Determination of Soluble MICA in Serum as a Novel Marker for Tumor Stage and Metastasis.

Abstract The human NKG2D ligands (NKG2DL) MICA and MICB have been shown to be expressed on tumors of epithelial and hematopoietic origin in vivo. Recently we reported that MICA is shed from the cell surface of tumor cells and is present in sera of tumor patients (J Immunol169:4098 (2002), Blood102:1389 (2003)). Since the strength of an anti-tumor response by NK cells and CD8 T cells is critically depending on NKG2DL expression levels, down-regulation of MICA-expression on tumor cells represents an immune escape mechanism that diminishes anti-tumor reactivity of NKG2D-bearing lymphocytes. However, no data are yet available regarding the correlation of soluble MICA (sMICA) levels with specific tumor entities, aggressiveness of the disease, and hence the potential implementation of sMICA as novel marker in differential diagnosis and prognosis of cancer. In this study, we determined sMICA levels in sera of 512 individuals including 296 patients with various cancers, 154 patients with benign disorders and 62 healthy individuals. Healthy individuals revealed significantly lower sMICA values (median<30pg/mL) than patients with benign diseases (84pg/mL; p=0.005) and cancer patients (161pg/mL; p<0.0001). In addition, sMICA levels differed significantly between cancer patients and patients with benign disorders (p<0.0001) that represent the most relevant control group for differential diagnosis. In cancer patients, while there was no association between sMICA levels and tumor size (p=0.456), cell differentiation (p=0.271), or lymph node involvement (p=0.674), sMICA correlated significantly with cancer stage and metastasis (p=0.015 and p=0.007, respectively). Our data indicate that release of MICA might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. Thus, determination of sMICA levels provides valuable information for cancer staging, and sMICA in serum seems to be an indicator for systemic manifestation of malignancy rather than for local tumor extent.

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IMMU-44. AN IMMUNOTHERAPEUTIC VACCINE COMPOSED OF IRRADIATED WHOLE TUMOR CELLS PULSED WITH MANNAN-BAM, TLR LIGANDS AND ANTI-CD40 ANTIBODY INDUCES POTENT IMMUNE RESPONSE IN PRECLINICAL GBM ANIMAL MODEL

Neuro-Oncology ◽

10.1093/neuonc/noab196.403 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi102-vi102

Author(s):

Herui Wang ◽

Rogelio Medina ◽

Juan Ye ◽

Pashayar Lookian ◽

Ondrej Uher ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Tumor Cells ◽

T Lymphocyte ◽

Cd8 T Cells ◽

Therapeutic Efficacy ◽

Poly I:C ◽

Control Group ◽

Complete Regression ◽

Adaptive Immune

Abstract Despite numerous therapeutic advances, the treatment of glioblastoma multiforme (GBM) remains a challenge, with current 5-year survival rates estimated at 4%. Multiple characteristic elements of GBM contribute to its treatment-resistance, including its low immunogenicity and its highly immunosuppressive microenvironment that can effectively disarm adaptive immune responses. Hence, therapeutic strategies that aim to boost T-lymphocyte mediated responses against GBM are of great therapeutic value. Herein, we present a therapeutic vaccination strategy that promotes the phagocytosis of tumor cells, enhances tumor antigen presentation, and induces a tumor-specific adaptive immune response. This strategy consists of vaccinations with irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists (Mannan-BAM), TLR ligands [LTA, Poly (I:C), and R-848], and anti-CD40 antibody (collectively abbreviated as rWTC-MBTA). We evaluated the therapeutic efficacy of rWTC-MBTA strategy in a mouse syngeneic GL261 orthotopic GBM tumor model. rWTC-MBTA or vehicle control were administered subcutaneously over the right foreleg three days after intracranial injection of GL261 cells. Complete regression (CR) of intracranial tumors was achieved in 70% (7/10) of rWTC-MBTA treated animals while none survived in the control group. Immunophenotyping analyses of peripheral lymph nodes and brain tumors of rWTC-MBTA treated mice demonstrated: (1) increased mature dendritic cells and MHC II+ monocytes; (2) increased effector (CD62L-CD44+) CD4-T and CD8-T cells; (3) increased cytotoxic IFNγ-, TNFα-, and granzyme B-secreting CD4-T and CD8-T cells. Of note, the therapeutic efficacy of rWTC-MBTA disappeared in CD4-T and/or CD8-T lymphocyte depleted mice. Three mice that achieved CR were rechallenged with 50k GL261 cells intracranially 14 months after the last rWTC-MBTA treatment and all rechallenged animals resisted GL261 tumor development, confirming the establishment of long-term immunological memory against GL261 tumor cells. Collectively, our study demonstrated that rWTC-MBTA strategy can effectively activate antigen presenting cells and induce more favorable T-cell signatures in the GBM tumors.

