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

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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Circulating immune markers predict the therapeutic effect in primary lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e21203 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e21203-e21203

Author(s):

Liangliang Xu ◽

Jitian Zhang ◽

Li Yang ◽

Guangqiang Shao ◽

Taiyang Liuru ◽

...

Keyword(s):

Lung Cancer ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Blood Lymphocyte ◽

Lymphocyte Subpopulations ◽

Significant Difference

e21203 Background: Radiotherapy (RT), surgical resection (SR), and immunotherapy (IT) as main therapies in lung cancer have either suppressive or stimulatory effects on the immune system. It’s still unclear the mechanism involved in the systemic changes of immune cells in the blood. Peripheral blood lymphocyte subpopulations were useful markers for evaluating immune response in tumor patients. Hence, we aimed to systematically investigate the alteration of lymphocyte subpopulations during the local therapies to evaluate antitumor treatment effects. Methods: Blood samples were obtained EDTA coated tubes and then centrifuged gently for white blood cell separation. The white blood cells in 10% DMSO and 90% FBS were frozen slowly in -80°C refrigerator. The following fluorochrome-conjugated surface and nuclear antibodies were used in the lymphocyte subtyping: CD11b, CD45, CD19, CD3, CD56, CD4, CD8a, CD25,CD127 and FOXP3. The staining cells were detected in the BD FACS machine and data were analyzed by the paired T-test. The percentage of Lymphocytes, Myeloid cells, B cells, T cells, Treg, CD8+ T cells, CD4+ T cells, NK cells, and NKT were examined. Results: Between July 2019 and January 2020, a total of 176 patients eligible, including 135 RT patients and 29 SR patients,12 IT patients, with both blood collection with both Pre, During and End therapies. Before local therapies, the percentage of total T cells in the RT group was significantly higher than SR (RT v.s SR mean:64.1 v.s 55.3, P = 0.02) while CD8+ T cells (RT v.s SR mean:28.2 v.s 34.5, P = 0.04)and Tregs (RT v.s SR mean:0.0 v.s 0.1, P = 0.055) were lower. The baseline level of T cells and their subtypes showed a significant difference in these two group patients. After local therapies, myeloid cells, lymphocytes, CD4+ T cells, CD8+ T cells, NK cells were significant different. There is no significant difference due to the smaller number of IT patients. In the RT group, lymphocytes (Pre-RT v.s End-RT mean:75.2 v.s 54.3, P = 0.004) and B cells (Pre-RT v.s End-RT mean:12.6 v.s 8.0, P = 0.03) were significantly decreased while other subpopulations didn’t show any significant difference after RT. Interestingly, in the SR group, there was a significant increase in CD4+ T cells (mean:59.0 v.s 62.1, p = 0.02) a trend of reduction in CD8+ T cells (mean:34.5 v.s 32.0, p = 0.055) after SR. In addition, there was an increased trend of Tregs after IT. Conclusions: There are some different patterns of distribution in subtypes of leukocytes in operable and inoperable patients and between different therapies. All RT, SR and IT changed the distribution of peripheral blood lymphocyte subpopulations. Further validation study is warranted to validate our findings particularly in circulating lymphocytes and B cells as a marker to evaluate immune status after RT, CD4+ T cells and CD8+ T cells after SR, Tregs after IT, as well as their relationship with tumor microenvironment and implication for personalized care.

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Effects of Morbid Obesity and Metabolic Syndrome on the Composition of Circulating Immune Subsets

Frontiers in Immunology ◽

10.3389/fimmu.2021.675018 ◽

2021 ◽

Vol 12 ◽

Author(s):

Leontine H. Wijngaarden ◽

Erwin van der Harst ◽

René A. Klaassen ◽

Martin Dunkelgrun ◽

T Martijn Kuijper ◽

...

