allogeneic hematopoietic stem cell transplantation. Measuring reconstituted natural killer cells expressing CD56(+)/CD16(+) post-transplant may have novel prognostic and therapeutic implications.

Phase 1 Dose-Escalation Study of CP-690 550 in Stable Renal Allograft Recipients: Preliminary Findings of Safety, Tolerability, Effects on Lymphocyte Subsets and Pharmacokinetics

CP-690 550 inhibits Janus kinase 3 with nanomolar potency. In this dose-escalation study, we assessed the safety, tolerability, effects on lymphocyte subsets, and pharmacokinetics of CP-690 550 when coadministered with mycophenolate mofetil in stable renal allograft recipients for 28 days. Twenty-eight patients were enrolled. Six patients received CP-690 550 5 mg twice daily (BID), 6 patients received 15 mg BID, 10 patients received 30 mg BID, and 6 patients received placebo. The most frequent adverse events were infections and gastrointestinal (abdominal pain, diarrhea, dyspepsia, and vomiting). CP-690 550 15 mg BID and 30 mg BID were associated with a mean decrease in hemoglobin from baseline of 11% and a mean decrease in absolute natural killer cell counts of 50%. CP-690 550 30 mg BID was also associated with a mean increase in absolute CD19(+) B-lymphocytes of 130%. There were no changes in the number of neutrophils, total lymphocytes, platelets, or CD4(+) or CD8(+) T cells; clinical chemistry; vital signs; or electrocardiograms from the pretreatment baseline. Administration of CP-690 550 without a concomitant calcineurin inhibitor resulted in CP-690 550 exposures consistent with previous studies in nontransplant subjects. Additional dose-ranging studies are warranted to evaluate the safety and efficacy of CP-690 550 in renal transplant recipients over longer treatment duration.

89: Early Lymphocyte Recovery Predicts Superior Survival after Autologous Stem Cell Transplantation in Non-Hodgkin Lymphoma: A Prospective Study

Background: Day 15 absolute lymphocyte count (ALC-15) ≥500 cells/μl after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) is a significant predictor for survival. Limitations of previous reports have been their retrospective nature and no ALC-15 lymphocyte subsets analysis. Thus, from 2/2002 until 2/2007, 50 non-Hodgkin lymphoma (NHL) patients were enrolled in a prospective study to address these limitations. Methods: APHSCT eligible NHL patients who signed written consent for additional blood collections participated in the study. The primary end point was to confirm the ALC-15 survival role and to identify the lymphocyte subsets of greatest impact on ALC-15 after APHSCT. Results: The median age of the cohort was 57.7 years (range: 23–73). The groups (ALC-15 ≥500 vs < 500 cells/μl) were balanced to lymphoma type (p = 0.4), gender (p = 0.5), age (p = 0.2), extranodal sites (p = 0.4), lactate dehydrogenase (p = 0.5), performance status (p = 0.5), stage (p = 0.5), international prognostic index (p = 0.9), number of prior chemotherapies (p = 0.1), clinical status at APHSCT (p = 0.2), and infused CD34/kg (p = 0.8). With a median follow-up of 22.2 months (range: 6–63.7 months), patients with an ALC-15 ≥500 cells/μl (n = 35) experienced superior overall survival (OS) and progression-free survival (PFS) compared with those who did not (median OS: not reached vs 19 months, 3 years OS rates of 80% vs 40%, p < 0.0004; median PFS: not reached vs 3.4 months, 3 years PFS rates of 60% vs 14%, p < 0.0001, respectively). By day 15 post-APHSCT 30% of patients achieved a normal CD3, 18% a normal CD4, 38% a normal CD8, 4% a normal CD19, and 76% a normal [CD16+/56+/CD3-] counts. In Univariate analysis CD16+/56+/CD3- natural killer (NK) cells were the only ALC-15 lymphocyte subset that predicted for survival. Patients with a normal absolute NK cell count (≥80 cells/μl, n = 38) experienced superior OS and PFS compared with those who did not (median OS: not reached vs 5 months, 3 years OS rates of 76% vs 36%, p < 0.0001; and median PFS: not reached vs 3 months, 3 years PFS rates of 57% vs 9%, p < 0.0001, respectively). Conclusion: This study confirms the prognostic ALC-15 survival role and identifies NK cells as the key lymphocyte subset affecting clinical outcomes after APHSCT in NHL. This study was supported in part by the Grant CA90628–04A from the National Institute of Health, and the University of Iowa/Mayo Clinic Lymphoma Spore CA97274.