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Deficit of Circulating CD19+CD24hiCD38hi Regulatory B Cells in Severe Aplastic Anemia

Blood ◽

10.1182/blood-2019-127053 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1219-1219

Author(s):

Yoshitaka Zaimoku ◽

Bhavisha A Patel ◽

Sachiko Kajigaya ◽

Xingmin Feng ◽

Lemlem Alemu ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Aplastic Anemia ◽

Nk Cells ◽

Cd8 T Cells ◽

Healthy Individuals ◽

Regulatory B Cells ◽

Significant Difference ◽

Cell Phenotypes

Background: Immune aplastic anemia (AA) is caused by cytotoxic T cells (CTLs) that destroy hematopoietic stem and progenitor cells. Regulatory T cells (Tregs) are reduced in AA and increase in response to immunosuppressive therapy (IST; Solomou E et al, Blood 2007). Recent studies suggested an immune regulatory role of regulatory B cells (Bregs). Human CD19+CD24hiCD38hi Bregs suppress Th1 response of CD4+ T cells as well as IFN-γ production by CD8+ CTLs (Mauri C, Menon M, J Clin Invest 2017). The quantity and/or function of Bregs are impaired in autoimmune diseases, malignancies, chronic graft-versus-host disease, and during rejection of transplanted organs. Methods: We investigated B cell phenotypes including CD24hiCD38hi Bregs in previously untreated severe AA (SAA) and very severe AA (VSAA) patients, and healthy individuals aged 18 years and older, and tested their correlation with severity and response to IST. Absolute numbers of lymphocyte subsets, including CD19+ B cells, CD8+ T cells, CD4+ T cells, and NK cell (TBNK), were quantified in fresh blood. Percentages of B cell subsets among total CD19+ B cells, including CD24hiCD38hi Bregs, CD24loCD38lo mature naïve B cells, CD24hiCD38lo memory B cells and CD24loCD38hi plasma cells/plasmablasts, were analyzed using cryopreserved peripheral blood mononuclear cells (PBMCs). Blood samples were obtained from patients close to time of diagnosis and before institution of definitive therapy. All patients were treated with horse anti-thymocyte globulin, cyclosporine, and eltrombopag between 2012 and 2018 at the Hematology Branch, NHLBI (clinicaltrials.gov NCT01623167). Results: TBNK analysis revealed no significant difference in total B cell counts in 104 AA patients compared to 40 healthy individuals (median, 137/μl [IQR, 73-212] vs 163/μl [106-242], P=.11); NK cells were significantly decreased in patients with AA, as previously reported (Gascon P et al, Blood 1986). Total B cell count did not correlate with severity of AA (P=.89) nor with overall response at six months (P=.93). CD8+ T cells and NK cells were lower in VSAA patients compared to SAA patients. None of the TBNK subsets was predictive of overall response in six months after IST. When we assessed the phenotype of B cells among 60 AA patients whose cryopreserved PBMCs were available, CD24hiCD38hi Bregs were markedly decreased as compared to 29 healthy individuals (0.31% [0.14-0.85%] vs 1.9% [1.3-3.6%], P=3×10-7; Figure, Table), while there was no significant difference in other B cell phenotypes. Among these 60 patients, the percentage of CD24hiCD38hi Bregs was especially decreased in VSAA patients compared to SAA (0.18% [0.11-0.34%] vs 0.50% [0.17-1.4%], P=.017). In contrast, CD24loCD38lo mature naïve B cells were higher in VSAA than in SAA (69% [58-86%] vs 60% [42-70%], P=.024). CD24hiCD38hi Breg frequency was positively associated with neutrophil and reticulocyte counts (correlation coefficients [r], 0.34 and 0.26, respectively), while the frequency of CD24loCD38lo mature naïve B cells was negatively correlated (r, -0.34 and -0.40). CD24loCD38lo mature naïve B cells before IST were significantly lower in 47 patients who achieved overall responses at six months compared to 13 non-responders (64% [42-71%), vs 73% [58-88%], P=.014), but CD24hiCD38hi Breg frequency was not correlated with IST responses. At six months after IST, CD24hiCD38hi Bregs in AA patients had recovered to levels present in healthy individuals (2.3% [0.98-4.8%]), in both 34 responders and five non-responders; non-responders showed non-significant increased CD24loCD38lo mature naïve B cells at six months (P=.068). Discussion: A deficit of circulating CD24hiCD38hi Bregs in immune AA with recovery after IST, as occurs with Tregs, suggests Bregs may contribute to the immune pathophysiology in AA. We unexpectedly observed a higher percentage of CD24loCD38lo mature naïve B cells to be associated with more severe disease and a lower probability of responses to IST. B cell phenotype analysis may be beneficial for monitoring of AA and predicting outcomes of therapy. Disclosures No relevant conflicts of interest to declare.