Keyword(s):

Metabolic Syndrome ◽

Morbid Obesity ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Monocyte Subsets ◽

The Absolute

Morbid obesity is characterized by chronic, low-grade inflammation, which is associated with ‘inflamm-aging’. The presence of metabolic syndrome (MetS) might accelerate this phenomenon of metaflammation. In this study, we assessed the effects of morbid obesity and MetS on the composition of a broad spectrum of immune cells present within the circulation. A total of 117 morbidly obese patients (MOP) without MetS (MetS-), 127 MOP with MetS (MetS+) and 55 lean controls (LC) were included in this study. Absolute numbers of T cell, B cell, NK cell and monocyte subsets were assessed within peripheral blood using flow cytometry. Both absolute cell numbers and proportion of cells were evaluated correcting for covariates age, body mass index and cytomegalovirus serostatus. Although the absolute number of circulating CD4+ T cells was increased in the MetS+ group, the CD4+ T cell composition was not influenced by MetS. The CD8+ T cell and B cell compartment contained more differentiated cells in the MOP, but was not affected by MetS. Even though the absolute numbers of NK cells and monocytes were increased in the MOP as compared to LC, there was no difference in proportions of NK and monocyte subsets between the three study groups. In conclusion, although absolute numbers of CD4+ and CD8+ T cells, B cells, NK cells and monocytes are increased in MOP, obesity-induced effects of the composition of the immune system are confined to a more differentiated phenotype of CD8+ T cells and B cells. These results were not affected by MetS.

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Expression of Tim-1 and Its Pathogenetic Role in Primary CNS Lymphoma

Blood ◽

10.1182/blood.v124.21.2961.2961 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2961-2961

Author(s):

Momoko Nishikori ◽

Wataru Kishimoto ◽

Hiroshi Arima ◽

Kotaro Shirakawa ◽

Toshio Kitawaki ◽

...

Keyword(s):

Central Nervous System ◽

T Cells ◽

Nervous System ◽

B Cells ◽

B Cell ◽

Cd8 T Cells ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Regulatory B Cells ◽

A Cell

Abstract Primary central nervous system lymphoma (PCNSL) is a subtype of non-Hodgkin’s lymphoma that arises within the central nervous system (CNS) as a primary lesion, most of which demonstrate diffuse large B-cell lymphoma (DLBCL) histology. However, CNS is recognized as an “immune sanctuary”, and it is not clear in what mechanism B cells develop tumor at this immunoprivileged site. In the past mouse models of multiple sclerosis and cerebral infarction, regulatory B cells, a population of B cells with high IL-10 producing capacity, were reported to have a function to migrate to CNS and suppress inflammation. As the IL-10 level is typically increased in the cerebrospinal fluid (CSF) of PCNSL patients, we hypothesized that PCNSL might originate from B cells that have a physiological role to produce IL-10 for suppressing unfavorable inflammation in CNS, such as regulatory B cells in mice. Recently, a cell surface molecule T cell immunoglobulin domain and mucin domain protein 1 (Tim-1) has been reported to be specifically expressed in the majority of regulatory B cells in mice. Tim-1 was originally identified as a costimulatory molecule on T cells that negatively regulates cellular immune response. Regulatory B cells in mice with defective Tim-1 mutation were reported to demonstrate a profound defect in IL-10 production, suggesting that Tim-1 plays an essential role in their IL-10 production. However, there has been no previous report on Tim-1 expression on human B-cells or B-cell lymphomas, or what function they may serve if it is present. We performed immunohistochemical staining of Tim-1 in various formalin-fixed paraffin embedded lymphoma samples. We observed strong expression of Tim-1 in PCNSL samples, in contrast to its lower expression in other DLBCL and follicular lymphoma samples. Expression of Tim-1 is also detected in a cell line derived from PCNSL (TK), as well as in several other B-cell lymphoma cell lines, by RT-PCR and western blot. As we detected spontaneous shedding of the ectodomain of Tim-1 in the culture media of Tim-1-expressing B-cell lymphoma cell lines, we examined whether Tim-1 can also be detected in the CSF of PCNSL patients. By ELISA, we detected soluble Tim-1 in the CSF of PCNSL patients with active disease, and found it undetectable after the successful treatment with chemo/radiotherapy. The level of soluble Tim-1 in the CSF was positively correlated with IL-10, and it is suggested that these two molecules are functionally related also in humans. In a patient with continuous Tim-1 detection in the CSF after chemotherapy while radiological examination could no longer detected any abnormality, subsequently manifested relapse in the brain. According to these findings, soluble Tim-1 in CSF may be expected to serve as a sensitive biomarker for PCNSL. To clarify the biological role of soluble Tim-1 in tumor microenvironment, we examined its effect on T cell function. We stimulated CD4+ and CD8+ T cells with or without soluble Tim-1 in vitro and compared their cytokine production. The result showed that, in the presence of soluble Tim-1, both IL-2 and IFN-g production was suppressed in CD8+ T cells. In conclusion, Tim-1 is expressed in PCNSL and shedding of extracellular domain of Tim-1, in addition to IL-10, may contribute to the immunosuppressive microenvironment of PCNSL. Disclosures No relevant conflicts of interest to declare.