Natural killer (NK) cells are associated with reduced relapse without increased graft-versus-host disease (GVHD) after allogeneic hematopoetic stem-cell transplantation (HSCT)

6542 Background: Natural killer cells have potential for anti-leukemia activity after haplo-identical HSCT. However, the role of reconstituted NK cells in relapse and survival after matched HLA-identical HSCT is unknown. We investigated the correlation between NK reconstitution and death, relapse, and GVHD after HSCT. Methods: We prospectively studied NK reconstitution in 123 patients with predominantly high-risk hematologic malignancies. Peripheral blood absolute NK cell count (ANK) was determined by flow cytometry analysis of lymphocytes co-expressing bright staining CD56+ and CD16+. Cluster analysis distinguished patients at engraftment with high ANK (&gt;12.2/mm3) (n=80) and low ANK (n=43), and at 60 days with high ANK (&gt;20/mm3) (n=64) and low ANK (n=26). Primary study endpoints were death, relapse and acute grade II-IV GVHD with median follow up of 360 days (28–1967). A cox proportional hazard model was adjusted for important covariates, including age, transplant conditioning (myeloablative vs. non-myeloablative), graft source (peripheral blood vs. bone marrow), and donor-type (HLA-matched related vs. unrelated), dendritic cell level and absolute neutrophil count. Results: A low ANK at engraftment was significantly associated with death (p=0.03) after non-myeloablative HSCT. Using a selection model, only NK remained in the model, and a low ANK was associated with a 2.5 hazard ratio for subsequent death (p=0.04). Additionally, for the overall cohort, ANK at 60 days was strongly associated with time to death (92% in high vs 65% in low group; LR p=0.0007), at 310 days after HSCT. ANK level at 60 days was strongly associated with time to relapse (n=82, (LR p=0.0001). In a multivariate model, low ANK at 60 days was associated with a 4.2 fold relative risk (RR) of death (1.9, 9.1) and 8.6 RR for time to relapse (3.1, 24.3). Conclusions: High NK cell reconstitution is associated with reduced relapse and death without increased incidence of GVHD after HLA-identical HSCT. Our study suggests that NK may have both a potential anti-tumor effect, as well as be tolerogenic. Measuring NK after HSCT may have novel prognostic and therapeutic implications. No significant financial relationships to disclose.

Changes in lymphocyte subsets after cardiac surgery in children.

Children undergoing cardiopulmonary bypass (CPB) operations have an increased risk of developing severe infections. Impairment of the immune system may contribute to the development of sequelae such as capillary leaks, pulmonary dysfunction and auto-immune reactions. The objective of this study was to investigate the impact of cardiac surgery with CPB on the immune system of infants and young children. We conducted a prospective study to investigate the changes in circulating lymphocyte subpopulations in a sample of 21 consecutive infants and young children undergoing cardiac surgery for congenital heart disease. The following statistically significant ( P<0.05) results were obtained: leucocyte counts rose 6 h after surgery due to the increase in neutrophils. Absolute T-cell number and absolute T-helper cell number decreased within 24 h after CPB. The proportion of T-cells expressing the T-cell receptor gammadelta as well as natural killer cells increased during CPB. In contrast, the proportion of T-cells expressing activation markers (CD25, CD45R0) decreased within 24 h after CPB, as did the number of cells expressing adhesion molecules (CD11b and ICAM). during cardiac surgery with cardiopulmonary bypass, absolute natural killer cell counts increase while T-cells decrease, presumably due to an extravasation or adhesion of activated T-cells. The relevance of this finding regarding the risk of infection is discussed.