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Correlation between driver mutation and immune microenvironment in colorectal cancer patients.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15513 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15513-e15513

Author(s):

Xiaochen Cai ◽

Yuezong Bai ◽

Xiaochen Zhao ◽

Longgang Cui ◽

Hui Chen

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

Nk Cells ◽

Cancer Patients ◽

Cd8 T Cells ◽

Braf Mutation ◽

Kras Mutation ◽

Tumor Stroma ◽

Wild Type ◽

Colorectal Cancer Patients

e15513 Background: Immune checkpoint inhibitor (ICI) therapy has made great achievements, but ICI monotherapies show less effectiveness in colorectal cancer patients. Biomarker exploration have carried out from PD-L1 expression, neo-antigen, gene mutation, etc., but no satisfactory results have been obtained. Methods: Patients, Colorectal cancer patients. Methods, Multi-color immunohistochemistry (multi-IHC) was used to evaluate CD8+ T cells, macrophages and natural killer cell (NK cell) in tumors and tumor stroma. The Shapiro-Wilk method was used to test the normality of the data, and the t-test or Mann-Whitney U test was used according to the test results. A two-sided P < 0.05 was considered a significant difference. Results: The study included 72 colorectal cancer patients, including 26 female (36.7%) and 46 male (63.8%), with a median age of 59.5 (50-67.3). There were 6 patients (8.3%) with BRAF mutation, 43 patients (59.7%) with KRAS mutation, and 56 patients (77.8%) with TP53 mutation. The results of immunohistochemistry showed that, BRAF mutation Vs BRAF wild-type or KRAS mutation Vs KRAS wild-type, the number or proportion of CD8+ T cells, macrophages or NK cells in tumor and tumor stroma were not statistically different. For TP53 mutation Vs TP53 wild-type, the number and proportion of CD8+ T cells or macrophages in tumor and tumor stroma were not statistically different. There was no statistical difference in the number and proportion of NK cells in tumor. But, the median number of NK cells in tumor stroma was 345 Vs 129, p = 0.06, and the proportion of NK cells was 5.2% Vs 1.39%, p = 0.02. Conclusions: There is no significant change in the immune microenvironment of colorectal cancer patients with BRAF mutation and KRAS mutation. There are more NK cells in tumor stroma of colorectal cancer patients with TP3 mutation.

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Evaluation of Donor CD4+CD25+Foxp3+ Regulatory T Cells and CD3-CD56+ NK Cells on Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2018-99-112735 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5726-5726

Author(s):

Fumihito Tajima ◽

Koji Adachi ◽

Takaya Nishio ◽

Toshio Kawatani ◽

Junji Suzumiya

Keyword(s):