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B-cell and T-cell values in peripheral blood in Polish mixed-breed rabbits with addition of blood of meet breeds

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2014-0060 ◽

2014 ◽

Vol 17 (3) ◽

pp. 421-426 ◽

Cited By ~ 1

Author(s):

B. Tokarz-Deptuła ◽

P. Niedźwiedzka-Rystwej ◽

B. Hukowska-Szematowicz ◽

M. Adamiak ◽

A. Trzeciak-Ryczek ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Laboratory Animals ◽

Immunological Parameters ◽

Four Seasons ◽

Mixed Breed ◽

The Impact

Abstract In Poland, rabbit is a highly valued animal, due to dietetic and flavour values of its meat, but above all, rabbits tend to be commonly used laboratory animals. The aim of the study was developing standards for counts of B-cells with CD19+ receptor, T-cells with CD5+ receptor, and their subpopulations, namely T-cells with CD4+, CD8+ and CD25+ receptor in the peripheral blood of mixed-breed Polish rabbits with addition of blood of meet breeds, including the assessment of the impact of four seasons of the year and animal sex on the values of the immunological parameters determined. The results showed that the counts of B- and T-cells and their subpopulations in peripheral blood remain within the following ranges: for CD19+ B-cells: 1.05 - 3.05%, for CD5+ T-cells: 34.00 - 43.07%, CD4+ T-cells: 23.52 - 33.23%, CD8+ T-cells: 12.55 - 17.30%, whereas for CD25+ T-cells: 0.72 - 2.81%. As it comes to the season of the year, it was observed that it principally affects the values of CD25+ T-cells, while in the case of rabbit sex, more changes were found in females.

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Engineered immunostimulatory cells can convert PBMCs from chronic lymphocytic leukemia (CLL) patients into potent tumor killing immune cells.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.7517 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 7517-7517

Author(s):

Joshua W. Keegan ◽

Frank Borriello ◽

Stacey M. Fernandes ◽

Jennifer R. Brown ◽

James A. Lederer

Keyword(s):

T Cells ◽

B Cells ◽

Tumor Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Immune Cells ◽

Killing Activity ◽

Tumor Killing ◽

Tumor Cell Killing ◽

Potent Tumor

7517 Background: Alloplex Biotherapeutics has developed a cellular therapeutic that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand populations of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). This process leads to a 300-fold expansion of NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells that are called SUPLEXA cells, which will be cryopreserved and transferred back into patients as an autologous immune cell therapy for cancer. In this study, PBMCs from CLL patients were used to generate SUPLEXA cells as a first approach to comparatively profile SUPLEXA cells from cancer patients and normal healthy volunteers (NHVs). Methods: ENLIST cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million (M) PBMCs from 10 CLL patients or 2 NHVs were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Results: Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF) using a 47-marker antibody panel. CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all 10 CLL patients showed expansion of NK cells (17%), CD8 T cells (11%), and CD4 T cells (7.5%) that were similar in phenotype to SUPLEXA cells from NHVs showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells (60% ± 9.5%) was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and NHV patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell (MEL-14 melanoma cells expressing RFP) ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients (77.8% ± 2.6% at 2:1) and NHVs (81.5% ± 0.3% at 2:1) were nearly identical at all effector to target ratios. Conclusions: We demonstrate for the first time that PBMCs from CLL patients can be converted into SUPLEXA cells despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

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Short-Term Immunopathological Changes Associated with Pulse Steroids/IVIG/Rituximab Therapy in Late Kidney Allograft Antibody Mediated Rejection

Kidney360 ◽

10.34067/kid.0001082019 ◽

2020 ◽

Vol 1 (5) ◽

pp. 389-398

Author(s):

Kenna R. Degner ◽

Nancy A. Wilson ◽

Shannon R. Reese ◽

Sandesh Parajuli ◽

Fahad Aziz ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Regulatory T Cells ◽