T Cells ◽

Nk Cells ◽

Normal Control ◽

Cd8 T Cells ◽

Research Funding ◽

Absolute Number ◽

Treg Cells ◽

Normal Control Group ◽

Control Group ◽

The Absolute

Abstract Background: It is important to determine the changes in both donor T cells and B cells on immune reconstitution following allogeneic hematopoietic stem cell transplantation (HCT). It is well-known that Treg cells play important roles in the prevention of T-cell-mediated graft-versus-host disease (GVHD) and in promoting tumor escape from T-cell-dependent immunosurveillance. However, very little is known about the number and function of both CD4+CD25+Foxp3+ regulatory T (Treg) cells and CD3-CD56+ natural killer (NK) cells and about NK activity during immune reconstitution. In this study, we examined changes in the number of circulating Treg and NK cells in the peripheral blood (PB) and the NK activity of HCT patients at various times after HCT, and we evaluated the clinical significance of these findings relative to patient survival. Patients and Methods: We evaluated 29 consecutive patients (ages >18) without GVHD who underwent HCT for different hematologic diseases between December 2008 and April 2017. Treg and NK cells were characterized and quantified by flow cytometry from these 29 patients and 15 healthy controls. Evaluated variables included recipient and donor characteristics, transplant-related factors, including conditioning regimen (myeloablative conditioning or reduced-intensity conditioning), donor type (matched sibling donor, unrelated donor, other relative (haploidentical), or cord blood transplant), degree of match (8/8, 7/8, 6/8, or haploidentical), and GVHD prophylaxis. Patients who were followed up for more than 12 months were enrolled in the study. After obtaining informed consent, blood was drawn from all patients on the day of engraftment and at day 100 and at 12-month intervals thereafter. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard 51Cr release assay at an effector: target (E: T) ratio of 20:1. Results: The median patient age was 58 years (range: 19-71 years), and 21 out of 29 of the patients were male. At 100 days, the percentage of B cells (2.5±6.0%) and absolute numbers of CD8+ T cells (269.7±284.8/μL), CD4+T (22.83±292.4/μL) cells and NK cells (248.3±229.1/μL) were significantly lower than those at 2 years (20.9±11.6%, 747.2±648.4/μL, 588.0±607.3/μL, 558.1±336.2/μL, p<0.01, p<0.01, p<0.01, p=0.027, respectively) ,and at 3 years (18.2±11.2%, 554.1±343.3/μL, 488.1±210.3/μL, 500.1±451.3/μL, p<0.01, p=0.020, p=0.017, p=0.043, respectively). The changes in Treg% values in peripheral lymphocytes within 100 days to 3 years after HCT significantly decreased from 11.57 ±11.39 to 3.65 ±2.59% (p=0,039). The % of NK cells in the PB at 1 year after HCT (13.95±10.34%) was significantly lower than that within 100 days after HCT (27.1±16.8%, p=0.01). However, the absolute number of NK cells did not differ. In comparison with the normal control group, significant difference was observed in the Treg cells. Both percentage and absolute number of Treg cells (0.70±0.29%, 40.2±17.3/μL) in the normal control group were significantly higher than those at 1 year (0.33±0.16%, 20.6±15.4/μL, p<0.01, p<0.01, respectively), at 2 years (0.44±0.16/%, 25.1±15.4/μL, p=0.35, p<0.01, respectively), and at 3 years (0.29±0.23%, 15.5±11.8/μL, p<0.01, p<0.01, respectively). In contrast, the absolute number of CD8+ T cells (380.3±128.5/μL) in the normal control group were significantly lower than those at 1 year (935.1±648.4/μL, p<0.01), at 2 years (747.2±648.4/μL, p<0.01), and then did not differ at 3 years (554.1±343.3/μL). Conclusion: The percentage and the absolute number of Treg cells in HCT patients were significantly lower than those in the normal control group, whereas CD8+ T cells was significantly higher in the patients within 2 years after HCT than that in the normal control group. The % Treg cells did not recover even at 3 years after HCT, rather it tended to be somewhat lower. In contrast, there was no change in the NK cells. These results suggest that the immunological reconstitution has not been achieved even at 3 years after transplantation and that the immunological consequences of GVHD are maintained. The antitumor effect is also maintained. On the other hand, NK cells recover at an early stage. The roles of these different immune cells after HCT require further investigation. Disclosures Suzumiya: Eisai: Research Funding, Speakers Bureau; Pfizer: Research Funding; Celltrion: Research Funding; Taiho: Research Funding, Speakers Bureau; Sumitomo Dainioppon: Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; SymBio: Research Funding; Bristol Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Chugai-Roche: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Zenyaku Kogyo: Consultancy; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Ono: Speakers Bureau; Ohtsuka: Speakers Bureau; Shire Japan: Speakers Bureau.

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Activation Levels of Natural Killer Cells and CD8+ T Cells Correlate Highly with Sustained Complete Molecular Response After Discontinuation of Imatinib in Chronic Myeloid Leukemia Patients

Blood ◽

10.1182/blood.v120.21.3745.3745 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3745-3745

Author(s):

Seiichiro Katagiri ◽

Izuru Mizoguchi ◽

Takayuki Yoshimoto ◽

Junichiro Mizuguchi ◽

Junko H Ohyashiki ◽

...

Keyword(s):

T Cells ◽

Chronic Myeloid Leukemia ◽

Nk Cells ◽

Cd8 T Cells ◽

Myeloid Leukemia ◽

Surface Antigens ◽

Control Group ◽

Molecular Response ◽

Cell Surface Antigens ◽

Complete Molecular Response

Abstract Abstract 3745 Introduction: Recently, it has been recognized that some chronic myeloid leukemia (CML) patients with a complete molecular response (CMR) are able to maintain the CMR after discontinuation of imatinib. Mahon et al. reported that among patients with a CMR lasting at least 2 years, CMR was sustained in 41% after discontinuation of imatinib (Lancet Oncol. 2010;11:1029). Similarly, Takahashi et al. reported that 47% of Japanese CML patients with CMR maintained CMR after imatinib discontinuation (Haematologica 2012;97:903). Moreover, Ohyashiki et al. have recently demonstrated that higher peripheral natural killer (NK) cell counts are associated with a reduced risk of relapse after halting imatinib (Br. J. Haematol. 2012;157:254). These findings suggest that, although CML stem cells may remain, even if CMR status is attained, some CML patients could discontinue imatinib therapy and maintain a stable condition, possibly owing to immune surveillance. Aim: To more precisely identify these patients who can safely discontinue imatinib, we characterized the immunophenotype profiles of CML patients. We compared the profiles among CML patients who received imatinib with CMR for more than 2 consecutive years (CMR group), those who could not sustain CMR but maintained a major molecular response (fluctuated CMR group), those who sustained CMR for more than 6 months after discontinuation of imatinib (STIM group), those who relapsed after discontinuation of imatinib (relapse group), and healthy volunteers (control group). Methods: Peripheral blood mononuclear cells (PBMCs) from CML patients and healthy volunteers were separated using a Ficoll density gradient, and immunophenotyping analysis was performed with a 5-color flow cytometry panel, including antibodies against the following cell surface antigens and effector molecules: CD3, CD8, CD45RO, CD56, CCR7, IFN-g, granzyme B, and perforin. After PBMCs were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 4 h in the presence of monensin, cell surface antigens were stained, fixed, and permeabilized. Resultant cells were then intracellularly stained and analyzed with the FACSCanto II flow cytometer. Results: The percentage of effector populations of NK cells, such as interferon (IFN)-g+CD3−CD56+ cells, was significantly higher in the CMR and STIM groups than in the control group. In contrast, the percentage of effector populations of CD8+ T cells, such as IFN-g+CD8+ T cells, was significantly higher in the STIM and control groups than in the CMR group. Moreover, the percentage of effector populations of NK cells, but not CD8+ T cells, was significantly higher in the CMR group than in the fluctuated CMR group. On the other hand, CML patients with a lower percentage of effector populations of NK cells and CD8+T cells, and those with a higher percentage of these effector populations, but whose percentage had decreased after imatinib cessation, had a tendency to relapse after discontinuation of imatinib. Conclusion: The percentage of effector populations of NK cells and CD8+ T cells, such as those expressing IFN-g, correlated highly with sustained CMR after discontinuation of imatinib. Moreover, the activation level of NK cells, but not CD8+ T cells, inversely correlated with the BCR-ABL1 transcript level. Taken together, these results suggest that whether imatinib treatment can be safely discontinued may depend greatly on the immunological activation status of both NK cells and CD8+ T cells in CML patients. Disclosures: No relevant conflicts of interest to declare.

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Cross Talk between the Immune Compartment and the Tumor Cells in Myelodysplastic Syndromes (MDS)

Blood ◽

10.1182/blood-2019-124887 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2986-2986

Author(s):