B Cell ◽

Cd8 T Cells ◽

Hla Class I ◽

B Cell Depletion ◽

Short Term ◽

Antibody Mediated Rejection ◽

Pulse Steroids

BackgroundB cell depletion is a common treatment of antibody-mediated rejection (ABMR). We sought to determine the specific immunopathologic effects of this therapeutic approach in kidney transplantation.MethodsThis was a prospective observational study of recipients of kidney transplants diagnosed with late ABMR (>3 months after transplant). Patients received treatment with pulse steroids, IVIG, and rituximab. Donor-specific HLA antibodies (DSA), kidney allograft pathology, renal function, immune cell phenotypes, and 47 circulating cytokines were assessed at baseline and at 3 months.ResultsWe enrolled 23 patients in this study between April 2015 and March 2019. The majority of patients were male (74%) and white (78%) with an average age of 45.6±13.8 years. ABMR was diagnosed at 6.8±5.9 years (4 months to 25 years) post-transplant. Treatment was associated with a significant decline in circulating HLA class I (P=0.003) and class II DSA (P=0.002) and peritubular capillaritis (ptc; P=0.04) compared to baseline. Serum creatinine, BUN, eGFR, and proteinuria (UPC) remained stable. Circulating B cells were depleted to barely detectable levels (P≤0.001), whereas BAFF (P=0.0001), APRIL (P<0.001), and IL-10 (P=0.02) levels increased significantly post-treatment. Notably, there was a significant rise in circulating CD4+ (P=0.02) and CD8+ T cells (P=0.003). We also noted a significant correlation between circulating cytotoxic CD8+ T cells and BAFF (P=0.05), regulatory T cells and IL-10 (P=0.002), and regulatory T cells and HLA class I DSA (P=0.005).ConclusionsShort-term pulse steroids/IVIG/rituximab therapy was associated with inhibition of ABMR (DSA and ptc), stabilization of kidney function, and increased regulatory B cell and T cell survival cytokines. Additional studies are needed to understand the implications of B cell depletion on the crosstalk between T cells and B cells, and humoral components that regulate ABMR.

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Lymphocyte aggregation in response to adrenergic stimulation

Blood ◽

10.1182/blood.v71.5.1470.1470 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1470-1474 ◽

Cited By ~ 5

Author(s):

DE Hammerschmidt ◽

C Jeanneret ◽

M Husak ◽

M Lobell ◽

HS Jacob

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Binding Studies ◽

Beta Adrenergic ◽

Cell Counts ◽

Significant Difference ◽

Induced Aggregation

Abstract A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

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

Blood ◽

10.1182/blood.v104.11.1344.1344 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1344-1344

Author(s):

Helmut R. Salih ◽

Petra Stieber ◽

Andrea Peterfi ◽

Dorothea Nagel ◽

Lothar Kanz ◽

...

Keyword(s):

T Cells ◽

Differential Diagnosis ◽

Nk Cells ◽

Cancer Patients ◽

Tumor Cells ◽

Cd8 T Cells ◽

Control Group ◽

Healthy Individuals ◽

Benign Disorders

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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Anti-GvHD Effect of Antithymocyte Globulin Is Mediated by Antibodies Binding to T Cells and Regulatory NK Cells, and Less So or Not at All by Antibodies Binding to Other Mononuclear Cell Subsets

Blood ◽

10.1182/blood.v116.21.2346.2346 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2346-2346

Author(s):

Mette Hoegh-Petersen ◽

Minaa Amin ◽

Yiping Liu ◽

Alejandra Ugarte-Torres ◽

Tyler S Williamson ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Antithymocyte Globulin ◽