Camille Bigenwald ◽

Amir Horowitz ◽

Shyamala C. Navada ◽

Rosalie Odchimar-Reissig ◽

Richa Rai ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Tumor Cells ◽

Cross Talk ◽

Cd8 T Cells ◽

Research Funding ◽

Nk Cell ◽

Myeloid Cells

Background: Abnormalities of the immune system and innate immune signaling have been described in patients (pts) with MDS, however the interaction between the MDS tumor cells and the immune compartment is poorly defined. Immune modulatory therapies are being explored in MDS and a better understanding of the interaction and potential cross-talk with immune components and MDS tumor cells is critical in developing new therapeutic strategies. Methods: We developed a multiscale immune profiling strategy to map the immune environment of treatment naïve pts with lower- and higher-risk MDS, using mass cytometry by time of-flight (CyTOF) combined with Olink proteomics analyses. We obtained bone marrow (BM) and peripheral blood (PB) from 33 pts with MDS, (17 higher-risk; 16 lower-) and 10 healthy donors. Pts were representative of the MDS distribution across age, gender, mutational status and histologic subtype. PB and BM were freshly processed and stained with an antibody panel for myeloid cells. For the lymphoid panel, all the pts' samples were stained simultaneously to avoid batch effect. We processed the BM and the PB plasma for Olink. Results: We profiled myeloid cells, NK cells and T cells in pts with MDS to determine differences in their constituents. Phenograph clustering across all pts revealed distinct T lymphoid metaclusters, that corresponded to known immune cell populations. We observed a robust immune response in the BM from MDS patients. T cells and myeloid cells were found in equal proportion in the BM from MDS and healthy patients. However, the B cell population were present at significantly lower frequency in the MDS BM (p<0.05), whereas gamma delta (gd)T cells and NK cells were at significantly higher frequency in the MDS pts (p <0.05). Paired mass cytometric analysis revealed a distinct composition and phenotype of T cell subsets. Among the T cell compartment, regulatory T cells (Treg) (p<0.001), cytolytic CD8 T cells (expressing perforin (p<0.01) or granzyme (p<0.001)) and exhausted CD8 T cells (expressing PD-1 (p= 0.09), LAG-3 (p<0.001) or TIM-3) seemed to accumulate in the MDS BM. Compared to healthy BM Treg in MDS BM expressed higher levels of TIGIT, TIM3, CD27, CD25, CD28 and OX40. Cytolytic NK cells accumulate in MDS BM (p<0.05). In addition to measuring adaptive lymphocyte responses to tumors, we also analyzed the distribution of innate lymphocytes and in particular NK cells in MDS BM. In the NK cell population, the cytotoxic NK cells (CD56dim CD16+) were found in a higher proportion (p<0.05) than the cytokinic counterpart (CD56bright) in the MDS BM. NK cells that accumulate in the MDS BM expressed higher level of CD45RA, CD38, granzyme B, CD56 suggesting their superior cytotoxic potential at a critical developmental stage of retaining enhanced sensitivity to pro-inflammatory cytokines derived from myeloid cells. We also analyzed a panel of 92 unique cytokines, chemokines and inflammatory markers produced in the tumor milieu using OLink Proteomics proximity extension assay. Many chemokines and cytokines were differently expressed in the MDS BM compared to the healthy BM, suggesting an important inflammatory reaction. Conclusion: These data demonstrate new findings suggesting interactions and cross-talk between Tregs, NK cells, CD8 T cells and gdT cells in the BM of pts with untreated MDS. The data also suggest that there is a strong immune response in the MDS BM, particularly an intense cytotoxic response with the accumulation of cytotoxic CD8 T cells, NK cells and gdT cells. These results are are contrary to those observed in most solid tumors. This cytotoxic response seemed to be counteracted by the presence of Treg. Each of the subgroups of T cells (as well as NK cells) have been implicated as possibly promoting immune tolerance and tumor growth in different model systems and other tumor types. NK cells and gdT cells are known to respond to virus-infected and transformed cells rapidly and without the need for prior sensitization. However, anecdotal evidence from numerous independent studies may suggest that a strong but prolonged NK cell- and gdT cell-mediated antiviral response may result in dysregulation and promote tumorigenesis and/or aggravate pathology. Additional studies are underway to further characterize the immune landscape in those untreated and treated and to study the interaction of the immune components with tumor cells. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

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Infiltration Patterns of Immune Cells in Gastric Cancer and Their Clinical Correlation with Prognosis

10.21203/rs.3.rs-18120/v1 ◽

2020 ◽

Author(s):

XiaoLi Wu ◽

Hongbo Ma ◽

YanYan Li

Keyword(s):

Gastric Cancer ◽

T Cells ◽

Mast Cells ◽

Nk Cells ◽

Cancer Patients ◽

Cd8 T Cells ◽

Immune Cells ◽

Immune Cell ◽

M1 Macrophages ◽

Gastric Cancer Patients

Abstract Abstract: Objective Gastric cancer is a malignant tumour that severely affects the health of patients. This study analyses the correlation between gastric cancer-infiltrating immune cell patterns and clinical prognosis and provides a scientific basis for the development of comprehensive tumour prevention and treatment strategies. Method Transcripts and related clinical data from 9-2019 for gastric cancer were downloaded from the TCGA database. The proportions of 22 kinds of immune cells were calculated by CIBERSORT software, and the correlation of each immune cell component ratio with tumour grade, clinical stage and overall survival (OS) was evaluated. Results A total of 413 gene transcript data sets were obtained from the TCGA database, including 381 for gastric cancer and 32 for normal tissues. The expression of various macrophages in tumour tissues was abundant. The immune cell composition, which included resting dendritic cells (p=0.02), M1 macrophages (p=0.031), resting mast cells (p=0.02), CD8 T cells (p=2.445e-04), M0 macrophages (p=6.353e-04), activated mast cells (p=0.006), neutrophils (p=0.003), resting NK cells (p=0.014), and gamma delta T cells (p=0.033), is related to the pathological grade. As the tumour stage of gastric cancer patients progresses, the proportion of some immune cells, including eosinophils (p=0.013), activated mast cells (p=0.042), neutrophils (p=0.007), and resting NK cells (p=0.036) gradually increases, while the proportion of other immune cells, for example, CD8 T cells (p=0.018), Tregs (p=0.039), M1 macrophages (p=0.018), and activated NK cells (p=0.042) gradually decreases. Higher expression of CD8 T cells suggests a better prognosis. Conclusion The composition of tumour-infiltrating immune cells differed greatly in different pathological grades and stages of gastric cancer. CD8 T cells can be used as a prognostic factor for gastric cancer patients.