Chronic Gvhd ◽

Wilcoxon Test ◽

Effector Memory

Abstract Abstract 2346 Introduction: Polyclonal rabbit-anti-human T cell globulin may decrease the likelihood of graft-vs-host disease (GVHD) without increasing the likelihood of relapse. We have recently shown that high levels of antithymocyte globulin (ATG) capable of binding to total lymphocytes are associated with a low likelihood of acute GVHD grade 2–4 (aGVHD) as well as chronic GVHD needing systemic therapy (cGVHD) but not increased likelihood of relapse (Podgorny PJ et al, BBMT 16:915, 2010). ATG is polyclonal, composed of antibodies for antigens expressed on multiple cell subsets, including T cells, B cells, NK cells, monocytes and dendritic cells. These cell subsets may play a role in the pathogenesis of GVHD. The anti-GVHD effect of ATG may be mediated through killing/inhibition of one or several of these cell subsets (eg, T cells) or their subsets (eg, naïve T cells as based on mouse experiments naïve T cells are thought to play a major role in the pathogenesis of GVHD). To better understand the mechanism of action of ATG on GVHD, we set out to determine levels of which ATG fraction (capable of binding to which cell subset) are associated with subsequent development of GVHD. Patients and Methods: A total of 121 patients were studied, whose myeloablative conditioning included 4.5 mg/kg ATG (Thymoglobulin). Serum was collected on day 7. Using flow cytometry, levels of the following ATG fractions were determined: capable of binding to 1. naïve B cells, 2. memory B cells, 3. naïve CD4 T cells, 4. central memory (CM) CD4 T cells, 5. effector memory (EM) CD4 T cells, 6. naïve CD8 T cells, 7. CM CD8 T cells, 8. EM CD8 T cells not expressing CD45RA (EMRA-), 9. EM CD8 T cells expressing CD45RA (EMRA+), 10. cytolytic (CD16+CD56+) NK cells, 11. regulatory (CD16-CD56high) NK cells, 12. CD16+CD56− NK cells, 13. monocytes and 14. dendritic cells/dendritic cell precursors (DCs). For each ATG fraction, levels in patients with versus without aGVHD or cGVHD were compared using Mann-Whitney-Wilcoxon test. For each fraction for which the levels appeared to be significantly different (p<0.05), we determined whether patients with high fraction level had a significantly lower likelihood of aGVHD or cGVHD than patients with low fraction level (high/low cutoff level was determined from ROC curve, using the point with maximum sum of sensitivity and specificity). This was done using log-binomial regression models, ie, multivariate analysis adjusting for recipient age (continuous), stem cell source (marrow or cord blood versus blood stem cells), donor type (HLA-matched sibling versus other), donor/recipient sex (M/M versus other) and days of follow up (continuous). Results: In univariate analyses, patients developing aGVHD had significantly lower levels of the following ATG fractions: binding to naïve CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients developing cGVHD had significantly lower levels of the following ATG fractions: capable of binding to naïve CD4 T cells, CM CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients who did vs did not develop relapse had similar levels of all ATG fractions. In multivariate analyses, high levels of the following ATG fractions were significantly associated with a low likelihood of aGVHD: capable of binding to naïve CD4 T cells (relative risk=.33, p=.001), EM CD4 T cells (RR=.30, p<.001), naïve CD8 T cells (RR=.33, p=.002) and regulatory NK cells (RR=.36, p=.001). High levels of the following ATG fractions were significantly associated with a low likelihood of cGVHD: capable of binding to naïve CD4 T cells (RR=.59, p=.028), CM CD4 T cells (RR=.49, p=.009), EM CD4 T cells (RR=.51, p=.006), naïve CD8 T cells (RR=.46, p=.005) and regulatory NK cells (RR=.55, p=.036). Conclusion: For both aGVHD and cGVHD, the anti-GVHD effect with relapse-neutral effect of ATG appears to be mediated by antibodies to antigens expressed on naïve T cells (both CD4 and CD8), EM CD4 T cells and regulatory NK cells, and to a lesser degree or not at all by antibodies binding to antigens expressed on B cells, cytolytic NK cells, monocytes or DCs. This is the first step towards identifying the antibody(ies) within ATG important for the anti-GVHD effect without impacting relapse. If such antibody(ies) is (are) found in the future, it should be explored whether such antibody(ies) alone or ATG enriched for such antibody(ies) could further decrease GVHD without impacting relapse. Disclosures: No relevant conflicts of interest to declare.

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Multidimensional Flow Cytometric (MFC) Analysis of the Immune System of Multiple Myeloma (MM) Patients Achieving Long Term Disease Control

Blood ◽

10.1182/blood.v118.21.810.810 ◽

2011 ◽

Vol 118 (21) ◽

pp. 810-810

Author(s):