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703 Favorable pre-clinical safety profile of the novel not-alpha IL-2 agonist ANV419 supports first in human clinical development

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.703 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A731-A731

Author(s):

Christoph Huber ◽

Guzman Alonso ◽

Elena Gerralda ◽

Christoph Bucher ◽

Philippe Jacqmin ◽

...

Keyword(s):

Blood Pressure ◽

T Cells ◽

Nk Cells ◽

Cancer Patients ◽

Cd8 T Cells ◽

Dose Escalation ◽

Safety Profile ◽

Serum Cytokines ◽

Dose Dependent Increase ◽

Dose Dependent

BackgroundANV419 is a novel interleukin-2 (IL-2)/anti-IL-2 fusion protein with preferential signaling through the IL-2 beta/gamma receptor that induces selective proliferation of CD8 T cells and NK cells in vivo for the treatment of cancer. The safety and pharmacodynamic effects of ANV419 were studied in a 4-week cynomolgus monkey GLP study to support the ongoing PhI dose escalation clinical trial.MethodsANV419 was administered by i.v. injection over 1 min at doses of 0.03, 0.1, 0.3 mg/kg, or vehicle control on days 1 and 15 of the 29-day study. Assessments included body weight, blood pressure, hematology, clinical pathology, serum cytokines, immunophenotyping, histopathology, and pharmacokinetics.ResultsThe pharmacokinetics of ANV419 were characterized by target mediated disposition, with a half-life of approximately 24h at concentrations not affected by target mediated clearance. Dose-dependent increases in WBC were observed after each injection, driven by preferential expansion of CD8 T cells and NK cells over Tregs. NK cells were more sensitive to ANV419 than CD8 T cells reaching maximal proliferation in blood at 0.03 mg/kg vs. 0.3 mg/kg for CD8 T cells. Hematological changes included: transient dose-dependent increase in basophils; elevation in eosinophils, up to 2.2-fold above control animals at > 0.03 mg/kg, remaining within the normal range for cynomolgus monkeys (<1.94 G/L); minor decrease in platelets at day 4 after each injection. There were no relevant treatment-related changes in inflammatory serum cytokines (IL-1b, IL-5, IL-6, IL-8, IFNg, TNFa, GM-CSF). A mild systemic inflammatory response was observed at 0.3 mg/kg evidenced by a transient increase of CRP on days 4 and 19, preceded after the first injection by a slight dose dependent increase in IL-1RA at 4h post injection, and an increase in IL-10 at 24h post treatment at 0.3mg/kg. No significant changes in body weights or blood pressure and no signs of capillary leak were observed during the entire study.A multi-part PhI dose-escalation study of ANV419 has been initiated in cancer patients. In the part A single patient escalation cohort, two patients have been dosed Q2W multiple times with 0.003mg/kg and 0.006mg/kg respectively with the expected PD profile and no DLT observed.ConclusionsConsistent findings, relating to expected effects of ANV419 as a not-alpha IL-2 agonist, demonstrated a favorable tolerability and safety profile at pharmacodynamically relevant doses that strongly support its translational development in cancer patients to identify clinical benefits.

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263 Pleiotropic effects of IL-7 in prostate cancer patients receiving Sipuleucel-T vaccination

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0263 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A287-A287

Author(s):

Caroline Duault ◽

Nirasha Ramchurren ◽

Russell Pachynski ◽

Lawrence Fong ◽

Sean Bendall ◽

...

Keyword(s):

Prostate Cancer ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Cancer Patients ◽

T Cell Activation ◽

Cd8 T Cells ◽

Therapeutic Potential ◽

Intracellular Cytokine Staining ◽

Γδ T Cells

BackgroundSipuleucel-T (Provenge) is the first therapeutic vaccination approved by the FDA so far, indicated for advanced metastatic prostate cancer patients. Despite an improvement of the overall survival, the benefits of the therapy are still short-term so increasing the duration of the efficacy is necessary. Specifically, T-cell anergy is one of the challenges that we need to overcome to improve the overall efficacy. IL-7 is known to promote the naive T cell activation and to increase the proliferation and activation of the T cell memory subsets. Therefore, in this phase II clinical trial, we tested the therapeutic potential of a human recombinant glycosylated IL-7 after completion of the Provenge therapy on asymptomatic advanced prostate cancer patients.MethodsTo get a comprehensive analysis of the immune landscape in these patients, we performed CyTOF analysis on PBMC samples obtained at week 1 (baseline) and week 6 after the beginning of the IL-7 therapy. After stimulation with PMA/Ionomycin, we proceeded to surface and intracellular cytokine staining before acquisition on the CyTOF. The data were then analyzed by expert gating on Cytobank.ResultsAt 6 weeks post therapy, our data showed an increase in the number of circulating T lymphocytes in the IL-7 cohort, especially CD8 T cells, in accordance with previous literature. Even though of the frequency of CD4 T cells did not increase, the cells showed greater functionalities, with increased expression of IL-2, TNFα and IL-6 upon stimulation by PMA-Ionomycine. Cytotoxic subsets were also positively affected, with increased expression of IFNγ in CD8 T cells, TNFα in NK cells and IL-2 in γδ T cells. Moreover, PD-1 expression was decreased on CD4, CD8 and γδ T cells while CD137 increased on CD4, CD8 and NK cells. In addition, despite a reduction in the pool of circulating monocytes, we observed higher TNFα expression in these cells.ConclusionsAltogether, our data revealed multiple effects of IL-7 in these patients, highlighting a complex set of in vivo mechanisms. In the future, knowledge of these effects may help in choosing the best agents to use in combination with IL-7 and/or the best patients to benefit from IL-7 as part of their therapeutic approach.Trial RegistrationNCT01881867Ethics ApprovalThe study was last approved by Fred Hutchinson Cancer Research Center Institutional Review Board, IR file 8037, on January 23, 2020

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Reduced frequency of perforin-positive CD8+ T cells in menstrual effluent of endometriosis patients compared to healthy controls

10.1101/2020.12.03.20243436 ◽

2020 ◽

Author(s):

Timo Schmitz ◽

Verena Hoffmann ◽

Elisabeth Olliges ◽

Alina Bobinger ◽

Roxana Popovici ◽

...

Keyword(s):

T Cells ◽

Cell Adhesion ◽

Nk Cells ◽

Adhesion Molecules ◽

Cd8 T Cells ◽

Cell Adhesion Molecules ◽

Nk Cell ◽

Control Group ◽

Healthy Controls ◽

Tnf Α

AbstractBackgroundEndometriosis is a widespread disease in women of reproductive age. The disease often reduces life quality of women affected by symptoms like dysmenorrhea, dyspareunia or infertility. Diagnosis remains challenging and no causal treatments exist until now. The scientific literature indicates many immunological changes like reduced cytotoxicity of natural killer cells and macrophages or altered concentrations of cytokines or cell adhesion molecules in women with endometriosis. Research has been concentrated on immunological alterations in peripheral blood, endometrial tissue and peritoneal fluid of women with endometriosis. Yet, knowledge on immunological differences in menstrual effluent is scarce. The aim of this study was to investigate menstrual effluent of hormone-free women with endometriosis in comparison to age matched healthy controls regarding selected immunological parameters.Methods12 women with endometriosis and 11 healthy controls were included in the study. Menstrual effluent (ME) was collected using menstrual cups over a time period of 24 h, and venous blood samples (PB) were taken. Mononuclear cells were obtained from ME (MMC) and PB (PBMC) using density gradient centrifugation and analyzed using flow cytometry. Furthermore, concentrations of cell adhesion molecules (ICAM-I and VCAM-I) and cytokines (IL-6, IL-8 and TNF-α) were measured in centrifugation supernatant of ME and plasma of PB.ResultsThe comparison of MMC from women with endometriosis and healthy controls revealed a significantly lower frequency of perforin-positive cells among CD8+ T cells in endometriosis patients compared to healthy controls. No additional differences regarding frequencies of CD8+ or CD4+ T cells, regulatory T cells (Tregs), NK cell subsets or monocytic subsets could be detected between MMC or PBMC, of endometriosis patients and healthy controls. Comparing MMC with PBMC revealed that MMC contained significantly more B cells and significantly less T cells than PBMC. Among the T cells, the CD4/CD8 ratio was significantly higher in MMC, and Tregs were significantly less common in MMC. T cells and NK cells expressed significantly more CD69 in MMC., NK cells of the MMC were predominantly CD56bright/CD16dim and the CD56dim NK cell subset had a low frequency of perforin+ cells, in contrast to CD56dim NK cells of PBMC, which were predominantly perforin-positive. However, NKp46 was significantly more expressed on NK cells from MMC. Finally, the endometriosis group had significantly lower plasma ICAM-I concentrations than the control group. There were no significant differences in VCAM-1, IL-6, IL-8 or TNF-α between the two groups.ConclusionMMC are distinctively different from PBMC and, thus, seem to be of endometrial origin. The CD8+ T cells from the ME were significantly less perforin-positive in endometriosis patients than in healthy controls, suggesting a reduced cytotoxic potential.

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