Roberto J. Pessoa Magalhaes ◽

María-Belén Vidriales ◽

Bruno Paiva ◽

Maria-Victoria Mateos ◽

Norma C. Gutierrez ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

B Cells ◽

Disease Control ◽

B Cell ◽

Nk Cells ◽

Cell Populations ◽

High Dose ◽

Newly Diagnosed

Abstract Abstract 810FN2 Increasing evidence shows that a small fraction of MM patients (pts) treated with high-dose therapy followed by autologous stem cell transplantation achieve long-term remission. Interestingly, this is not restricted to pts in complete response (CR), since those that revert to a monoclonal gammopathy of undetermined significance (MGUS) profile may also achieve long-term remission, despite the persistence of residual myeloma plasma cells (PCs). These results suggest that in addition to the anti-myeloma therapy, other factors may play a role in the control of the disease. Herein, we used 8-color MFC for detailed characterization of the structural components of the immune system and hematopoietic precursor cells (HPC) in paired bone marrow (BM) and peripheral blood (PB) samples from 26 MM patients in long-term disease control (LTDC): 9 in continuous CR and 17 who reverted to an MGUS profile and that subsequently showed stable disease without treatment for ≥5 years (median of 9 years; range, 5–19). As controls, paired BM and PB samples from 23 newly-diagnosed MGUS and 16 MM pts, together with 10 healthy adults (HA), were studied in parallel. In all BM and PB samples the distribution of the major T- (CD4, CD8, Tregs and γδ), NK- (CD56dim and CD56bright) and B-cell subsets (Pro-B, Pre-B, naïve and memory), in addition to normal PCs, dendritic cell (DC) subsets (plasmacytoid, myeloid and monocytic), monocytes, and CD34+ HPC (myeloid and lymphoid), were studied. The percentage and absolute count of each cell population was analysed in the BM and PB, respectively. Comparison of the two groups of MM pts with LTDC (9 CR vs. 17 MGUS-like) showed similar (p>.05) cellular profiles in PB and BM, except for an increased number of BM and PB normal PCs in CR patients (P≤.04). Consequently, for all subsequent analyses, LTDC myeloma pts were pooled together. When compared to HA, patients with LTDC had increased numbers of CD8 T-cells and CD56dim NK-cells in both the BM and PB (p≤.03 and p≤.01, respectively). Despite this, the distribution of BM and PB CD4, CD8 and γδ T-cells among LTDC patients was similar (p>.05) to that of both newly-diagnosed MM and MGUS cases; in contrast, BM and PB Tregs were significantly decreased vs newly-diagnosed MM (P=.03) and MGUS (P=.04). Regarding B-cells and normal PCs, LTDC patients showed increased numbers of BM B-cell precursors (both Pro-B and Pre-B cells) and normal PCs vs. newly diagnosed MM (P≤.05), but not MGUS, together with increased numbers of naïve B-cells vs. both MM and MGUS pts (P≤.01); all such cell populations returned to levels similar (p>.05) to those of HA. As expected, this also included the number of CD34+ B-cell HPC which was increased among patients who achieved LTDC vs MM (p=.02), at levels similar (p>.05) to those of MGUS and HA. Regarding DC, LTDC patients showed normal DC numbers in PB (but with higher PB myeloid-DC numbers vs. MM; p=.02), in association with decreased numbers of plasmacytoid DC and increased monocytic-DC in the BM vs. HA (p≤.04). No differences were found for the numbers of BM and PB monocytes. In summary, here we investigated for the first time the immune cell profile of MM patients who achieve long-term disease control. Our results show that, as newly-diagnosed MM, patients that achieve long-term disease control also show increased numbers of cytotoxic T-cells and CD56dim NK-cells; however, in contrast to newly-diagnosed MM, among LTDC patients such increase is associated with lower numbers of T-regs and an almost complete recovery of the normal PC, B-cell precursor and naïve B-cell compartments both in BM and PB. Further investigations on the activation and functional status of these cell populations are warranted.MO (%)/SP (cels./μl)HA N= 10MGUS N= 23MM N= 16LTDC-MM N= 26T cells9.588110.6117313113711926 CD4+4.85004.6624^6*5085463 CD8+3.7∼216∼4.63865.32645.3431 TCR γδ.2426.3230.2428.3421 Treg.4137.4141^.54*38.3432NK cells.7∼87∼1.51982.11721.6212 CD56 dim.65∼79∼1.41922.21681.6202B cells2.81471.8104.97*68*1.9160 Pro B.11—.06—.02*—.07— Pre B.6—.4—.08—.23— Naive SP—80—57^—36*—118 Normal-PCS.18.9.11.7.008.72*.11.84DCs.3449.3653.6848.558 Monocytes2.22472.42853.43023.1315 m-DC SP—11—14—8*—12 MO-DC.11∼29.2036.434.2837 p-DC.2∼4.1.145.112.8.123.8CD34+.9∼1.46.61.1.261.4.431.4 Mie-HPC.8∼—.53—.26—.36— Linfo-HPC.1—.07—.03*—.05—*p≤.05 LTDC vs MM: ^ p≤.05 LTDC vs MGUS; ∼ p≤.05 LTDC vs HA Disclosures: Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Martinez:Janssen: Honoraria; Celgene: Honoraria. Maiolino:Centocor Ortho Biotech Research & Development: Research Funding.

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